Accelerating Decarboxylation by Acid Association
A R T I C L E S
Scheme 1. Intermediates in Benzoylformate Decarboxylase
Scheme 2. Decarboxylation of MT; the Conjugate of Thiamin and
Benzoylformate
argon and cooled to -5 °C. A solution of ethyl benzoylformate (8.2
mL, 3.5 equiv) and anhydrous magnesium chloride (0.6 g, 0.4 equiv)
in 50 mL of ethanol was deoxygenated and immediately added under
nitrogen to the thiamin solution. A sodium ethoxide solution (0.7 g of
sodium in 40 mL of ethanol) was deoxygenated and added to the
mixture with stirring. After 10 min at -5 °C, the solution was acidified
with 12 M hydrochloric acid. The precipitate, consisting of sodium
chloride and unreacted thiamin, was removed by filtration. The filtrate
was concentrated by rotary evaporation at 25 °C to a minimal volume,
which was then dissolved in 25 mL of water and washed with 3 × 50
mL portions of dichloromethane. The aqueous layer was stirred with
Chelex resin (sodium form) for 1-2 h at pH 6 to remove magnesium.
After filtration, the aqueous solution was acidified and lyophilized to
dryness. The resulting yellow solid was dissolved in ethanol, filtered,
and eluted on cellulose with 1:4 (v/v) ethanol/ethyl acetate to give 0.33
g (5%) of the ethyl ester of MT (attempts to increase this amount have
not yet been successful).
thiazolium ring). The diphosphate side chain of TDP is not
involved in catalysis but does bind the cofactor to the protein
through coordination to a magnesium ion, allowing precise
positioning in the active site.14,17 The simplified but chemically
correct analogue of MTDP, 2-(2-mandelyl)thiamin (MT), the
conjugate of benzoylformate and thiamin, undergoes decar-
boxylation at a relatively rapid rate compared to most carboxylic
1
8
1H NMR (400 MHz, DCl in D
(3H, t, J ) 7.2 Hz, CH CH OCO), 2.41(3H, s, CH
-thiazole), 3.25 (2H, t, J ) 5.8 Hz, CH
.94 (2H, t, J ) 5.8 Hz, CH CH OH), 4.43 (2H, q, J ) 7.2 Hz, CH
CN), 5.86 (1H, d, J ) 18 Hz,
CN), 6.81 (1H, s, H-C6′ pyrimidine), 7.32 (3H, m, aromatic),
acids (Scheme 2).
2
O relative to internal DSS): δ 1.30
-C2′ pyrimidine),
CH OH),
2
CH -
Yet, the rate constants for the decarboxylation of MT are
much smaller than that for the comparable enzymic reaction:
kcat for BFD exceeds the unimolecular decarboxylation rate
3
2
3
2
3
.49 (3H, s, CH
3
2
2
2
2
3
6
a b
OCO), 5.36 (1H, d, J ) 18 Hz, H H
constant of MT by a factor of about 10 . Another critical point
a b
H H
of divergence is the observation that the product of decarbox-
ylation of MT, the conjugate base of 2-(1-hydroxybenzyl)-
thiamin (HBnT), undergoes a fast fragmentation reaction that
cleaves the thiamin-derived portion of the conjugate base of
13
7
.54 (2H, m, aromatic). C NMR (100 MHz, DCl in D
2
O relative to
internal DSS): δ 194.53, 169.71, 161.74, 160.85, 145.57, 137.99,
1
4
36.25, 135.15, 130.07, 129.92, 126.14, 107.55, 65.72, 60.36, 49.38,
7.49, 29.63, 20.94, 13.39, 11.90. IR: 3390 (broad), 1631. ESIMS of
2
+
HBnT. The rate constant for fragmentation is about 10 times
parent peak [C H N O S] , calcd 443, found 443.
22
27
4
4
larger than the enzymic kcat (Scheme 3). Protonation of the
The ethyl ester was hydrolyzed in concentrated hydrochloric acid
for 6 days at room temperature. After concentration under a vacuum
and lyophilization, the chloride hydrochloride salt of MT was obtained
(stored dry at -20 °C).
HBnT carbanion at C2R competes with fragmentation and
1
9
produces HBnT. Thus, the protein accelerates the decarbox-
ylation step while reducing the rate of the fragmentation process.
The extent to which the fragmentation process occurs provides
a measure or “clock” versus the protonation reaction that
produces HBnT. In this paper we present evidence for a
previously undetected form of acid catalysis in decarboxylation
that was the subject of a preliminary report.2 This mechanism
provides a basis for explaining the enigmatic distinctions of the
enzymic and nonenzymic reactions of the intermediates. It also
provides more general insights into how decarboxylation may
be catalyzed in other systems.
1H NMR (400 MHz, DCl in D
3H, s, CH -C2′ pyrimidine), 2.49 (3H, s, CH
t, J ) 5.8 Hz, CH CH OH), 3.94 (2H, t, J ) 5.8 Hz, CH
.41 (1H, d, J ) 18 Hz, H CN), 5.90 (1H, d, J ) 18 Hz, H
O relative to internal DSS): δ 2.39
-thiazole), 3.23 (2H,
CH OH),
CN),
2
(
3
3
2
2
2
2
5
6
a
H
b
a
H
b
.82 (1H, s, H-C6′ pyrimidine), 7.30 (3H, m, aromatic), 7.55 (2H, m,
0
+
aromatic). ESIMS [C20
H
23
N
4
O
4
S] , calcd 415, found 371 (MT loses
CO upon ionization, calcd 371).
2
Kinetics. Measurements of the rate of decarboxylation of MT were
conducted using buffer solutions maintained at 25.0 °C in a jacketed
beaker with a circulating water bath. In solutions whose acidity was
pH < 4.0, the reaction was followed at 295 nm. Between pH 4.5 and
Experimental Section
8
.5, the decarboxylation was followed at 328 nm (the absorbance of a
fragmentation product, PTK, see below). Under these conditions the
reactions gave an isosbestic point. Above pH 8.5, no isosbestic point
was observed. H NMR analysis indicated that the ring-opened
1
Materials and Methods. Spectra. H NMR spectra were recorded
at 400 MHz in deuterium oxide. Chemical shifts are relative to DSS
2,2-dimethyl-2-silapentane-5-sulfonate sodium salt). C NMR spectra
1
13
(
derivative of thiamin (from addition of hydroxide)21 and benzoylformate
are the principal products (decarboxylation is not base catalyzed so
that it becomes insignificant in these solutions; see Figure 1). Under
these conditions, thiamin undergoes ring opening at a rapid rate,
comparable to the rate of elimination of benzoylformate from MT. The
two processes were followed at 248 nm and 274 nm, and the rate
were recorded at 100 MHz in deuterium oxide with chemical shifts
relative to DSS. IR: FT spectra were recorded with samples in KBr
pellets. Mass spectra were recorded on an ESI instrument at medium
resolution.
Synthesis of MT. Thiamin chloride hydrochloride (5.0 g, 0.015 mol)
was suspended in 80 mL of ethanol. The mixture was purged with
2
2
constants were obtained using a multiwavelength algorithm.
(18) Hu, Q.; Kluger, R. J. Am. Chem. Soc. 2002, 124, 14858-14859.
(19) Hu, Q.; Kluger, R. J. Am. Chem. Soc. 2004, 126, 68-69.
(20) Hu, Q.; Kluger, R. J. Am. Chem. Soc. 2005, 127, 12242-12243.
(21) Maier, G. D.; Metzler, D. E. J. Am. Chem. Soc. 1957, 79, 4386-4391.
(22) Kluger, R.; Chin, J.; Smyth, T. J. Am. Chem. Soc. 1981, 103, 884-8.
J. AM. CHEM. SOC.
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VOL. 128, NO. 49, 2006 15857