Polyoxometalate-Based Catechol Dioxygenase
J. Am. Chem. Soc., Vol. 121, No. 42, 1999 9841
Supporting Information. The final reaction mixture (after completion
of a typical DTBC oxygenation, vide supra) was transferred into a
separatory funnel and extracted three times with 10 mL of aqueous
X-ray crystallographically, see the Supporting Information, section S11,
X-ray Structures and Crystallographic Tables, Figure S11-D for a
thermal ellipsoid drawing and crystallographic tables.
1
saturated solution of KHCO
Cl layer was saved for further manipulation to be detailed in a moment.
The aqueous layer containing the RCO
acidified with 6 M HCl to reform the acids, RCO
twice with 5 mL of CHCl . The chloroform extracts were combined
and dried over ∼2 g of anhydrous MgSO for 8 h at room temperature.
The MgSO was then filtered off, rinsed with 5 mL of fresh CHCl
and the combined filtrate was evaporated in vacuo at 40 °C. Ap-
3
to separate acid products; the 1,2-C
2 4
H -
3,5-Di-tert-butyl-5-(carboxymethyl)-2-furanone, 5: H NMR (CDCl )
3
1
3
2
δ 0.98 (s, 9 H), 2.83 (d, 9H), 2.93 (d 1 H), 6.95 (s, 1 H); C NMR
-
+
2
K
products was separated,
H, and then extracted
(CDCl ) δ 25.5 (q), 28.1 (q), 31.7 (s), 37.5 (s), 37.9 (s), 88.4 (s), 144.1
3
1
5a,c
2
(s), 145.9 (s), 171.5 (s), 174.9 (s), in comparison to literature data
3
(see also Table S20, Supporting Information).
4
3,5-Di-tert-butyl-1,2-benzoquinone, 6: GC-MS data found for
1
+
4
3
,
C H O , m/e 220 (M ); H NMR (CDCl ) δ 1.18 (s, 9 H), 1.22 (2,
1
4
2
2
3
9H), 6.16 (s 1 H), 6.89 (s, 1 H); 13C NMR (CDCl ) δ 27.8 (q), 29.1
3
proximately 20-40 mg of organic, RCO
2
H product was obtained in
(q), 35.4 (s), 35.9 (s), 122.0 (s), 133.4 (s), 149.8 (s), 163.2 (s), also
characterized X-ray crystallographically, see the Supporting Information,
section S11, X-ray Structures and Crystallographic Tables, Figure S11-E
for a thermal ellipsoid drawing and crystallographic tables.
this step.
2 4 2
The 1,2-C H Cl layer (containing the nonacid products) was then
concentrated in vacuo at 40 °C by rotary evaporation to yield a dark-
brown, oily residue. Separation and purification of the main products
was accomplished by column chromatography. (A picture of the column
with approximate retention times of the various products is provided
as Figure S18 of the Supporting Information.) The chromatography
column (450 × 30 mm) was filled with a slurry of silica gel
Solvent Effects on the Oxygenation of DTBC. The oxygenations
were carried out following the experimental procedure discribed above
for the catalytic oxygenations of DTBC. The reactions were carried
out at 1 atm dioxygen and 65 °C in 20 mL of the solvent (N,N-dimethyl
formamide, acetonitrile, 1,2-dichloroethane, 1,1,2,2-tetrachloroethane,
and benzene), with 4.98 mg (n-Bu N) [(CH CN) Fe‚SiW V O ], III
(Mallinckrodt, 100 mesh, 100 g, suspended in 200 mL n-hexane).
4
5
3
x
9
3
40
-
6
-3
Approximately 500 mg of the brown, oily residue was redissolved in
-2 mL C Cl , placed on top of the chromatography column and
eluted with 1100 mL of CHCl
at a flow rate of ∼1.5 mL/min.
The first 225 mL were discarded, then, a total of 25, ∼25 mL
eluant); and these
(1.33 × 10 mol, 0.07 mM), and 1.00 g (4.5 × 10 mol, 0.225 M)
DTBC. A catalyst/substrate ∼1:3400) was maintained in all reactions.
The solvents were dried by standing for at least 48 h over ∼25 vol %
3-Å molecular sieves previously activated under vacuum at 170 °C.
1
2
H
4
2
3
fractions were collected, (∼870 mL total CHCl
3
(This is important, since control reactions revealed that added H
2
O has
fractions were then analyzed by GC on a DB-1 capillary column, using
the temperature program given above, to ascertain which fractions
contained what products. On the basis of the GC results, the 25 mL
fractions 1 and 2 were combined (and contained primarily product 4,
Scheme 1), fraction 3 was discarded; the fractions 4-8 were combined
a significant effect on at least the induction period and associated
20
catalyst evolution process. ) The reaction’s progress was then followed
periodically by sampling via a gastight syringe and analyzing the
mixture by gas chromatography [DB-1 capillary column, temp (initial)
200 °C for 2 min, 2 °C per min temperature ramp, temp (final) 240 °C
for 3 min, He carrier gas flow 1-2 mL per min and 15 psig head
pressure]. The identities of the observed products were established by
co-injection of authentic materials, vide supra, as well as by GC-MS.
A summary of the results is provided in Table 2.
(and contained primarily product 2, Scheme 2), fractions 9-12 were
combined (and contained primarily product 2 and 3, Scheme 2),
fractions 13-16 were combined (and contained primarily products 3
and 6, Scheme 2), and fractions 17-22 were combined (and contained
primarily the red autoxidation product, 1,2-di-tert-butyl-1,2-benzo-
quinone, 6, Scheme 2). The remaining fractions 23-25 were discarded.
The resulting five main fractions were evaporated to dryness in a
vacuum at room temperature, and the residues were dissolved in 2.0
X-ray Crystallography. Single crystals of products 2, 3, 4, and 6
were grown under the conditions detailed in the Supporting Information.
X-ray diffraction data were collected on a Bruker AXS SMART CCD
X-ray diffractometer equipped with a graphite crystal monochromator
using Mo KR (λ ) 0.71073 Å) radiation. The structures were solved
by direct methods using SHELXTL (Sheldrick, G. M.; SHELXTL,
1
13
mL CDCl and then analyzed by GC, GC-MS, H and C NMR
18
3
spectroscopy, and further purified by fractional crystallization (see
Figures S17 and S18 in the Supporting Information for details). A
summary of spectroscopic and analytical data for 13 known oxidation
products of DTBC are provided in Table S20 of the Supporting
Information; the five products (2-6) identified following the chroma-
tography detailed above are presented next. The net mass balance for
the five products 2-6, vs the amount of initial DTBC, was ∼95%.
2
version 5.03, 1994) and refined by full-matrix least-squares on F to
the final R values. X-ray crystallographic tables, bond lengths and angles
for 3,5-di-tert-butyl-1-oxacyclohepta-3,5-diene-2,7-dione (muconic acid
anhydride), 2, 4,6-di-tert-butyl-2H-pyran-2-one, 3, spiro[1,4-benzo-
dioxin-2(3H), 2′-[2H]pyran]-3-one, 4′,6,6′,8-tetrakis(1,1-dimethylethyl),
4, and 3,5-di-tert-butyl-1,2-benzoquinone, 6, are provided as Supporting
Information, section S11, X-ray Structures and Crystallographic Tables.
Oxygen-Uptake Experiments. Oxygen uptake experiments were
performed using a 50 mL round-bottomed flask with sidearm as the
reaction vessel which was connected to a gas-uptake line equipped with
a mercury manometer (Figure S21, Supporting Information). In the
3
,5-Di-tert-butyl-1-oxacyclohepta-3,5-diene-2,7-dione, 2: GC-MS
+
1
data found for C14
H), 1.22 (2, 9H), 6.16 (s 1 H), 6.55 (s, 1 H); C NMR (CDCl ) δ
8.6 (q), 29.1 (q), 36.4 (s), 36.9 (s), 116.0 (dd), 124.4 (dd), 149.8 (s),
60.2 (s), 160.9 (s), 162.1 (s); also characterized X-ray crystallographi-
H
20
O
3
, m/e 236 (M ); H NMR (CDCl
3
) δ 1.18 (s,
1
3
9
2
1
3
-
3
cally, see the Supporting Information, section S11, X-ray Structures
and Crystallographic Tables, Figure S11-B for a thermal ellipsoid
drawing and crystallographic tables.
drybox, 400 mg (1.80 × 10 mol) DTBC was placed in the 50 mL
round-bottomed flask equipped with a 10 mm Teflon-coated stir bar
and dissolved by adding 8 mL of 1,2-C H Cl . In a separate 5 mL
2
4
2
-
6
4
,6-Di-tert-butyl-2H-pyran-2-one, 3: GC-MS found for C13
H
20
O
2
,
glass vial, 5.66 × 10 mol of catalyst (a catalyst concentration of
+
1
m/e 208 (M ); mp 111-112 °C; H NMR (CDCl
.22 (s, 9H), 6.00 (d 1 H), 6.01 (d, 1 H); C NMR (CDCl ) δ 28.0 (q),
8.9 (q), 35.3 (s), 36.1 (s), 98.5 (s), 107.1 (s), 164.0 (s), 167.8 (s),
71.3 (s); also characterized X-ray crystallographically, see the Sup-
3
) δ 1.18 (s, 9 H),
0.71 mM was maintained in all experiments) was dissolved in 200 µL
1
3
1
2
1
3
2 4 2
of 1,2-C H Cl and then transferred into a 1 mL gastight syringe. The
reaction flask was then sealed with a stopcock, brought out of the
drybox, and connected to the gas-uptake line; the gastight syringe
containing the solution of the catalyst was brought out of the drybox
with its needle stuck in a septum-caped vial. The reaction flask was
porting Information, section S11, X-ray Structures and Crystallographic
Tables, Figure S11-C for a thermal ellipsoid drawing and crystal-
lographic tables.
then placed in liquid N (-196 °C) for 10 min and the uptake line,
2
Spiro[1,4-benzodioxin-2(3H), 2′-[2H]pyran]-3-one, 4′, 6, 6′, 8-tet-
plus the calibration flask and the reaction flask, was then evacuated.
The connection to the vacuum pump was then closed, and the system
was refilled with 1 atm dioxygen. The liquid N bath was then replaced
2
with a temperature-controlled, paraffin-oil bath, and the reaction mix-
ture was stirred for 25 min to equilibrate at the reaction temperature of
40 °C.
After 20 min, the solution containing the catalyst was then injected
through the septum-capped sidearm into the reaction vessel, the pressure
rakis(1,1-dimethylethyl), 4: GC-MS data found for C28
H
40
O
4
, m/e
) isomer
no. 1 (∼60%) δ 0.79 (s, 9 H), 1.18 (s, 9H), 1.22 (s, 9 H), 1.45 (s, 9H),
.51 (d 2 H), 7.03 (m, 2 H); isomer no. 2 (∼40%) δ 0.86 (s, 9 H), 1.15
s, 9H), 1.25 (s, 9 H), 1.27 (s, 9H), 5.56 (d 2 H), 6.86 (m, 2 H). The
+
1
4
40 (M ); mp 141-144 °C; H NMR (both isomers, CDCl
3
5
(
13
C NMR spectrum for the combined isomers is given in the Supporting
Information as Figure S19. The spiro product, 4, was also characterized
Weiner
