Glycine enolates: The effect of formation of iminium ions to simple ketones on α-amino carbon acidity and a comparison with pyridoxal iminium ions

Article abstract of DOI:10.1021/ja078006c

Equilibrium constants in D₂O were determined by ¹H NMR analyses for formation of imines/iminium ions from addition of glycine methyl ester to acetone and from addition of glycine to phenylglyoxylate. First-order rate constants, also determined by ¹H NMR, are reported for deuterium exchange between solvent D₂O and the α-amino carbon of glycine methyl ester and glycine in the presence of increasing concentrations of ketone and Bronsted bases. These rate and equilibrium data were used to calculate second-order rate constants for deprotonation by DO^- and by Bronsted bases of the α-imino carbon of the ketone adducts. Formation of the iminium ion between acetone and glycine methyl ester and between phenylglyoxylate and glycine is estimated to cause 7 unit and 15 unit decreases, respectively, in the pK_a's of 21 and 29 for deprotonation of the parent carbon acids. The effect of formation of iminium ions to phenylglyoxylate and to 5′-deoxypyridoxal (DPL) [Toth, K.; Richard, J. P. J. Am. Chem. Soc. 2007, 129, 3013-3021] on the carbon acidity of glycine is similar. However, DPL is a much better catalyst than phenylglyoxylate of deprotonation of glycine, because of the exceptionally large thermodynamic driving force for conversion of the amino acid and DPL to the reactive iminium ion.

Full text of DOI:10.1021/ja078006c

Published on Web 01/17/2008
Glycine Enolates: The Effect of Formation of Iminium Ions to
Simple Ketones on r-Amino Carbon Acidity and a
Comparison with Pyridoxal Iminium Ions
Juan Crugeiras,# Ana Rios,*,# Enrique Riveiros,# Tina L. Amyes,† and
John P. Richard*,†
Departamento de Qu´ımica F´ısica, Facultad de Qu´ımica, UniVersidad de Santiago, 15782
Santiago de Compostela, Spain and Department of Chemistry, and UniVersity at Buffalo, SUNY,
Buffalo, New York 14260
Received October 18, 2007; E-mail: qfarr2cn@usc.es; jrichard@chem.buffalo.edu
Abstract: Equilibrium constants in D2O were determined by 1H NMR analyses for formation of imines/
iminium ions from addition of glycine methyl ester to acetone and from addition of glycine to phenylglyoxylate.
First-order rate constants, also determined by 1H NMR, are reported for deuterium exchange between
solvent D2O and the R-amino carbon of glycine methyl ester and glycine in the presence of increasing
concentrations of ketone and Brønsted bases. These rate and equilibrium data were used to calculate
second-order rate constants for deprotonation by DO- and by Brønsted bases of the R-imino carbon of the
ketone adducts. Formation of the iminium ion between acetone and glycine methyl ester and between
phenylglyoxylate and glycine is estimated to cause 7 unit and 15 unit decreases, respectively, in the pKa’s
of 21 and 29 for deprotonation of the parent carbon acids. The effect of formation of iminium ions to
phenylglyoxylate and to 5′-deoxypyridoxal (DPL) [Toth, K.; Richard, J. P. J. Am. Chem. Soc. 2007, 129,
3013-3021] on the carbon acidity of glycine is similar. However, DPL is a much better catalyst than
phenylglyoxylate of deprotonation of glycine, because of the exceptionally large thermodynamic driving
force for conversion of the amino acid and DPL to the reactive iminium ion.
putational,10 and X-ray crystallographic studies11 are converging
to show that the conversion of the protein-bound amino acid to
the zwitterionic carbanion reaction intermediate is strongly
favored by catalysis at a nonpolar enzyme active site.5
1. Introduction
We are interested in characterizing the kinetic and thermo-
dynamic barriers for deprotonation of the R-amino carbon of
amino acids1-3 and peptides4 in water and in understanding the
mechanism by which enzymes lower these barriers in catalysis
of deprotonation of amino acids.1,5 There are enzymes which
catalyze deprotonation of amino acids such as alanine,6,7
glutamate,8-10 and diaminopimelate11 without any assistance
from an electrophilic cofactor to stabilize negative charge at
the R-amino carbon. The results of experimental,1,5,12,13 com-
Pyridoxal 5′-phosphate (PLP) is an extraordinary electrophilic
catalyst of carbon deprotonation of R-amino acids in water,14,15
and at enzyme active sites.16 The first step in the mechanism
for covalent catalysis by PLP is formation of an imine between
the amino acid and PLP. Formation of this adduct labilizes all
of the bonds of the R-imino carbon, because heterolytic bond
cleavage with loss of H+, CO2, or R+ gives a carbanion that is
strongly stabilized by delocalization of negative charge onto
the pyridine ring of the cofactor.
# Universidad de Santiago.
† University at Buffalo, SUNY.
(1) Rios, A.; Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 2000, 122, 9373-
9385.
The presence of the pyruvoyl prosthetic group at enzymes
that catalyze decarboxylation of amino acids suggests that this
ketone is an effective electrophilic catalyst of reactions that
proceed through R-amino carbanion intermediates.17-20 How-
(2) Rios, A.; Crugeiras, J.; Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc.
2001, 123, 7949-7950.
(3) Rios, A.; Richard, J. P. J. Am. Chem. Soc. 1997, 119, 8375-8376.
(4) Rios, A.; Richard, J. P.; Amyes, T. L. J. Am. Chem. Soc. 2002, 124, 8251-
8259.
(5) Richard, J. P.; Amyes, T. L. Bioorg. Chem. 2004, 32, 354-366.
(6) Spies, M. A.; Toney, M. D. Biochemistry 2003, 42, 5099-5107.
(7) Spies, M. A.; Woodward, J. J.; Watnik, M. R.; Toney, M. D. J. Am. Chem.
Soc. 2004, 126, 7464-7475.
(13) Price, W. D.; Jockusch, R. A.; Williams, E. R. J. Am. Chem. Soc. 1998,
120, 3474-3484.
(8) Gallo, K. A.; Tanner, M. E.; Knowles, J. R. Biochemistry 1993, 32, 3991-
(14) Dixon, J. E.; Bruice, T. C. Biochemistry 1973, 12, 4762-4766.
(15) Toth, K.; Richard, J. P. J. Am. Chem. Soc. 2007, 129, 3013-3021.
(16) Toney, M. D. Arch. Biochem. Biophys. 2005, 433, 279-287.
(17) Tolbert, W. D.; Zhang, Y.; Cottet, S. E.; Bennett, E. M.; Ekstrom, J. L.;
Pegg, A. E.; Ealick, S. E. Biochemistry 2003, 42, 2386-2395.
(18) Graham, D. E.; Xu, H.; White, R. H. J. Biol. Chem. 2002, 277, 23500-
23507.
3997.
(9) Glavas, S.; Tanner, M. E. Biochemistry 1999, 38, 4106-4113.
(10) Puig, E.; Garcia-Viloca, M.; Gonzalez-Lafont, A.; Lluch, J. M. J. Phys.
Chem. A 2006, 110, 717-725.
(11) Pillai, B.; Cherney, M. M.; Diaper, C. M.; Sutherland, A.; Blanchard, J.
S.; Vederas, J. C.; James, M. N. G. Proc. Nat. Acad. Sci. U.S.A. 2006,
103, 8668-8673.
(19) Bach, R. D.; Canepa, C. J. Am. Chem. Soc. 1997, 119, 11725-11733.
(20) Gallagher, T.; Rozwarski, D. A.; Ernst, S. R.; Hackert, M. L. J. Mol. Biol.
1993, 230, 516-528.
(12) Patrick, J. S.; Yang, S. S.; Cooks, R. G. J. Am. Chem. Soc. 1966, 118,
231-232.
9
10.1021/ja078006c CCC: $40.75 © 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 2041-2050
2041

A R T I C L E S
Chart 1
Crugeiras et al.
clidine and 3-chloroquinuclidine and the hydroxyl proton of 3-quinu-
clidinol were exchanged for deuterium, as described previously, before
preparing solutions in D2O.1,4 Phenylglyoxylic acid, methoxyacetic acid,
HFIP, and TFE were dissolved directly in D2O, which resulted in <1
atom % increases in the protium content of this solvent. Phosphate
buffers were prepared by mixing stock solutions of K2DPO4 and KD2-
PO4 in D2O at I ) 1.0 (KCl) to give the desired acid/base ratio. Acetate
buffers were prepared by dissolving the basic form of the buffer in
D2O that contains KCl followed by addition of DCl to give the desired
acid/base ratio at I ) 1.0 (KCl). Buffers of methoxyacetate, HFIP, and
TFE were prepared by dissolving their acidic forms and KCl in D2O
followed by addition of KOD to give the desired acid/base ratio at I )
1.0 (KCl). Solutions of quinuclidine, 3-quinuclidinol, and 3-chloro-
quinuclidine cations in phosphate buffer at pD ) 7.6 were prepared
by mixing the tertiary ammonium ion (I ) 1.0, KCl) and phosphate
buffer (I ) 1.0, KCl) and 1 M KCl to give 0.1 M buffer.
The pH and pD were determined at 25 °C using an Orion model
720A pH meter equipped with a Radiometer GK2321C combination
electrode (γOL ) 0.79) or an Orion model 350 pH meter equipped with
a Radiometer pHC4006-9 electrode (γOL ) 0.78). Values of pD were
obtained by adding 0.4 to the observed pH meter reading.23 The
concentration of deuterioxide ion at any pD was calculated using eq 1,
where Kw ) 10-14.87 is the ion product of deuterium oxide at 25 °C
and γOL is the apparent activity coefficient of DO- under our
experimental conditions.24,25
ever, there is little known about the relative advantage for
electrophilic catalysis of deprotonation of amino acids by simple
carbonyl compounds and by PLP, because there have been few
model studies of such catalysis by the former electrophiles.21,22
We have reported that the simple ketone acetone possesses a
significant fraction of the power of PLP as a catalyst of the
deprotonation of amino acids, because of the strong carbon
acidity of the iminium ion adduct 1-H (Chart 1).2 We report
here the full results from our earlier communication and
extensive new data for Brønsted base catalysis of deprotonation
of 1-H and for catalysis of deprotonation of glycine by
phenylglyoxylate (Chart 1). A comparison of data for catalysis
of the deprotonation of glycine by phenylglyoxylate and for
catalysis by 5′-deoxypyridoxal (DPL)15 shows that the R-amino
carbon acidity of adducts to phenylglyoxylate (2-H, Chart 1)
and to DPL are similar. In other words, the problem of
increasing the acidity of the R-amino carbon of amino acids
may be solved by formation of iminium ion adducts to carbonyl
compounds with structures much simpler than that for PLP. On
the other hand, we find that DPL is a much better catalyst of
carbon deprotonation than phenylglyoxylate at pD 7, so that
there are other properties of PLP, which confer upon it a unique
role as a cofactor in catalysis of bioorganic reactions.
10pD-pK
γOL
w
[DO-] )
(1)
2.3. 1H NMR Analyses. 1H NMR spectra at 500 MHz were recorded
in D2O on a Varian Unity Inova 500 NMR spectrometer or a Bruker
AMX500 NMR spectrometer as described in previous work.1,24,26,27 In
all cases, the relaxation delay between pulses was at least 10-fold longer
than the longest relaxation time of the protons of the substrates being
examined (T1 ) 4 s for glycine methyl ester and glycine). Spectra were
obtained with a sweep width of 2600 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 HOD
at 4.67 ppm or (CH3)4N+ at 2.94 ppm.
2.4. Determination of Equilibrium Constants. The position of the
equilibrium for formation of imines to glycine and glycine methyl ester
1
was determined by H NMR analysis at 25 °C. The formation of the
imine of glycine methyl ester was monitored in solutions that contained
0.1 M glycine methyl ester and 3.0 M acetone at I ) 1.0 (KCl). The
formation of the imine of glycine was monitored in solutions that
contained 0.1-2.0 M glycine, 0.8 M phenylglyoxylate at I ) 1.0 (KCl).
The pD was maintained by use of 0.10 M of the following buffers:
methoxyacetic acid, pD 3.3-4.8; acetic acid, pD 4.5-6; phosphate,
pD 6.2-8; HFIP, pD 8.9, and TFE, pD 12-13. Glycine methyl ester
served as the buffer for experiments at pD > 8. Hydrolysis of the ester
at pD < 8.8 was not significant (<6%) during the ca. 1 h time needed
to record NMR spectra, but the breakdown of the ester at higher pD
prevented the determination of equilibrium constants for imine/iminium
ion formation.
2. Experimental Section
2.1. Materials. Deuterium chloride (37 wt %, 99.5% D), potassium
deuterioxide (40 wt %, 98 + % D), deuterium oxide (99.9% D),
acetone-d6 (99.9 atom % D), glycine methyl ester hydrochloride,
quinuclidine hydrochloride, 3-quinuclidinol, 3-chloroquinuclidine hy-
drochloride, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and 2,2,2-trif-
luoroethanol (TFE) were from Aldrich. Glycine and phenylglyoxylic
acid were purchased from Fluka. The 3-substituted quinuclidines were
purified by recrystallization from the following solvents: quinuclidine
hydrochloride, ethanol; 3-quinuclidinol, acetone and 3-chloroquinucli-
dine hydrochloride, 1:1 (v:v) methanol/propanol. All other chemicals
were reagent grade and were used without further purification.
2.2. General Methods. The acidic protons of glycine, glycine methyl
ester hydrochloride, K2HPO4, KH2PO4, the hydrochlorides of quinu-
X-D
A
[X - D]T
CH2
(Kadd obsd
)
)
)
X ) 1, 2 (2)
Gly
CH2
[Gly]T[Ketone]
A
[Ketone]
(23) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190.
(24) Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 1996, 118, 3129-3141.
(25) Rios, A.; O’Donoghue, A. C.; Amyes, T. L.; Richard, J. P. Can. J. Chem.
2005, 83, 1536-1542.
(26) Richard, J. P.; Williams, G.; Gao, J. J. Am. Chem. Soc. 1999, 121, 715-
(21) Owen, T. C.; Young, P. R., Jr. FEBS Lett. 1974, 43, 308-312.
(22) Young, P. R.; Howell, L. G.; Owen, T. C. J. Am. Chem. Soc. 1975, 97,
6544-6551.
726.
(27) Amyes, T. L.; Richard, J. P. J. Am. Chem. Soc. 1992, 114, 10297-
10302.
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2042 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Effect of Iminium Ions on Carbon Acidity of Glycine
A R T I C L E S
Scheme 1
Values of the observed equilibrium constant (Kadd obsd
(M-1) for imine
)
formation were determined from the ratio of the integrated areas of
X-D
CH2
the peaks for the methylene protons of the imine product (A ), and
of the reactant glycine or glycine methyl ester (AGCHly , eq 2). The
2
relative equilibrium concentration of the glycine-phenylglyoxylate
imine at pD > 8 was determined 24 h after mixing the reactants in
order to ensure that chemical equilibrium had been reached. The rate
of phenylglyoxylate-catalyzed exchange of the R-CH2 hydrogen of
glycine for solvent deuterium at pD 12-13 is similar to the rate of
1
imine formation. However, H NMR analysis shows that deuterium
Figure 1. The dependence on pD of (δCH
)
obsd
(ppm) for the -CH2-
2
enrichment of the R-CH2 groups of glycine and of the phenylglyoxylate
imine are the same within experimental error. Therefore, there is no
detectable enrichment of either species with deuterium, and the ratio
of the concentrations of glycine and its imine to phenylglyoxylate anion
may be determined as the ratio of the sum of integrated peak areas for
hydrogen of the imine formed in D2O that contains: (9), 0.1 M glycine
methyl ester and 3 M acetone; (b), glycine (0.1-2.0 M, depending upon
the pD), and 0.8 M phenylglyoxylate at 25 °C and I ) 1.0 (KCl). The solid
lines show the fit of the data to eq 7 derived for Scheme 2 (9), or to eq 8
derived for Scheme 3 (b).
1
the -CH2- and -CHD- groups from H NMR analyses.
2.5. Kinetic Measurements. All deuterium-exchange reactions were
carried out in D2O at 25 °C and I ) 1.0 (KCl). The reactions were
initiated by preparing solutions of the carbon acid, ketone catalyst, and
the buffer at the same pD and I ) 1.0 (KCl) and mixing these reagents
to give a final carbon acid concentration of 15 mM. A slow downward
drift in the pD was observed during the deuterium exchange reactions
of glycine methyl ester, due to the competing hydrolysis of the ester.2
The pD of these solutions was monitored and maintained within 0.05
unit of the initial value by the addition of an aliquot of a 2 M KOD
solution. An even slower increase (0.03 units in two weeks) in pD was
observed during deuterium exchange of glycine catalyzed by phenylg-
lyoxylate in the presence of lowest concentrations of buffer. No new
major signals were detected by 1H NMR, and no significant deviation
was observed in the kinetic plots of data for this reaction.
halftimes for the hydrolysis reaction. The values of kex ) 2kobsd
(Scheme 1) were determined from the integrated areas of the singlet
due to the R-CH2 group of glycine and the triplet due to the R-CHD
group of glycine using eq 5, where khyd is the first-order rate constant
for hydrolysis of glycine methyl ester under the conditions of the
+
experiment. The values of khyd were calculated from eq 6, where fND
3
is the fraction of substrate present in the reactive N-protonated form,
and using the second-order rate constants (kB)hyd for general base
catalysis of the hydrolysis reaction (Table S1 of the Supporting
Information) calculated from data reported in part in earlier work.2 A
control experiment at pD 6.6 showed that there is no change in khyd
((5%), for reaction in the presence of a fixed concentration of
phosphate buffer, as the concentration of acetone is increased from 0
to 0.2 M. This shows that there is no significant catalysis of the
hydrolysis reaction of glycine methyl ester by acetone.
The exchange for deuterium of the first R-H of glycine and of glycine
methyl ester was followed by monitoring the disappearance of the
singlet due to the -CH2- group of the substrate and the appearance
khyd
1
of the upfield-shifted triplet due to the -CHD- group by H NMR
kex
)
)
(5)
(6)
spectroscopy at 500 MHz, as described in previous work.2 Reactions
were monitored during the exchange for deuterium of 20-90% of the
first R-H of the substrate, and the reaction progress, R, was calculated
A
CH2
1
2
-
(
)
2ACHD
using eq 3, where ACH and ACHD are the integrated areas of the peaks
khyd
((kB)hyd[B])fN
2
∑
+
D
3
for the R-CH2 and the R-CHD groups, respectively. Observed first-
order rate constants for exchange for deuterium of a single proton of
the R-CH2 group of the substrate, kobsd (s-1), were determined from
the slopes of linear semilogarithmic plots of R against time (eq 4).
The values of the first-order rate constant for exchange of the first
3. Results
3.1. Equilibrium Constants for Imine Formation. Figure
1
obsd
1 (9) shows the change with changing pD in (δCH
)
(ppm)
2
R-CH2 proton to give monodeuterated product, kex (s-1), were deter-
of the signal for the R-CH2 group of 1/1-D (Scheme 2) that
forms in D2O that initially contains 0.1 M glycine methyl ester
and 3 M acetone at 25 °C and I ) 1.0 (KCl). The signal for the
R-CH2 group moves from 4.18 ppm at pD 8.8 to 4.73 ppm at
pD 4.5, because of protonation of the imino nitrogen. The solid
2
mined as kex ) 2 kobsd
.
A
CH2
R )
(3)
(4)
A
+ ACHD
CH2
line in Figure 1 shows the nonlinear least-squares fit of the data
ln R ) -kobsdt
1-D
CH2
to eq 7, derived for Scheme 2, where (a) δ
) 4.73 and δ1CH
2
) 4.18 ppm are the chemical shifts of the R-CH2 group observed
at extreme low and high pD, and (b) (Ka)1-D ) 10-7.3 is
determined by treating this acidity constant as a variable
parameter (Scheme 2).
The rates of the deuterium exchange and hydrolysis reactions of
glycine methyl ester in the presence of acetone are similar at pD 7.6.
The deuterium enrichment of the glycine product of the hydrolysis
reaction was determined by 1H NMR analysis after more than 10
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 2043

A R T I C L E S
Scheme 2
Crugeiras et al.
Scheme 3
Table 1. Equilibrium Constants in D2O for Addition of Glycine Methyl Ester to Acetone (Scheme 2) and for Addition of Glycine to
Phenylglyoxylate (Scheme 3)a
b
c
1
ketone
amino compound
imine
p(Ka)Gly
p(Ka)X
Kadd d/M-
-D
(CH3)2CO e
+D3NCH2COOCH3
D2NCH2COOCH3
+D3NCH2COOD
+D3NCH2COO-
D2NCH2COO-
1-D
1
2-D2
2-D
2
8.48
7.30 ( 0.04
(3.3 ( 0.4) × 10-3
(5.0 ( 0.8) × 10-2
C6H5COCOO- f
2.76 ( 0.09
6.19 ( 0.04
10.35
(1.0 ( 0.1) × 10-4
1.50 ( 0.01
a At 25 °C and I ) 1.0 (KCl). b The acidity constant of the amino acid in D2O at 25 °C and I ) 1.0 (KCl) determined by potentiometric titration.1
c Apparent acidity constants in D2O at 25 °C and I ) 1.0 (KCl), determined by NMR titration as described in the text. d Equilibrium constant for the addition
of the amino acid to the corresponding carbonyl compound. e Equilibrium constants defined by Scheme 2. f Equilibrium constants defined by Scheme 3.
Figure 1 (b) also shows the change with changing pD in
for the sake of simplicity. However, there are insufficient data
to distinguish this from protonation of the R-imino carboxylate.
2
(δCH )obsd (ppm) of the signal for the R-CH2 group of 2/2-D/2-
2
D2 (Scheme 3) that forms in D2O that initially contains glycine
(0.1-2.0 M depending upon the pD) and 0.8 M phenylglyoxy-
late at 25 °C and I ) 1.0 (KCl).28 The chemical shift of the
R-CH2 group changes because of protonation of the imine
nitrogen and of the amino acid carboxylate groups. The solid
δ1CH (Ka)1-D + δ1-DaD
CH2
2
1
obsd
(δCH
)
)
(7)
2
((Ka)1-D + aD)
2
(δCH
)
)
obsd
2
δ2CH (Ka)2-D(Ka)2-D + δ2-D(Ka)2-D aD + δ2-D2a2D
line shows the fit of these data to eq 8, derived for Scheme 3,
CH2
CH2
2
2
2
2-D2
) 4.51, δ2C-HD ) 4.33, and δC2 H ) 4.11 ppm are the
(8)
(9)
where δ
CH2
((Ka)2-D(Ka)2-D + (Ka)2-D aD + a2D)
2
2
1H NMR chemical shifts for the -CH2- group of the different
2
2
ionic forms of 2, and (Ka)2-D and (Ka)2-D are the acidity
2
aD + (Ka)X-D
aD + (Ka)GlyD
(Kadd obsd ) (Kadd)X-D
)
constants for 2-D2 and 2-D (Table 1) determined from the
(
)
nonlinear least-squares fit of the experimental data to eq 8. There
is a relatively large uncertainty in the value of (Ka)2-D because
2
The apparent equilibrium constants (Kadd obsd
)
(M-1) for
of the limited data for imine formation at low pD (Figure 2).
addition of glycine methyl ester to acetone, or for addition of
glycine to phenylglyoxylate, to form the corresponding imines
Scheme 3 shows protonation of the glycine carboxylate of 2-D,
9
2044 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Effect of Iminium Ions on Carbon Acidity of Glycine
A R T I C L E S
Figure 2. Logarithmic dependence of (K )
add obsd (M-1) on pD for conversion
of glycine and glycine methyl ester to the corresponding imines in D2O at
25 °C and I ) 1.0 (KCl). (Key) Reaction of glycine with phenylglyoxylate
(b). The solid line was calculated using eq 9 (X ) 2) and the equilibrium
constants from Table 1. Reaction of glycine methyl ester with acetone (2).
The solid line was calculated using eq 9 (X ) 1) and the equilibrium
constants from Table 1. Reaction of glycine with 5′-deoxypyridoxal in H2O
(9).15
1
in D2O at 25 °C were determined by H NMR analysis as
described in the Experimental Section. Figure 2 shows the
change, with changing pD, in log(Kadd)obsd for addition of glycine
methyl ester to acetone (2) or of glycine to phenylglyoxylate
(b). The line through the triangles shows the fit of data for the
first reaction to eq 9 (X ) 1), derived for Scheme 2, using
(Ka)MeOGlyD ) 3.3 × 10-9 M,1 (Ka)1-D ) 5.0 × 10-8 M (Table
1) and (Kadd 1-D
) 0.0033 M-1 determined for the formation
)
of the iminium ion at low pD. The line through the circles in
Figure 2 shows the fit of data for the second reaction to eq 9
(X ) 2), derived for Scheme 3, using (Ka)GlyD ) 4.5 × 10-11
M,1 (Ka)2-D ) 6.5 × 10-7 M (Table 1) and values of (Kadd)2)
1.50 M-1 and (Kadd 2-D
for the reaction at low and high pD, respectively. Figure 2 (9)
)
) (1.0 ( 0.1) × 10-4 M-1 determined
Figure 3. (A) Dependence of kex (s-1) for exchange of the first R-proton
of glycine methyl ester in the presence of acetone on the total concentration
of acetate buffer (pD ) 5.56) in D2O at 25 °C and I ) 1.0 (KCl). (Key)
(b) 0.05 M acetone; (1) 0.10 M; (9) 0.25 M; (2) 0.50 M. (B) Dependence
of the intercepts of the linear correlations in Figure 3A (ko) on [acetone].
The slope of Figure 3B gives (kw)obsd (M-1 s-1) for the acetone-catalyzed
deuterium exchange reaction at pD 5.56. (C) Dependence of the slopes of
the linear correlations in Figure 3A (kB)obsd on [acetone]. The slope gives
also shows values of (Kadd obsd for addition of glycine to 5′-
)
deoxypyridoxal determined in earlier work.15
3.2. Rate Constants for Deuterium Exchange. 3.2.1.
Catalysis by Acetone. The exchange for deuterium of the first
R-proton of the methylene group of glycine methyl ester in D2O
1
at 25 °C and I ) 1.0 (KCl) was followed by H NMR (500
(kT)obsd (M-2 s-1) for a termolecular acetone and buffer-catalyzed deute-
MHz) as described in previous work.1,4 Figure 3A shows the
linear dependence of kex ) 2kobsd (Scheme 1) on the total
concentration of acetate buffer (pD 5.56) for reactions in the
presence of increasing [acetone]. The intercepts of these
correlations are equal to ko ) kw f1-D (s-1, Table 2) for
deuterium exchange catalyzed by the solvent, where f1-D is the
fraction of glycine methyl ester present as the iminium ion
adduct 1-D (eq 10, X )1, derived for Scheme 2)29 and kw is
the apparent first-order rate constant for the solvent reaction.
The plot of ko (s-1, Table 2) against [acetone] shown in Figure
3B is linear. The slope is equal to the apparent rate constant
B
rium exchange reaction at pD 5.56.
(kw)obsd ) 2.7 × 10-8 M-1 s-1 for acetone-catalyzed deproto-
nation of glycine methyl ester by solvent at pD 5.56 (eq 11).
Equation 12, which can be simply derived from eq 10 and 11,
shows the relationship between (kw)obsd and kw for deprotonation
of 1-D by the solvent D2O. Substitution of (kw)obsd ) 2.7 ×
10-8 M-1s-1, (Kadd 1-D
)
) 0.0033 M-1, (Ka)Gly-D ) 3.3 × 10-9
M and aD ) 10-5.56 M into eq 12 gives kw ) 8.2 × 10-6 s-1
.
The same analysis of data for reactions in phosphate buffers at
pD 7.64 and 6.61 (Table 2) gives kw ) 9.3 × 10-4 s-1 and kw
) 1.0 × 10-4 s-1, respectively. Figure 4 (b) shows the pD-
rate profile of log kw for deprotonation of 1-D in D2O. The
(28) There is little hydration of the carbonyl group of phenylglyoxalate (,1%)
because only 5% of pyruvate anion is hydrated in water [Esposito, A.;
Lukas, A.; Meany, J. E.; Pocker, Y. Can. J. Chem. 1999, 77, 1108-1117]
and the phenyl for methyl group substitution at pyuvate is expected to cause
the same ca. 130-fold decrease in the equilibrium constant for hydration
observed for acetaldehyde [Guthrie, J. P. J. Am. Chem. Soc. 2000, 122,
5529-5538].
(29) Derived by assuming that the concentration of imine/iminium ion is
negligible because less than 1% of the amino acid is converted to the imine/
iminium ion adduct in the presence of 0.5 M acetone at pD < 8.
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 2045

A R T I C L E S
Crugeiras et al.
Figure 4. pD-rate profiles of kw (s-1) for exchange for deuterium of: (a)
(b), the first R-CH2- hydrogen of 1-D; (b) (2), the first R-CH2- hydrogen
of 2-D. (c) (9), the first R-CH2- hydrogen of N-protonated glycine methyl
ester;1 (1), the first R-CH2- hydrogen of N-protonated glycine.1
Table 2. Rate Constants for Exchange of the First R-Proton of
Glycine Methyl Ester in the Presence of Acetone and Phosphate
or Acetate Buffers in D2Oa
Figure 5. Dependence of kex (s-1) for exchange for deuterium of the first
R-proton of glycine in the presence of phenylglyoxylate on the total
concentration of phosphate buffer at pD ) 7.65 in D2O at 25 °C and I )
1.0 (KCl). Key: (b), 0.1 M phenylglyoxylate; (1), 0.20 M; (9), 0.25 M;
(2), 0.40 M; ([), 0.50 M.
[acetone] /
M
ko1 c
/
(kB)obsd
/
(kw)obsd
/
(kT)obsd
/
B
1 d
1 e
1 f
base
pD
s-
M-1 s-
M-1 s-
M-2 s-
DPO4
7.64
0.01
3.7 × 10-7 2.9 × 10-6 2.7 × 10-6 1.1 × 10-4
2-
pKBD ) 7.0b
0.02
0.05
0.10
0.02
0.05
0.10
0.20
0.05
4.1 × 10-7 3.9 × 10-6
4.9 × 10-7 7.4 × 10-6
reaction catalyzed by buffer at the given concentration of
acetone, where (kB)1-D (M-1 s-1) is the second-order rate
constant for deprotonation of 1-D by the buffer base and fB is
the fraction of the catalyst in the basic form. The values of
(kB)obsd increase linearly with [acetone], as shown in Figure 3C.
The slope of this correlation is the apparent third-order rate
constant for deprotonation of glycine methyl ester catalyzed by
6.2 × 10-7 1.3 × 10-5
6.61
5.56
4.7 × 10-8 1.1 × 10-6 3.3 × 10-7 4.5 × 10-5
6.3 × 10-8 2.3 × 10-6
8.0 × 10-8 4.1 × 10-6
1.1 × 10-7 9.1 × 10-6
CH3COO-
3.7 × 10-9 8.2 × 10-8 2.7 × 10-8 1.6 × 10-6
pKBD ) 5.0b
0.10
0.25
0.50
5.6 × 10-9 1.4 × 10-7
8.5 × 10-9 3.8 × 10-7
1.6 × 10-8 8.1 × 10-7
acetone and acetate anion, (kT)obsd ) 1.6 × 10-6 M-2 s-1
B
a
b
At 25 °C and I ) 1.0 (KCl). Apparent pKa’s of the conjugate acids
in D2O at 25 °C and I ) 1.0 (KCl).24 c Apparent first-order rate constants
(Table 2). Substitution of (kT)obsd into eq 14 along with
B
(Kadd 1-D
)
) 0.0033 M-1, fB ) 0.75, (Ka)Gly-D ) 3.3 × 10-9
M
for deuterium exchange, determined as the intercepts of plots of kex (s-1
)
against [buffer]T. d Apparent second-order rate constants for buffer-catalyzed
deuterium exchange, determined as the slopes of plots of kex (s-1) against
[buffer]T. e Apparent second-order rate constants for acetone-catalyzed
deprotonation of glycine methyl ester determined as the slopes of plots of
ko against [acetone]. f Observed third-order rate constants for buffer-
catalyzed deprotonation of glycine methyl ester, determined as the slopes
and aD ) 10-5.56 M gives (kB)1-D ) 6.5 × 10-4 M-1 s-1 for
deprotonation of 1-D by acetate ion (Scheme 2).
(Ka)Gly-D
(kTB)obsd 1 +
(
)
aD
of plots of (kB)obsd against [acetone].
(kB)1-D
)
(14)
solid line of unit slope shows that deprotonation of 1-D is by
fB(Kadd 1-D
)
DO- ((kDO 1-D
, Scheme 2). The nonlinear least-squares fit of
)
the data to eq 13, where Kw ) 10-14.87 M2 is the ion product of
D2O at 25 °C, and γOL ) 0.79 is the apparent activity coefficient
A similar treatment of the data for the reactions catalyzed by
phosphate buffers at pD 7.64 and 6.61 (Table 2) gives the values
of DO- under our experimental conditions, gives (kDO 1-D
)
)
of (kT)obsd reported in Table 2. Substitution of these values of
13,000 M-1 s-1 for deprotonation of 1-D by DO- (Scheme 2).2
B
(kT)obsd into eq 14, as described above for the reactions
B
catalyzed by acetate ion, gives values of (kB)1-D ) 4.7 × 10-2
and 4.8 × 10-2 M-1 s-1 for reactions at pD 7.64 and 6.61,
respectively, for deprotonation of 1-D by phosphate dianion.
Values of kex (s-1) for the exchange for deuterium of the first
R-proton of glycine methyl ester at pD 7.64 (0.1 M phosphate
buffer) in the presence of acetone and quinuclidine, 3-quinu-
clidinol, or 3-chloroquinuclidine are reported in Table S2 of
the Supporting Information. Table 3 reports values of (kB)obsd
(M-1 s-1) for catalysis by these tertiary amines of deuterium
exchange into glycine methyl ester that were determined from
the slopes of linear plots (not shown) of kex against the total
concentration of the acidic and basic forms of the amine. Values
of (kB)1-D (M-1 s-1) (Table 3) were calculated from the values
of (kB)obsd as described above for the acetate anion-catalyzed
reaction.
(Kadd)X-D[Ketone]
fX-D
)
(10)
(11)
(Ka)Gly-D
1 +
(
)
aD
ko ) (kw)obsd[Ketone] ) kwfX-D
(Ka)Gly-D
(kw)obsd 1 +
(
)
aD
kw )
(12)
(13)
(Kadd X-D
)
(kDO 1-DKw
)
log kw ) log
+ pD
(
)
γOL
The slopes of the correlations in Figure 3A are equal to
(kB)obsd ) (kB)1-D fB f1-D (Table 2) for the deuterium exchange
9
2046 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Effect of Iminium Ions on Carbon Acidity of Glycine
A R T I C L E S
Table 3. Rate Constants for Exchange of the First R-Proton of Glycine Methyl Ester in the Presence of Acetone and 3-Substituted
Quinuclidines at pD 7.64a
[acetone]/
M
(kB)obsd
/
(kT)obsd
/
(kB)1
/
-D
1 f
B
b
c
1
d
1
e
base
pKBD
fB
M-1 s-
M-2 s-
M-1 s-
quinuclidine
12.1
10.7
9.7
3.5 × 10-5
8.7 × 10-4
8.6 × 10-3
0.01
0.02
0.05
0.10
0.01
0.02
0.05
0.10
0.01
0.02
0.05
0.10
8.0 × 10-7
1.4 × 10-6
2.4 × 10-6
5.0 × 10-6
1.9 × 10-6
2.3 × 10-6
3.9 × 10-6
8.4 × 10-6
1.4 × 10-6
2.6 × 10-6
6.5 × 10-6
9.5 × 10-6
4.6 × 10-5
7.3 × 10-5
9.0 × 10-5
460 ( 30
3-quinuclidinol
29 ( 3
3-chloroquinuclidine
3.6 ( 0.5
a In D2O buffered with 0.1 M phosphate at 25 °C and I ) 1.0 (KCl). b Apparent pKa’s of the 3-substituted quinuclidinium cations.24 c Fraction of tertiary
amine present in the basic form at pD 7.64. d Observed second-order rate constants for quinuclidine-catalyzed deuterium exchange at pD 7.64, determined
as the slopes of plots of kex against the total concentration of quinuclidine. e Observed third-order rate constants for acetone-catalyzed deprotonation
of glycine methyl ester by the 3-substituted quinuclidines at pD ) 7.64, determined as the slope of a plot of (kB)obsd against [acetone]. f Second-order rate
constants for deprotonation of 1-D by 3-substituted quinuclidines determined using eq 14.
Table 4. Rate Constants for Exchange of the First R-Proton of Glycine Catalyzed by Phenylglyoxylate in D2Oa
[PhCOCOO-]/
1
d
1
e
1
f
1 g
base
pD
M
ko/s-
kw/s-
(kB)obsd/M-1 s-
(kB)/M-1 s-
2-
DPO4
7.65
0.10
0.20
0.25
0.40
0.50
1.7 × 10-8
3.0 × 10-8
3.3 × 10-8
6.5 × 10-8
7.6 × 10-8
1.70 × 10-3
1.50 × 10-3
1.32 × 10-3
1.63 × 10-3
1.52 × 10-3
2.1 × 10-7
4.6 × 10-7
5.2 × 10-7
1.0 × 10-6
1.2 × 10-6
2.6 × 10-2
2.9 × 10-2
2.6 × 10-2
3.1 × 10-2
3.0 × 10-2
(fBc ) 0.8)
pKBD ) 7.0 b
(kw)av ) (1.5 ( 0.2) × 10-3
(kB)av ) (2.8 ( 0.3) × 10-2
7.05
0.25
0.50
1.5 × 10-8
6.0 × 10-4
3.7 × 10-7
3.0 × 10-2
(fBc ) 0.5)
3.3 × 10-8
6.6 × 10-4
1.0 × 10-6
5.2 × 10-7
4.0 × 10-2
(kw)av ) 6 × 10-4
(kB)av ) 3.5 × 10-2
6.49
0.50
2.0 × 10-8
4 × 10-4
5 × 10-2
( fBc ) 0.2)
a At 25 °C and I ) 1.0 (KCl). b Apparent pKa of the conjugate acid in D2O at 25 °C and I ) 1.0 (KCl).1 c Fraction of the buffer present in the basic form.
d The intercept of plots of kex against [B]T (Figure 5). e Apparent first-order rate constants for solvent-catalyzed deprotonation of 2-D, calculated from the
values of ko as described in the text. f The slope of plots of kex against [B]T (Figure 5). g Apparent second-order rate constants for buffer-catalyzed deprotonation
of 2-D, calculated from the values of (kB)obsd as described in the text.
3.2.2. Catalysis by Phenylglyoxylate. The exchange for
deuterium of the first R-proton of glycine was studied in D2O
at 25 °C and I ) 1.0 (KCl) in solutions buffered with potassium
phosphate (pD 6.49-7.65). Table S3 of the Supporting Infor-
mation gives the observed first-order rate constants kex (s-1
)
for deuterium exchange in the presence of various concentrations
of phenylglyoxylate and phosphate buffer at pD 6.49, 7.05, and
7.65, determined as described in the Experimental Section.
Figure 5 shows the effect of increasing concentrations of
phosphate buffer at pD 7.65 on kex (s-1) for reactions at different
fixed concentrations of phenylglyoxylate. The intercepts of these
linear plots are the first-order rate constants ko ) kw f2-D (s-1
Table 4) for solvent-catalyzed exchange at pD 7.65, where f2-D
) (Kadd 2-D[phenylglyoxylate] is the fraction of glycine present
,
)
as 2-D calculated using eq 10 [X ) 2, 1 . ((Ka)Gly-D/aD)].
Table 4 reports values of kw, calculated using eq 15, for reactions
at different constant [phenylglyoxylate]. Less data is reported
for reactions pD 7.05 and pD 6.49, because the slow deuterium
exchange reactions could only be detected at high [phenylgly-
oxylate]. The linear plot (not shown) of kw (s-1) against [DO-]
Figure 6. Statistically corrected Brønsted correlations for deprotonation
of 1-D (b) and N-protonated glycine methyl ester (2) by buffer bases and
DO- in D2O, where q and p are equal to the number of chemically
equivalent basic sites in the reactant and equivalent acidic hydrogen in the
product, respectively.
was fit to eq 16 to give (kDO 2-D
)
) 1.7 × 104 M-1 s-1 for
deprotonation of 2-D by DO- (Scheme 3) and (kw)o ) 3.1 ×
10-4 s-1 for either pD-independent deprotonation of 2-D or the
kinetically equivalent deprotonation of 2-D2 by DO- (see
Discussion section).
The slopes of the linear correlations in Figure 5 are apparent
second-order rate constants, (kB)obsd ) kB fB f2-D (M-1 s-1) for
buffer-catalyzed deprotonation of glycine, where kB is the
kw ) ko/f2-D
kw ) (kw)o + (kDO 2-D
(15)
(16)
2-
second-order rate constant for deprotonation of 2-D by DPO4
)
[DO-]
(Scheme 3) and fB is the fraction of the buffer in the basic form
9
J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 2047

A R T I C L E S
Crugeiras et al.
This suggests that DO- is normally so much more reactive than
DOD toward deprotonation of R-amino carbon that the reaction
remains first order in [DO-] at pD 5-7. We therefore suggest
that the new pathway observed at low pD is for deprotonation
of a second ionic form or the substrate (2-D2) by DO- (Scheme
3). The observed rate constant for this reaction is pD-
Table 5. Second-Order Rate Constants for Carbon Deprotonation
of 1-D, 2-D and 2-D2 in D2Oa
b
1 c
base catalyst
pKBD
iminium ion
kB/M-1 s-
DO-
16.6
1-D
2-D
2-D2
1-D
1-D
1-D
1-D
2-D
2-D2
1-D
(1.3 ( 0.1) × 104
(1.7 ( 0.1) × 104
∼(3.0 ( 0.7) × 108
460 ( 30
quinuclidine
3-quinuclidinol
3-chloroquinuclidine
12.1
10.7
9.7
independent at pD > p(K ) (Table 1) because the decrease
a 2-D2
29 ( 3
in DO- at decreasing pD is balanced by the increase in the
concentration of the strong carbon acid 2-D2. Equation 17 gives
the relationship between the apparent pD-independent rate
3.6 ( 0.5
2-
DPO4
7.0
(4.7 ( 0.2) × 10-2
(2.8 ( 0.1) × 10-2
∼(1.1 ( 0.3) × 102
(6.5 ( 0.2) × 10-4
CH3COO-
5.0
constant (kw)o and (k
)
for the deprotonation of the minor
DO 2-D2
ionic form 2-D2 by deuterioxide ion. A value of (kDO 2-D
)
≈ 3
2
a At 25 °C and I ) 1.0 (KCl). The quoted errors are standard deviations.
b Apparent pKa of the conjugate acid of the base catalyst in D2O at 25 °C
and I ) 1.0 (KCl).24 c Second-order rate constants for deprotonation of
the iminium ion by the base catalyst, calculated from kinetic data as
described in the text.
× 108 M-1 s-1 (Table 5) was calculated using eq 17, the
experimental value for (kw)o, Kw ) 10-14.87 M2, γOL ) 0.78
and (Ka)2-D ≈ 1.7 × 10-3 M (Table 1).
2
(kDO)2-D Kw
2
(kw)o )
(17)
(18)
(Table 4). The values for kB reported in Table 4 were calculated
from (kB)obsd using the appropriate values of fB calculated
(Ka)2-D γOL
2
from the pD and pKa of the buffer catalyst, and f2-D
)
(kB)2-D aD
2
kB ) (kB)2-D
+
(Kadd 2-D[phenylglyoxylate] (see above). The second-order rate
)
(K )
a 2-D2
constants determined for deprotonation of 2-D are also sum-
marized in Table 5.
The increase with decreasing pD in the apparent second-order
rate constant kB for deprotonation of 2-D by DPO4 is also
2-
4. Discussion
due to the appearance of a second reaction pathway for
deprotonation of the strong carbon acid 2-D2 by DPO4
(Scheme 3). Equation 18 gives the relationship between the
The exchange of the R-CH2 hydrogen of glycine methyl ester
for deuterium from D2O catalyzed by acetone was monitored
at 25 °C and neutral pD, as described in earlier work.1,4 There
is no detectable (<1%) catalysis by acetone of the exchange
for deuterium of the R-CH2 hydrogen of glycine after 1-month
incubation of acetone (1 M) with glycine in D2O at pD 7.8 and
25 °C. Experiments to characterize catalysis of the exchange
for deuterium of the R-CH2 hydrogen of glycine by pyruvate,
an analogue for the pyruvoyl prosthetic group used in some
enzyme-catalyzed decarboxylation reactions,17,18,20 failed be-
cause of the competing bimolecular aldol condensation reaction
of pyruvate. Phenylglyoxylate was examined as a model for
pyruvate that lacks acidic R-hydrogen atoms.
Reaction Pathways. The kinetic data for acetone-catalyzed
deuterium exchange reactions of glycine methyl ester were fit
to a mechanism in which the imine 1-D undergoes irreversible
deprotonation by DO-, the conjugate base of solvent, and by
other Brønsted bases. Two pathways are observed for depro-
tonation of 2-D by solvent and buffer bases. The dominant
pathway at pD 7.65 is for deprotonation of 2-D by DO- and
phosphate dianion. The apparent rate constant for the “solvent”
reaction approaches a limiting value of kw ) 3.1 × 10-4 s-1
and the apparent rate constant for the buffer-catalyzed rate
constant increases as the pD is decreased to 6.49 (Table 4).
These observations show that additional pathways are important
for the reactions of these bases with 2 at low pD.
2-
apparent rate constants kB and the rate constants for deproto-
nation of 2-D ((kB)2-D) and 2-D2 ((kB)2-D ) by DPO42-. The
2
plot (not shown) of kB (M-1 s-1) against aD is linear. The
intercept of this plot gives the second-order rate constant for
deprotonation of the iminium ion 2-D by DPO42-, (kB)2-D
)
2.8 × 10-2 M-1 s-1 (Table 5). Combining the slope of this
plot with (Ka)2-D ) 1.7 × 10-3 M (Table 1) gives the second-
2
2-
order rate constant for deprotonation of 2-D2 by DPO4
,
(kB)2-D ) 1 × 102 M-1 s-1 (Table 5). There is a very large
2
uncertainty in the values of the rate constants estimated for
deprotonation of 2-D2 by DO- and by DPO42-. First, there is
the inherent uncertainty in the value for (Ka)2-D determined
2
by fitting experimental data from Figure 1 to Scheme 3. Second,
there are two carboxylates at 2-D of uncertain relative basicity
which may undergo protonation, and our limited experimental
data is not sufficient to distinguish between the two different
forms of 2-D2.
Brønsted Correlations. The Brønsted correlation of second-
order rate constants kB (M-1 s-1) for deprotonation of 1-D (b)
shown in Figure 6 was obtained using data from Table 5. The
slope of this correlation, â ) 0.83 ( 0.01, is smaller than the
value of â ) 0.92 ( 0.04 for deprotonation of N-protonated
glycine methyl ester (2) determined in earlier work.1 The
smaller Brønsted â observed for 1-D compared with N-
protonated glycine methyl ester is consistent with a Hammond-
type shift to an earlier transition state for deprotonation of the
more strongly acidic carbon acid.31
Figure 4 (2) shows a logarithmic pD-rate profile of rate
constants kw for deprotonation of 2-D. The solid line shows the
fit of the data obtained using values of (kDO 2-D
)
) 1.7 × 104
M-1 s-1 and (kw)o ) 3.1 × 10-4 s-1 for the DO--catalyzed
and the pD-independent reactions, respectively. No similar
upward breaks at pD 5-7 are observed in the pD-rate profiles
for carbon deprotonation of simpler amino acid derivatives.1,30
Electrophilic Catalysis of Deprotonation of Glycine. A
broad goal of this work is to determine the effect of different
electrophilic catalysts on the carbon acidity of glycine. Chart 2
shows that formation of an imine between acetone and glycine
(30) Richard, J. P.; Williams, G.; O’Donoghue, A. C.; Amyes, T. L. J. Am.
Chem. Soc. 2002, 124, 2957-2968.
(31) Hammond, G. S. J. Am. Chem. Soc. 1955, 77, 334-338.
9
2048 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008

Effect of Iminium Ions on Carbon Acidity of Glycine
A R T I C L E S
Chart 2
of glycine methyl ester and of glycine, from the relative carbon
acidity of the iminium ion adducts to these compounds (Chart
2). In particular, 5′-deoxypyridoxal (DPL) is a much better
catalyst of deprotonation of glycine than is phenylglyoxylate
anion, despite the weaker carbon acidity of the adduct to glycine
(Chart 2). Chart 3 shows a comparison of the rate constants for
solvent-catalyzed deprotonation of glycine methyl ester and
glycine, and the rate constants observed upon addition of a 0.01
M ketone catalyst. The first row shows that, despite the strong
carbon acidity of the iminium ion adduct, 0.010 M acetone
provides only modest catalysis of deprotonation of glycine
methyl ester at pD 7.6, because of the unfavorable equilibrium
constant for imine formation. A 250-fold rate acceleration is
observed for catalysis of deprotonation of glycine by 0.010 M
of the much more effective catalyst phenylglyoxylate. However,
this rate acceleration pales in comparison to the 3,000,000-fold
acceleration observed for catalysis of deprotonation of glycine
by 0.010 M DPL at pH 7.1, where the estimated rate constant
for solvent-catalyzed deprotonation of glycine is only 5 × 10-12
methyl ester causes a healthy 2200-fold increase in the second-
order rate constant kDO for deprotonation of the R-amino carbon
by deuterioxide ion. This reflects the ca. 7 unit difference in
the carbon acid pKa of 21 for N-protonated glycine methyl ester
and of 14 for 1-H.2
The formation of the imine between glycine and phenylgly-
oxylate causes a much larger 2.2 × 108-fold increase in the
second-order rate constant kDO for deprotonation of the R-amino
carbon by deuterioxide ion. This is 50-fold larger than the effect
of formation of an imine to the oxygen-ionized form of
5′-deoxypyridoxal (Chart 2). Combining kDO ) 1.7 × 104 M-1
s-1 for deprotonation of 2-D with an estimated solvent deuterium
isotope effect of kDO/kHO ) 1.532 gives kHO ) 1.1 × 104 M-1
s-1 for deprotonation of 2-H by hydroxide ion in H2O (Scheme
3). Equation 19 is the linear logarithmic correlation between
kHO and the carbon acidity pKCH for deprotonation of cationic
ketones and esters.2
s-1 1
.
The large difference in the observed catalytic activity of DPL
and phenylglyoxylate is due to the more favorable equilibrium
constant for formation of the imine to the former electrophile
(Figure 2). The imine 2-D is only barely detectable by 1H NMR
in solutions that contain molar concentrations of glycine and
0.80 M phenylglyoxylate anion, while the reaction of 0.10 M
glycine with 0.010 M DPL at pH 7.1 results in conversion of
80% of the cofactor to 3-H2 at chemical equilbrium.15 These
data show that one imperative for the strong preference of
enzyme catalysts to use pyridoxal 5′-phosphate rather than the
pyruvoyl prosthetic group17,18,20 as a coenzyme for catalysis of
the reactions of amino acids is the larger affinity of the pyridine
cofactor compared with R-keto acids for addition to the amino
group to form an imine. This imperative was noted in an earlier
study of imine formation to PLP.33
10.2 - log kHO
pKCH
)
(19)
(
)
We conclude that imines between pyridoxal and amino acids
are strongly stabilized by intramolecular interaction between
the pyridine ring, the ring substitutents, and the imino nitrogen,33
compared to the corresponding intramolecular interactions at
imines between amino acids and simple ketones and aldehydes.
There is an extensive literature on imine formation in water.
However, to the best of our knowledge there has been no
systematic study reported to rationalize the high stability of
imines to PLP compared to imines to simple aldehydes and
ketones. This study is currently in progress in the laboratory at
the Universidad de Santiago.
0.44
This correlation and kHO ) 1.1 × 104 M-1 s-1 gives a value of
pKa ) 14 for the carbon acidity of 2-H. The carbon acid pKa
for this iminium ion is 15 units smaller than pKa of 29 for the
parent carbon acid glycine,1 and smaller even than the pKa of
17 estimated for 3-H2.15 By comparison, formation of an imine
to acetone causes only a 7 unit reduction in the pKa of glycine
methyl ester.2 The strong carbon acidity for 2-H shows the
extensive stabilization of negative charge of the carbanion 4
that is presumably due to delocalization of negative charge onto
the electron-deficient substituents at 4.
Acknowledgment. We acknowledge the National Institutes
of Health (Grant GM 39754 to J.P.R.), and the Ministerio de
Educacio´n y Ciencia and the European Regional Development
It is important to distinguish the relative absolute reactivity
of the parent carbonyl compounds as catalysts of deprotonation
(32) This is the secondary solvent deuterium isotope effect determined for
deprotonation of acetone by hydroxide ion at 25 °C [Pocker, Y. Chem.
Ind. 1959, 1383-1384].
(33) Gout, E.; Zador, M.; Beguin, C. G. NouV. J. Chim. 1984, 8, 243-250.
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J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008 2049

A R T I C L E S
Chart 3
Crugeiras et al.
Fund (ERDF) (Grant CTQ2004-06594 to A.R. and J.C.) for
generous support of this work.
N-protonated glycine methyl ester; Table S2 of first-order rate
constants, kex, for exchange for deuterium of the first R-proton
of glycine methyl ester in the presence of acetone and buffer
catalysts; Table S3 of first-order rate constants, kex, for exchange
for deuterium of the first R-proton of glycine in the presence
of phenylglyoxylate and buffer catalysts. This material is
available free of charge via the Internet at http://pubs.acs.org.
Note Added after ASAP Publication. Reference 27 was
changed after ASAP publication of this article on January 17,
2008. The current version shows the correct reference.
Supporting Information Available: Table S1 of second-order
rate constants (kB)hyd for general base-catalyzed hydrolysis of
JA078006C
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2050 J. AM. CHEM. SOC. VOL. 130, NO. 6, 2008