Rearrangement in Coenzyme B -Dependent Mutase
A R T I C L E S
12
cooling for 3 h. Acetic acid was added, the hydrobromide salt of DBU
was filtered off, and the solvent was removed in vacuo. The residue
was purified by column chromatography (silica, elution with 10% ethyl
acetate in petrol) to afford the title compounds (2.63 g, 86%) as an
Hz, 3H), 3.78 (q, J 7.2, 0.9 Hz, 1H), 7.12 (d, J 0.9 Hz, 1H), 11.15 (br,
1H); 13C NMR (125 MHz, d6-acetone) δ 173.8, 166.5, 139.1, 113.5,
45.0, 16.4.
(Z)-3-Methyl[2′-2H1]itaconic acid (racemic 2c). (Z)-2′-Bromo-3-
methylitaconic acid 15 (290 mg, 1.29 mmol) was dissolved in D2O,
lyophilized, and redissolved in D2O (11 mL). To the rapidly stirred
solution was added 5% sodium amalgam (9.1 g, British Drug Houses),
and the mixture was stirred for 50 min. The aqueous phase was decanted
from the mercury, which was washed with water. The combined
aqueous solutions were acidified with 2 M HCl to pH 1 and extracted
with ethyl acetate (5×). The organic phases were washed with brine
and dried with sodium sulfate. The solvent was removed in vacuo to
afford the title compound as a white crystalline solid (181 mg, 97%).
An analytical sample was obtained by recrystallization with acetonitrile;
mp 148-149 °C (lit. value for unlabeled 3-methylitaconic acid: 152-
1
inseparable 1:1 mixture; Rf 0.32 (petrol/ethyl acetate, 9:1); H NMR
(300 MHz, CDCl3) δ 1.15-1.26 (m, 18H)x,y, 2.13 (s, 3H)y, 4.07-4.53
(m, 12H)x,y, 4.53 (s, 1H)x, 5.78 (s, 1H)x, 6.43 (s, 1H)x; HRMS: found
258.1100, C12H18O6 requires 258.1097.
Triethyl But-1-ene-2,3,3-tricarboxylate (12). To a suspension of
sodium hydride (60% w/w NaH in mineral oil, 640 mg, 16 mmol) in
anhydrous THF (40 mL) was added 11a/11b (4.0 g, 15.5 mmol) with
ice-cooling. The mixture was stirred for 1 h, after which it was cooled
in an ice bath. Methyl iodide (2.3 g, 16 mmol) was added dropwise
within 5 min, and the mixture was stirred at 60 °C for 2.5 h. The solvent
was removed, and the residue was partitioned between water and ether.
The ethereal phases were washed with brine and dried over magnesium
sulfate. The solvent was removed, and the oily residue was purified
by chromatography (silica, elution with a mixture of 20% ethyl acetate
in petrol) to afford 3.41 g (12.5 mmol, 80%) of a colorless oil; Rf 0.30
1
154 °C5); H NMR (200 MHz, d6-acetone): δ 1.50 (d, J 7.3 Hz, 3H),
3.78 (q, J 7.3 Hz, 1.1 Hz, 1H), 5.75 (s, 1H); 13C NMR (125 MHz,
d6-acetone): δ 174.9, 167.5, 142.6, 125.4 (t, 1JD-C 24.6 Hz), 41.4, 16.5.
(Z)-3-[2′-2H1,methyl-2H3]Methylitaconate (racemic 2e). This was
prepared in a manner similar to that described for (Z)-3-methyl[2′-2H1]-
itaconate 2c, except that trideuteriomethyl iodide was used in the
methylation step: mp 147-148 °C; 1H NMR (200 MHz, d6-acetone):
3.55 (s, 1H), 5.83 (s, 1H); 13C NMR (125 MHz, d6-acetone): δ 174.9,
1
(15% ethyl acetate in petrol); H NMR (200 MHz, CDCl3) δ 1.23 (t,
J ) 7.1 Hz, 6H), 1.27 (t, J ) 7.1 Hz, 3H), 1.70 (s, 3H), 4.18 (q, J )
7.1 Hz, 6H), 5.67 (s, 1H), 6.32 (s, 1H); 13C NMR (50 MHz, CDCl3) δ
170.4, 165.6, 140.1, 125.9, 61.8, 61.8, 61.1, 57.4, 21.6, 14.1, 13.1.
Triethyl 1,2-Dibromobutane-2,3,3-tricarboxylate (13). To a solu-
tion of triethyl but-1-ene-2,3,3-tricarboxylate 12 (830 mg, 3.01 mmol)
in dry dichloromethane (15 mL) was added bromine (2.9 mL, 50 mmol).
The mixture was stirred for 3 days in the dark. After cooling at 0 °C,
a saturated solution of sodium hydrogen sulfite was carefully added,
and the sulfur formed was removed by filtration. The aqueous phase
was extracted with dichloromethane, and the combined organic phases
were dried over magnesium sulfate and concentrated in vacuo. The
yellowish residue was purified by chromatography (silica, elution with
dichloromethane) to afford the title compound as a colorless oil (1.24
g, 2.84 mmol, 94%); Rf 0.48 (dichloromethane); 1H NMR (200 MHz,
CDCl3) δ 1.23-1.40 (m, 9H), 1.74 (s, 3H), 3.88 (d, J ) 11.3 Hz, 1H),
4.15-4.31 (m, 6H), 4.73 (d, J ) 11.3 Hz, 1H); 13C NMR (50 MHz,
CDCl3) δ 168.8, 168, 167.2, 71.5, 63.5, 63.2, 62.5, 62.3, 38.7, 20.0,
14.0, 13.8.
1
167.6, 141.6, 125.4 (t, JD-C ) 23.6 Hz), 41.4.
2-Methylene[4-2H2]glutaric acid (1g). Sodium metal (144 mg, 6.25
mmol) was reacted with dry ethanol (60 mL). Diethyl malonate (1.0 g,
6.25 mmol) was added, and the solution was cooled in an ice bath.
Ethyl R-bromoacrylate (1.21 g, 6.25 mmol) was added and the reaction
stirred at room temperature for 1 h. The solvent was removed, and the
resulting oil was dissolved in ether. The solution was filtered, and the
ether was removed. The crude product was purified by flash column
chromatography on silica, eluting with 15% ethyl acetate/petrol to afford
triethyl but-3-ene-1,1,3-tricarboxylate as a colorless oil (912 mg,
53%);1H NMR (200 MHz, CDCl3): δ 1.20-1.32 (m, 9H), 2.87 (d, J
) 7.8 Hz, 2H), 3.70 (t, J ) 7.8 Hz, 1H), 4.11-4.25 (m, 6H), 5.62 (s,
1H), 6.20 (s, 1H); 13C NMR (50 MHz, CDCl3): δ 168.6, 166.3, 136.8,
127.6, 61.5, 60.9, 50.9, 31.4, 14.2, 14.1.
A mixture of triethyl but-3-ene-1,1,3-tricarboxylate (1.0 g, 3.67
mmol) and 20% DCl in D2O (8 mL) was refluxed for 7 days. The
solvent was removed to yield a yellow solid. The crude product was
recrystallized from acetonitrile to afford the title compound as a white
crystalline solid (335 mg, 62%): mp 128-129°C; 1H NMR (200 MHz,
(E)-Triethyl 1-Bromo-but-1-ene-2,3,3-tricarboxylate (14). To a
solution of tetra-n-butylammonium fluoride trihydrate (4.9 g, 15.5
mmol) in hexamethylphosphorus triamide (24 mL) was added molecular
sieve beads (3 Å, ca. the volume of the solvent). The mixture was stirred
for 30 min. Triethyl 1,2-dibromobutane-2,3,3-tricarboxylate 13 (9.2
mmol) was added under nitrogen, and the reaction mixture was stirred
overnight. The molecular sieves were filtered off and washed with ether.
After addition of water to the filtrate, the solution was acidified to pH
1 with 1 M sulfuric acid. The aqueous layers were extracted three times
with ether, and the combined organic phases were washed with brine
and dried over magnesium sulfate. The solvent was removed, and the
residue was purified by chromatography (silica, elution with dichloro-
methane) to afford the title compound as a colorless oil (690 mg, 1.94
mmol, 21%); Rf 0.22 (dichloromethane); 1H NMR (200 MHz, CDCl3)
δ 1.18-1.33 (m, 9H), 1.68 (s, 3H), 4.13-4.30 (m, 6H), 6.76 (s, 1H);
13C NMR (50 MHz, CDCl3) δ 171.5, 167.8, 143.1, 129.8, 61.8, 61.9,
61.3, 54.4, 21.9, 14.2, 13.3.
d6-acetone): δ 2.63 (s, 2H), 5.75 (s, 1H), 6.23 (s, 1H); 13C NMR (50
1
MHz, d6-acetone): δ 174.7, 168.6, 140.7, 126.4, 32.9 (septet, JCD
)
19.6 Hz), 28.1.
Enzymology. Apo-2-methyleneglutarate mutase and the 3-methyl-
itaconate isomerase were overproduced and purified from E. coli as
described recently.6 Holo-2-methyleneglutarate mutase was reconstituted
just by addition of an excess of coenzyme B12. For the determination
of the primary deuterium isotope effect exhibited by 2-methylene[4-
2H2]glutarate in the reaction catalyzed by 2-methyleneglutarate mutase,
the standard assay was used. It is based on the subsequent isomerization
of the product 3-methylitaconate to 2,3-dimethylmaleate, the absorbance
of which is measured at λ ) 256 nm, using 3-methylitaconate isomerase
as an auxiliary enzyme.27 The assay mixture contained in a total volume
of 500 µl: 100 mM potassium phosphate, pH 7.4, 10-20 units of
3-methylitaconate isomerase, 0.05-0.2 units of apo-2-methyleneglu-
tarate mutase, and either unlabeled 1a or 2-methylene[4-2H2]glutarate
1g. The reaction was started by addition of 10 µM adenosylcobalamin.
The initial velocity was plotted against the substrate concentration. The
data were fitted to the Michaelis-Menten equation using the method
of nonlinear least-squares.
(Z)-2′-Bromo-3-methylitaconic acid (15). A mixture of (E)-triethyl
1-bromo-but-1-ene-2,3,3-tricarboxylate 14 (105 mg, 0.29 mmol) and
20% hydrobromic acid (4 mL) was heated for 4 days at 80 °C. The
solvent was removed in vacuo, and water (2 mL) was added to the
residue. The solution was extracted with ethyl acetate (5×), and the
combined organic extracts were dried over magnesium sulfate. The
solvent was removed, and the residual solid was purified by chroma-
tography [silica, elution with petrol-ethyl acetate-acetic acid (7:2:
1)] to afford the title compound as a white crystalline solid (37 mg,
0.166 mmol, 57% yield); mp 108 °C; Rf 0.26 (petrol-ethyl acetate-
The activity of 2-methyleneglutarate mutase with (R)-2-methylita-
conate as substrate was measured in an incubation containing in 50
mM potassium phosphate, pH 7.4, 0.05-0.2 units of apo-2-methyl-
eneglutarate mutase, and either unlabeled 2a or 3-[methyl-2H3]-
1
acetic acid, 7:2:1); H NMR (200 MHz, d6-acetone) δ 1.52 (d, J 7.2
9
J. AM. CHEM. SOC. VOL. 124, NO. 47, 2002 14047