1936 J . Org. Chem., Vol. 66, No. 6, 2001
Lee et al.
and evaporated, and the residue was dissolved in CD3CN. The
1H NMR spectrum showed a mixture of 1 and a byproduct21
in a ratio of 3:1. The same reaction described here occurred
over the pH range 7.0 to 11.0, giving the same ratio of
products, except that the reaction was slower at lower pH. The
addition of 1 equiv of 2,2′-bipyridine (based on copper) to the
reaction did not alter the results. However, Cu(II) was found
to be essential for this reaction, because a separate reaction
performed without Cu(II) for 1.5 h at pH 10 resulted in
recovery of starting 4-tert-butylresorcinol (5).
Rea ction of w ith Hyd r a zin e in DMSO-d 6. To a 5 mm
NMR tube containing 1 (50 mg, 0.028 mmol) in degassed
DMSO-d6 (0.6 mL) was added hydrazine (70 µL, 2.24 mmol)
via syringe. The 1H NMR spectrum recorded 20 min after
mixing showed generation of 5-tert-butyl-2-hydroxy-1,4-ben-
zoquinone-1-hydrazone (3) and 5-tert-butyl-1,2,4-benzenetriol
(2) in a ratio of 6:1. Over time, signals corresponding to
compound 5 increased with a concomitant decrease of signals
corresponding to compound 3. Integration of the 1H NMR
recorded after 5 weeks indicated a mixture of 3, 2, and 5 in a
ratio of 2:1:12. 3: 1H NMR (DMSO-d6) δ 1.22 (s, 9H), 5.14 (s,
1H), 6.41 (s, 1H); 13C NMR (DMSO-d6) δ 29.8 (3C, -), 33.9
(+), 106.2 (-), 129.0 (-), 134.3 (+), 139.8 (+), 174.7 (+), 181.8
(+); HRMS (sample prepared by evaporating the mixture of 3
and 2 in a ratio of 6:1) calcd for C10H14N2O2 m/z (rel intensity)
194.1055, found 194.1059 (11.0%). The same experiment run
without degassing showed that conversion of 3 to 5 was about
3 times faster than that of the reaction conducted under
anaerobic conditions.
to a solution of 1 (18 mg, 0.1 mmol) in CD3CN (0.5 mL) in a 5
mm NMR tube. Once each day for 5 days, the seal was opened
briefly to admit air, to induce oxidative recycling of 2 to 1,
thereby optimizing the yield of the 1,1-dimethylhydrazine-
derived products. After 5 days, the red crystals formed in the
reaction tube were filtered and washed with CH3CN, giving
pure 2,2′-methylenebis(4-methylazo-6-tert-butylresorcinol)
(12): 1H NMR (CDCl3) δ 1.39 (s, 18H), 3.69 (d, J ) 3.18 Hz,
6H), 3.80 (s, 2H), 7.10 (s, 2H), 12.1 (s, 2H); 13C NMR (CDCl3)
δ 17.8 (+), 29.9 (-), 34.6 (+), 43.4 (-), 116.6 (+), 129.0 (-),
131.9 (+), 133.4 (+), 166.3 (+), 168.5 (+); HRMS calcd for
C
23H32N4O4 m/z (rel intensity) 428.2426, found 428.2408 (65%).
An a er obic Rea ction of 1 w ith Meth ylh yd r a zin e or 1,1-
Dim eth ylh yd r a zin e in DMSO-d 6. To a 5 mm NMR tube
containing a solution of 1 (3.6 mg, 0.02 mmol) in degassed
DMSO-d6 (0.5 mL) was added methylhydrazine (2.1 µL, 0.04
mmol). 1H NMR signals corresponding to triol 2 and what
appears to be 2-hydroxy-5-tert-butyl-1,4-benzoquinone 1-(meth-
ylhydrazone) (8) [1H NMR (DMSO-d6) δ 1.27 (s, 9Η), 2.18 (s,
3Η), 6.38 (s, 1H), 6.62 (s, 1H)] were found in a ratio of 1:1
along with a trace of 9. Integration of 2 increased with a
concomitant decrease of 8 over the course of several hours at
room temperature. After 24 h, 8 disappeared completely,
leaving triol 2 as the major component (80%) derived from 1.
Traces of 9 and methane (δ 0.2) were also seen initially and
survived at least for 2 weeks in the solution without any sign
of decomposition or change.
The same reaction performed instead with 0.04 mmol of 1,1-
1
dimethylhydrazine exhibited in the earliest H NMR spectrum
Rea ction of 1 w ith Hyd r a zin e in CD3CN a n d th e Effect
of Ad d ed Cu (II). To a 5 mm NMR tube containing 1 (25 mg,
0.14 mmol) in CD3CN (0.5 mL) was added hydrazine (35 µL,
recorded (5 min) the same signals as seen above, tentatively
assigned to 8, as the major component (ca. 70%) along with a
trace of triol 2. After 16 h at room temperature, 1H NMR
indicated that 8 had disappeared completely, and triol 2 was
found to be the major component (ca. 60%) along with several
minor unidentified products.
1
1.14 mmol). The H NMR spectrum recorded 2 h after mixing
showed a mixture of compounds 3, 2, and 5 in a ratio of 10:
2:1. The signals corresponding to 5 increased with time, and
after 7 days, 5 was obtained as a sole product in quantitative
yield. In an identical experiment except that at the 2 h time
point Cu(ClO4)2‚6H2O (1 mg, 0.028 mmol) was added, rapid
In ter a ction of BP AO w ith Hyd r a zin e a n d Its An a -
logu es. Solutions (0.9 mL) of hydrazine, methylhydrazine, 1,1-
dimethylhydrazine, 3-phenylpropylhydrazine, or formaldehyde
(1-200 µM final concentration, as appropriate) in 100 mM
sodium phosphate buffer, pH 7.2, were incubated with 0.1 mL
of a BPAO suspension (1.2-2.4 µM final concentration) at 30
°C, with or without maintenance of anaerobic conditions by
argon bubbling. Aliquots (0.1 mL) were periodically withdrawn
and diluted with 1.0 mL of benzylamine (10 mM in 50 mM
sodium phosphate buffer, pH 7.2) in a 1 cm cuvette (1.5 mL).
The rate of oxidation of benzylamine to benzaldehyde was
measured by recording the increase in absorbance at 250 nm
using a UV-vis spectrometer for 1 min and compared to the
rate of benzylamine oxidation in a companion control solution
of enzyme without hydrazine. In some experiments, where only
up to four aliquots were required, the incubations were scaled
to half-volume (0.5 mL). In some anaerobic experiments, an
O2 “scrubber” was used, consisting of the addition of 10 units
of glucose oxidase, 500 units of catalase, and 15 mg of glucose
per 1 mL of primary incubation mixture.
In one experiment, the enzyme (1.5 µM) was incubated with
or without methylhydrazine (4 µM) in a 1 mL cuvette with or
without argon bubbling (using a needle projecting through a
serum cap along the side of the cuvette to the bottom, and
another needle for gas escape), and a concentrated solution of
phenylhydrazine‚HCl (8 µL of 2 mM) in degassed water was
added either after 3 min or after 100 min. The spectrum was
then recorded from 300 to 600 nm, and the A450 was deter-
mined for each of the (+) methylhydrazine cases relative to
the (-) methylhydrazine cases.
1
gas (N2) bubbling from the solution was noticed. The H NMR
spectrum recorded 15 min after addition of Cu(II) showed that
compound 5 was a single major product along with a trace of
compound 3.
Rea ction of 1 w ith Meth ylh yd r a zin e in CD3CN. To a 5
mm NMR tube containing a solution of 1 (9 mg, 0.05 mmol)
in degassed CD3CN (1 mL) were added tert-butylamine (5.2
µL, 0.1 mmol) and methylhydrazine (5.3 µL, 0.1 mmol) via
syringe. After 40 min, the 1H NMR spectrum indicated
complete disappearance of 1 and the formation of 4-(methyl-
azo)-6-tert-butylresorcinol (9) and 2 in a ratio 4:1 along with
a signal due to methane (δ 0.20), as confirmed by bubbling
methane into CD3CN. No further change in the product
distribution was noticed over the course of 4 days. 9: 1H NMR
(CD3CN) δ 1.38 (s, 9H), 3.83 (s, 3H), 6.29 (s, 1H), 7.51 (s, 1H);
13C NMR (CD3CN) δ 29.9 (3C), 34.7, 54.1, 104.8, 130.1, 131.4,
131.6, 154.4, 162.5. In another experiment showing nearly
pure 9 (and CH4) after 30 min, the CD3CN was removed in
vacuo and the spectra were re-recorded in DMSO-d6 for better
characterization. 9: 1H NMR (DMSO-d6) δ 1.33 (s, 9H), 3.84
(s, 3H), 6.29 (s, 1H), 7.41 (s, 1H); 13C NMR (DMSO-d6) δ 29.4
(3C), 33.7, 53.6, 103.7, 127.6, 128.3, 130.3, 153.2, 161.0; HRMS
calcd for
C11H16N2O2 m/z (rel intensity) 208.1212, found
208.1215 (100%).
Rea ction of 1 w ith 1,1-Dim eth ylh yd r a zin e in CD3CN.
To a 5 mm NMR tube containing a solution of 1 (9 mg, 0.05
mmol) in degassed CD3CN (1 mL) were added tert-butylamine
(5.2 µL, 0.05 mmol) and 1,1-dimethylhydrazine (7.6 µL, 0.1
mmol) via syringe. 1H NMR signals due to 9, 2-hydroxymethyl-
4-methylazo-6-tert-butylresorcinol (11, tentatively identified),
and 2 grew as the reaction proceeded for several hours.
Compounds 9 and 2 slowly disappeared from the solution
during the course of 2 days, leaving compound 11 as the major
product in about 25% yield along with several unidentified
Concentrations of the enzyme were estimated from the
absorbance change at 250 nm for benzaldehyde formation of
the control reaction, using an activity of 0.48 units/mg of
protein for the pure enzyme of molecular weight 8500028 and
∆ꢀ250 ) 12800 M-1 cm-1 for benzaldehyde. The values obtained
were consistent with the results of titration of active cofactor
with phenylhydrazine and measuring the inflection at 450 nm.
1
compounds. Apparent H NMR signals for 11: (CD3CN) δ 1.26
(s, 9H), 3.32 (s, 3H), 4.92 (s, 2H), 6.88 (s, 1H). In another
reaction, 1,1-dimethylhydrazine (23 µL, 0.3 mmol) was added
(28) J anes, S. M.; Klinman, J . P. Biochemistry 1991, 30, 4599-4605.