10030 J . Org. Chem., Vol. 63, No. 26, 1998
Notes
glass tubing (o.d. 12 mm; wall thickness 1 mm) was purchased
from either ChemGlass or Ace Glass. Reactions were irradiated
with a hand-held long-wave UV lamp (UVP model UVGL-25,
λmax ) 366 nm). In these reactions the Pyrex reaction tubes serve
as a filter for < 280 nm light. Densitometry of ethidium-stained
agarose gels was performed using an Alpha Innotech IS-1000
digital imaging system. Compounds 1, 3, 5, and 1,2,4-benzotri-
azine (fully deoxygenated 1) were prepared as previously
described.49
photolysis of 1. Again, the major product of this reaction is
unchanged starting material (1).
P h otolysis of 3-Aceta m id o-1,2,4-ben zotr ia zin e 1,4-Diox-
id e (5) in th e P r esen ce of p-An isid in e: Isola tion of 3-(4-
Meth oxya n ilin o)-1,2,4-ben zotr ia zin e 1,4-Dioxid e (6). To a
stirred solution of 5 (1.12 g, 5.2 mmol) in a mixture of acetonitrile
(30 mL, HPLC grade) and aqueous sodium phosphate buffer (12
mL, 50 mM, pH 8) at 24 °C, a solution of p-anisidine (2.58 g, 21
mmol) in acetonitrile (3 mL) was added. The resulting light
brown solution was photolyzed (>280 nm) with stirring at 25
°C for 90 h. After 90 h, TLC analysis of the reaction mixture
revealed that unreacted starting materials and N-acetylated
p-anisidine were major components of the reaction mixture. More
interestingly, TLC analysis indicated that several other products
had been formed. Control experiments revealed that the forma-
tion of these additional products is light-dependent. The reaction
was filtered, the filtrate was extracted with diethyl ether (5 ×
15 mL), and the combined extracts was dried over sodium
sulfate. The dried diethyl ether extract was filtered and evapo-
rated under reduced pressure to yield an oil. The oil was placed
on a silica gel column and eluted with a hexane/ethyl acetate
gradient (0f100% ethyl acetate) followed by elution with 95:5
ethyl acetate/methanol. From this column, 20 mg (1.5% yield)
of the pure p-anisidine adduct 6 was isolated as a light red
solid: Rf ) 0.28 (100% ethyl acetate); mp 210-213 °C; 1H NMR
(500 MHz, CDCl3) δ 8.98 (bs, 1H), 8.35 (d, 2H), 7.90 (t, 1H), 7.52
(m, 3H), 6.94 (d, 2H), 3.81 (s, 3H); 13C NMR (125.9 MHz, CDCl3)
δ 157.34, 147.79, 138.04, 136.01, 131.17, 128.46, 127.64, 123.01,
121.81, 117.72, 114.63, 55.55; HRMS (EI) m/z calcd for C14H12N4O3
284.0909, found 284.0915. Crystals of 6 suitable for X-ray
diffraction were obtained by slow evaporation of a chloroform
solution. Crystal data for 6: monoclinic, space group P21/n, a )
9.976(1) Å, b ) 11.353(1) Å, c ) 23.319(2) Å, â ) 100.065(1)°, V
) 2600.4(3) Å3, Fcalcd ) 1.45 mg/cm-3, 2θmax ) 45°, Mo KR
radiation (λ ) 0.710 69 Å) for Z ) 8. Intensity data were collected
with use of the Siemans SMART system at 298 K. Least-squares
refinement based on 2483 reflections with Inet > 2.0σ(Inet) (out
of 3371 unique reflections) and 379 parameters on convergence
gave a final value of R ) 0.050. All crystallographic calculations
were conducted using SHELXL-9351 locally implemented on an
IBM-compatible PC.
P h otoclea va ge of P la sm id DNA by 3-Am in o-1,2,4-ben -
zotr ia zin e 1,4-Dioxid e (1). For anaerobic reactions, a Pyrex
tube (sealed with a conical closure at one end) containing
plasmid DNA (pBR322, 37 µM bp) and 1 (250 µM) in a solution
of sodium phosphate (50 mM, pH 7.0) and acetonitrile (HPLC
grade, 10 vol %) was subjected to three freeze-pump-thaw
cycles followed by flame sealing under vacuum. The reaction was
then photolyzed with >280 nm light at room temperature (fan
cooled) for 5.5 h. For aerobic reactions, a Pyrex tube containing
plasmid DNA (pBR322, 37 µM bp) and 1 (250 µM) in a mixture
of sodium phosphate buffer (50 mM, pH 7.0) and acetonitrile
(10 vol %) was sealed with a rubber septum under ambient
atmosphere and photolyzed as described above. Following pho-
tolysis, the reaction vessels were opened, 5 µL of 50% glycerol
loading buffer (containing 0.1% bromophenol blue, 150 mM
EDTA, 1% SDS in 2 M Tris, 1 M acetate, pH 8) was added to
the reactions, and the resulting mixture was loaded onto a 0.9%
agarose gel. The gel was electrophoresed for approximately 4 h
at 80 V in TAE buffer (40 mM Tris, 20 mM acetate, 1 mM EDTA,
pH 8) and then stained in an aqueous ethidium bromide solution
(0.3 µg/mL) for 1-2 h. DNA in the gel was visualized by UV-
transillumination and the gel image recorded using an Alpha
Innotech IS-1000 digital imaging system. The values reported
are uncorrected for the differential staining of form I and II
plasmid DNA.50
In vestiga tion of P r od u cts Resu ltin g fr om Aer obic a n d
An a er obic P h otolysis of 3-Am in o-1,2,4-ben zotr ia zin e 1,4-
Dioxid e (1). A solution of 1 (250 µM) in a mixture of sodium
phosphate (50 mM, pH 7.0) and acetonitrile (HPLC grade, 10
vol %) was freeze-pump-thaw degassed (3×) and sealed under
vacuum in a Pyrex tube. The solution was photolyzed with >280
nm light for 5.5 h at room temperature. The reaction was
analyzed by HPLC (C18 reverse-phase Microsorb-MV column,
100 Å sphere size, 5 µM pore size, 25 cm length × 4.6 mm i.d.
eluted with a 74:25:1 mixture of water/methanol/acetic acid) with
monitoring at 254 nm. The formation of 3 in the photolysis of 1
was indicated by the presence of a peak eluting at ∼17.5 min (1
elutes at ∼5.5 min). Under the conditions employed here neither
1 or 3 undergoes detectable conversion to the fully deoxygenated
3-amino-1,2,4-benzotriazine (retention time ∼16.5 min). The
identity of the mono-N-oxide (3) was confirmed by comparison
of retention times to that of an authentic sample and by
co-injection with an authentic sample of 3. Yields of 3 in the
photolysis of 1 were determined by comparison of HPLC peak
areas to a calibration curve prepared by measuring the HPLC
peak areas resulting from injection of known concentrations of
authentic 3. Under the anaerobic conditions described above,
photolysis of 1 produces approximately a 5% yield of the mono-
N-oxide 3. The major component of the reaction mixture is
unchanged starting material (1). Under aerobic conditions, no
detectable amounts (<0.5% yield) of 3 are obtained in the
Ack n ow led gm en t. We thank Professor Shon Pulley
(University of Missouri) for the use of some facilities
and Professor Steven Keller (University of Missouri) for
technical assistance. We are grateful to the University
of Missouri Research Board for partial financial support
of this work and to the National Science Foundation for
partial support of the NMR facilities at the University
of MissourisColumbia (Grants 9221835 and 8908304).
Su p p or tin g In for m a tion Ava ila ble: Tables of complete
X-ray data and methods for 6 (7 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
J O981314W
(51) Sheldrick, G. M. SHELXL-93: program for crystal structure
refinement, University of Gottingen, Germany, 1993.
(52) Hertzberg, R. P.; Dervan, P. B. Biochemistry 1984, 23, 3934.
(49) Mason, J . C.; Tennant, G. J . Chem. Soc. B 1970, 911-916.
(50) Bauer, W.; Vinograd, J . J . Mol. Biol. 1968, 33, 141-171.