First synthesis of C-phenyl N-tert-butyl [15N]nitrone (PBN-15N) for the EPR spin trapping methodology

Article abstract of DOI:10.1515/znb-1996-0125

As compared to normal PBN, about fifty percent increase of electron paramagnetic resonance (EPR) spin trapping sensitivity has been gained by using a new 100% ¹⁵N-enriched spin trap, C-phenyl N-tert-butyl[¹⁵N]nitrone (PBN-¹⁵N). PBN-¹⁵N has been prepared by a convenient four-step route using ammonium-¹⁵N chloride as the starting material. This synthetic method produces 2-methyl-2-[¹⁵N]nitropropane which is useful for the synthesis of many other PBN-¹⁵N type spin traps for the purpose of increasing spin trapping sensitivity. EPR spin trapping with PBN-¹⁵N in benzene and in phosphate buffer has been investigated. The ¹⁵N hyperfine splitting constant (¹⁵N-hfsc) is larger than ¹⁴N-hfsc by 40%. The larger ¹⁵N-hfsc gives more opportunity to identify different radical addends within the same system.

Full text of DOI:10.1515/znb-1996-0125

First Synthesis of C-Phenyl N-tert-Buty\ [15N]nitrone (PBN-15N)
for the EPR Spin Trapping Methodology
Yong-Kang Zhang
OMRF Spin Trap Source, Free Radical Biology and Aging Research Program, Oklahoma
Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, Oklahoma 73104,USA
Z. Naturforsch. 51b, 139-143 (1996); received June 7, 1995
C-Phenyl N-tert-Butyl [15N]nitrone, Nitrone, Spin Trap, EPR Spin Trapping,
Aminoxyl (Nitroxide)
As compared to normal PBN, about fifty percent increase of electron paramagnetic reso-
nance (EPR) spin trapping sensitivity has been gained by using a new 100% 1:>N-enriched
spin trap, C-phenyl /V-rm-butyl[15N]nitrone (PBN-15N). PBN-15N has been prepared by a
convenient four-step route using ammonium-15N chloride as the starting material. This syn-
thetic method produces 2-methyl-2-[15N]nitropropane which is useful for the synthesis of
many other PBN-15N type spin traps for the purpose of increasing spin trapping sensitivity.
EPR spin trapping with PBN-15N in benzene and in phosphate buffer has been investigated.
The 15N hyperfine splitting constant (15N-hfsc) is larger than 14N-hfsc by 40%. The larger
15N-hfsc gives more opportunity to identify different radical addends within the same system.
h
c h 3
Several 15N-enriched aminoxyls and nitrones
have been synthesized for the purpose of increas-
ing the sensitivity of the electron paramagnetic
resonance (EPR) spin labelling and spin trapping
methodologies [1]. A 15N-enriched DMPO spin
trap, 5,5-di([2H3]methyl) [15N]-l-pyrroline 7V-oxide
(DMPO-d6-15N), has been prepared, but no spin
trapping results from this spin trap have been re-
ported [Id]. Although the perdeuterated DMPO-
15N has also been synthesized [Id], the wide-
spread use of this nitrone for enhancement of the
spin trapping sensitivity is limited because the ß-
deuterium hyperfine splitting is so small that it is
rarely useful for the diagnosis of free radical ad-
dends [Id]. For PBN (C-phenyl N-tert-buty\ nit-
rone) [2] type spin traps, no 15N-enriched com-
pounds have been synthesized before and no spin
trapping results are available. This communication
C = n N-C-CH3
O' CH3
n = 14, PBN-14N
n = 15, PBN-15N
As one of the most common spin traps, PBN-
14N has been used for about 30 years to detect
reactive free radicals involved in chemical and bio-
logical processes [3];
H
C
CH3
—14N — C —C H
O’ CH3
PBN-14N
+
R"
----------
►
\
—
3
'
--
'
R
The nuclear spin (/) of 14N is equal to 1 which
describes the first synthesis of a new 100% 15N- produces the triplet hyperfine splitting for the spin
enriched spin trap, C-phenyl N-tert-buty\ [15N]nit-
rone (PBN-15N), and spin trapping results with
adducts of PBN-14N in addition to the doublet due
to theß-H. Fig. 1(a) givesthe EPR spectrumfor the
this spin trap are also presented. About 50% n-butoxyl radical adduct of PBN-14N as an exam-
increase of spin trapping sensitivity has been ple. When I4N is replaced with 15N, 15N (/ = 1/2)
gained as compared to normal PBN which consists gives a doublet pattern as shown in the EPR
of 99.63% of PBN-14N and 0.37% of PBN-15N [2],
spectrum of the «-butoxyl radical adduct of PBN-
15N in Fig. 1(b).
PBN-15N has been synthesized by a convenient
four-step route using 100% 15N-enriched ammo-
nium-'^N chloride as a starting material. As il-
lustrated in Scheme 1, ammonium-15N chloride
* Reprint requests to Dr. Yong-Kang Zhang.
0932-0776/96/0100-0139 $06.00 © 1996 Verlag der Zeitschrift für Naturforschung. All rights reserved.
D
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140
Y.-K. Zhang • Synthesis of C-Phenyl N-tert-Butyl [15N]nitrone (PBN-15N)
15
H3+0/CH2CI2 15
15NH4CI + Ca(OCI)2 — -------- NCI3
(a)
1.(CH3)3CCI,
AICl3/CH2CI2
2. H jO /H a
? Hs
H=c- ? - ,s»°= T i S
1. NaOH
? Ha
h>c- ^- “ nh. er
c h 3
c h 3
(b)
CßHsCHO,
Zn/ChfeCOOH,
95%C2HsOH
ch3
1
5n+- c - ch3
O' ch3
Fig. 1. (a) EPR spectrum for the «-butoxyl radical spin
adduct of PBN-14N (14N-hfsc=13.70 G and H-hfsc=2.00
G), generated by photolysis of «-butyl nitrite (0.25 v.%)
and PBN-14N (10 mM) in benzene; (b) EPR spectrum
of the /7-butoxyl radical spin adduct of PBN- N (15N-
hfsc=19.21 G and H-hfsc=2.00 G), generated by photoly-
sis of «-butyl nitrite (0.25 v.%) and PBN-15N (10 mM)
in benzene. EPR spectrometric conditions: modulation
frequency = 100.0 kHz; modulation amplitude = 1.0 G;
----------** \ J T
Scheme 1.
was reacted with calcium hypochlorite to generate
trichloramine-15N(15NCl3) which was treated with
2-chloro-2-methylpropane in the presence of alu- sweep speed=2.39 G/s; center field=3476.0 G; microwave
frequency=9.65 GHz; microwave power=20 mW.
minum chloride providing 2-[15N]amino-2-methyl-
propane after hydrolysis [4,5]. Oxidation [6]of the
[15N]-labeled amine with potassium permanganate
contained in the same solution to trap the same
free radical generated in situ. Fig. 2shows the EPR
spectrum of the z-amyloxyl adducts of PBN-15N
and PBN-14N in the same benzene solution. The
relative intensities of the peaks are clearly greater
produced 2-methyl-2-[15N]nitropropane which is
expected to be widely useful for the synthesis of
PBN-15N type spin traps. ,/V-te/t-Butylhydroxyl-
[15N]amine generated in situ by reduction of the
[15N]nitro compound with zinc and acetic acid re-
acted further with benzaldehyde giving the PBN-
15N spin trap which was purified by the combina-
tion of column chromatography and repeated sub-
limations with 12% overall yield. Impurity EPR
signals are not detected from a solution of the
PBN-15N product (50 mM) in benzene when the
receiver gain of the EPR spectrometer is equal to
1.0 x 106 (for other spectrometric conditions, see
Fig. 1).
Table I. EPR hyperfine splittings for radical spin adducts
of PBN-15N in benzene.3
H
CH3
H
c h 3
c— ,5n - c - c h 3
c = 15n- c - ch 3
o- ch3
O
R
O' ch3
15N-hfsc H-hfsc
Radical (R»)
Source
18.55
18.62
19.16
19.55
19.21
19.21
19.21
19.45
20.01
20.09
19.95
20.04
20.19
1.50
1.52
1.36
2.02
2.00
1.91
1.96
2.05
3.13
3.18
3.81
4.50
2.20
c6h ,c o o *
c 6h 5c o o
r-BuOO
?-BuO*
(C6H,COO)2
Pure PBN-15N was used to trap twelve different
reactive free radicals in benzene (see Table I) and
nine in phosphate buffer (see Table II). The hy-
drogen hyperfine splitting constants (H-hfsc) for
the PBN-15N radical adduct should be identical to
the adduct of PBN-14N because the difference in
N-isotopes should have no EPR observable influ-
ence on the property of the /3-hydrogen of the rad-
ical adduct [7], Theoretically, as compared to
PBN-14N, the sensitivity of the adduct of PBN-15N
should be 50% greater because the triplet pattern
due to the 14N hfs is divided into the doublet due
to 15N hfs. This sensitivity improvement has been
tested by using 50% PBN-15N and 50% PBN-14N
C6H,COOOHb
f-BuOOH+UV
(r-BuO)2+UV
«-BuONO+UV
z-BuONO+UV
/-AmylONO+UV
a iB n +O 2+u v
Me(CH2)4CHO+(r-BuO),+UV
EtCHO+(r-BuO)2+UV
c-C6H„CHO+(r-BuO)2+UV
C6HsCHO+(f-BuO)2+UV
(C6H,COO)2+UV
/7 -B u O
«
i-BuO •
/-AmylO*
Me2C(CN)0*
Me(CH7)4C(0)*
EtC(O)«
c-C6HuC(0)*
Q,H5C(0)*
c6h 5-
3 The spectra were recorded at room temperature. The
hyperfine splittings are given in Gauss (1 G = 0.1 mT).
The error is estimated to be ±0.05 G. Photolysis with
UV was usually for a number of seconds; b Benzalde-
hyde was used; C6H5COOOH is present in benzalde-
hyde as an impurity from air oxidation.
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Y.-K. Zhang • Synthesis of C-Phenyl TV-tert-Butyl [15N]nitrone (PBN-15N)
141
Table II. EPR hyperfine splittings for radical spin ad-
ducts of PBN-15N in phosphate buffer.3
AA
AA
Source
Radical (R*)
15N-hfsc H-hfsc
•OH
•CH2OH
i% h 2o 2+uv
c h 3oh+ i% h2o 2+uv
21.70
22.39
22.43
22.58
22.48
22.92
22.97
22.39
21.24
2.74
3.81
3.27
3.13
3.62
3.67
3.62
4.20
2.34
•CH(CH3)OH c 2h 5oh+i % h2o 2+uv
•CH(C2H5)OH «-c3h 7oh+i % h2o 2+uv
•C(CH3)2OH
•c h 3
•c d 3
•c 6h 5
• n 3c
i_1_0_.0_G_
i
(CH3)2CHOH+l % h2o 2+uv
CH3S(0)CH3+1% h2o 2+uv
CD3S(0)CD3+1%H20 2+UV
(C6H5COO)2b
Fig. 2. EPR spectrum of the /-amyloxyl radical spin ad-
ducts of PBN-15N (labelled with “A”, 15N-hfsc=19.16 G
and H-hfsc=1.96 G) and PBN-14N (14N-hfsc=13.69 G
and H-hfsc=1.96 G), generated by photolysis of /-amyl
nitrite (0.25 v.%), PBN-15N (5 mM) and PBN-14N
(5 mM) in benzene. EPR spectrometric conditions are
identical to in Fig. 1.
NaN3+l%H20 2
a The spectra were recorded at room temperature. The
hyperfine splittings are given in Gauss (1 G = 0.1 mT).
The error is estimated to be ±0.05 G. Photolysis with
UV was usually for a number of seconds. The concentra-
tion of the phosphate buffer is 0.1 M; b the phenyl ad-
duct was generated in benzene and the residue after re-
moval of benzene was dissolved in phosphate buffer;
cthe splitting from one of the nitrogens in 14N3 addend
is equal to 1.98 G consistent with the value in ref. 7.
type of spin traps to increase spin trapping sensi-
tivity. PBN-15N is the first example of this type of
spin trap which has been synthesized and used to
trap reactive free radicals. It has been shown that
about 50% sensitivity improvement isrealized and
40% more spacing between lines is found as com-
pared to normal PBN. Other PBN-15N type spin
traps are being developed for further improve-
ment in spin trapping sensitivity.
for PBN-15N as compared to PBN-14N spin
adduct.
Six free radicals have been investigated for com-
parison and the results are organized in Table III.
This Table shows 42-55% sensitivity increase of
the PBN-15N adduct over PBN-14N. The 15N-hfsc
is also larger than 14N-hfsc by 40% which is ex-
pected from the difference of their nuclear mag-
netic moments (ju, -0.283 for 15N and +0.404 for
14N) [8]. When more than one radical adduct is
present in the same solution, the larger 15N-hfsc
(i.e. more spacing) might give more opportunity to
identify different radical addends.
Experimental
EPR spectra were measured on a Bruker ESP
300E or 300 spectrometer. MS spectra were deter-
mined on a FISONS/VG QUATTRO MS/MS tri-
ple quadrupole spectrometer with the condition of
70 eV (El). Ammonium-15N chloride was
purchased from ISOTEC, Inc. Other chemicals
were purchased from Aldrich Chemical Com-
pany, Inc.
In conclusion, a synthetic route has been devel-
oped to produce 2-methyl-2-[15N]nitropropane
which is useful for the preparation of PBN-15N
Table III. Comparison of EPR spin trapping of PBN-15N with PBN-14N in the same solution.3
PBN-,5N
PBN-14N
EPR intensity increaseb
Solvent
Radical (R#)
15N-hfsc H-hfsc
14N-hfsc H-hfsc
[(15N-14N)/14N]xl00%
benzene
benzene
benzene
bufferc
buffer0
buffer0
19.26
19.16
20.19
22.39
22.53
22.94
n-BuO*
/-AmylO*
C6H5*
• c h 2O h
•CHMeOH
• c d 3
1.96
1.96
2.20
3.81
3.27
3.62
13.73
13.69
14.42
16.03
16.03
16.37
1.96
1.96
2.22
3.81
3.30
3.62
42%
48%
42%
55%
45%
43%
3The spectra were recorded at room temperature. The hyperfine splittings are given in Gauss (1 G = 0.1 mT). The
error of the splitting is estimated to be ±0.05 G; b the average intensity of PBN-15N adduct subtracts the average
intensity of PBN-14N adduct, then divided by the average intensity of PBN-14N adduct. The obtaining result times
100% to give the percentage of the EPR intensity increase; c the concentration of phosphate buffer is 0.1 M.
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142
Y.-K. Zhang • Synthesis of C-Phenyl /V-rm-Butyl [15N]nitrone (PBN-15N)
EPR spin trapping
chloric salt of 2-[1>N]amino-2-methylpropane. The
solution was cooled to 0-5 °C and another solu-
The concentration of PBN-15N and PBN-14N
used in spin trapping experiments was 1x 10"2 M.
When ultraviolet(UV)-light was utilized to gener-
ate free radicals, the UV-light beam from a 75 W
high-pressure mercury UV lamp was directly fo-
cused into an EPR cavity. Within the cavity, an
EPR sample tube containing the interested solu-
tion had been placed. Generally, the sample solu-
tion was irradiated with UV-light just for a few
seconds.
tion of sodium hydroxide in water (50%, 30 g) was
added. The resulting basic solution was steam-dis-
tilled immediately. The distillate (ca. 100 ml) was
carefully collected in a precooled flask containing
potassium permanganate (10 g, 63.3 mmol). An-
other portion of potassium permanganate (10 g)
was added and the solution was stirred for 2 h at
0-5 °C and overnight at 20 °C. Again, potassium
permanganate (10 g) was added and the mixture
was stirred for 3 h at 20 °C. Steam-distillation was
conducted and the distillate (ca. 100 ml) was col-
lected in a flask cooled to 0-5 °C. The distillate
was extracted with diethyl ether (2 x 40 ml). The
The preparation of C-phenyl N-tert-butyl
[15N]nitrone (PBN-15N)
The literature procedure for the preparation of ether solution was dried over anhydrous magne-
trichloramine (NC13) [3,4] was adapted for the sium sulfate. After filtration, ether was removed
synthesis of trichloramine-13N (15NC13) in our sys- by slow fractional distillation to give 1.6 g of clear
tem. A 250-ml three-necked flask, equipped with a liquid. TLC (thin layer chromatography) analysis
mechanical stirrer, a thermometer and an addition showed that the Rf value of the liquid was the
funnel, was cooled to 1-2 °C. Calcium hypochlo- same as a known sample of 2-methyl-2-nitropro-
rite (70%, 12.35 g, 58.7 mmol), water (27 ml) and pane (Rf=0.5 with hexane as eluent). This liquid
methylene chloride (41 ml) were added succes- product is the desired intermediate of 2-methyl-2-
sively. With vigorous stirring, a solution of ammo- [15N]nitropropane which was used directly for fur-
nium-15N chloride (2.0 g, 36.7 mmol) in a mixed
solvent of 37% hydrochloric acid (7 ml) and water
ther preparation.
This crude liquid was mixed with benzaldehyde
(21 ml) was added dropwise for 20 min at < 5°C. (3.26 g, 30.76 mmol) in 95% ethanol (25 ml). After
The solution was stirred for additional 25 min with cooling to 0-5 °C, zinc dust (2.0 g, 30.76 mmol)
cooling. The two layers were separated and methy- was added with magnetic stirring. Acetic acid
lene chloride (20 ml) was used to wash the flask. (3.67 g, 61.52 mmol) was added dropwise for 20
The combination of the methylene chloride was min at < 9°C. The reaction mixture was stirred for
washed with cold water (2 x 40 ml) and dried over additional 7.5 h at < 3°C, then stored in a refriger-
anhydrous sodium sulfate. The obtained solution ator overnight. The mixture was filtered and the
contained the desired intermediate of trichlor- cake was washed with ethanol (2 x 30 ml). The
amine-15N (15NC13) and was kept below -50°C for residue after rotaevaporation was dissolved in
2 h before use.
chloroform (75 ml) and washed with water (2 x 30
The solution after immediate filtration was ml). The organic solution was dried over anhy-
added in one-portion to a precooled dry flask con- drous magnesium sulfate, filtered and rotaevapor-
ated. The residue was chromatographed on silica
taining aluminum chloride (7.33 g, 55 mmol) and
methylene chloride (15 ml) under an atmosphere gel eluted successively with methylene chloride
of pure nitrogen at -10°C. With magnetic stirring, and chloroform to give 1.18 g of sticky solid. One
a solution of 2-chloro-2-methylpropane (10.18 g, sublimation at 60-65 °C/0.1 torr gave 0.79 g of
110 mmol) in methylene chloride (10 ml) was white crystals with 12% overall yield. This product
added dropwise over 20 min to the reaction flask was further purified by repeated sublimations (5-
times more) at 50-55 °C/0.1 torr affording 0.65 g
while the temperature was controlled between
-10°C and -5°C. After the reaction mixture was of nice white crystals of C-phenyl N-tert-buty\
stirred for an additional hour at this temperature, [15N]nitrone (PBN-15N). TLC analysis with UV
detection method showed only one spot. The Rf
value of the product is the same as a known sam-
ple of normal PBN. Rf=0A2 (silica gel plate,
it was poured over a mixture of 37% hydrochloric
acid (5 ml) and ice (50 g). The mixture was stirred
for several minutes, then left to stand overnight.
The two layers were separated and the organic CHC13); m.p. 70-71 °C.
layer was washed with water (2 x 40 ml). The com-
bined aqueous solution was extracted with diethyl
!H NMR (300 MHz, CDC1/TMS, 298 K) <3
8.31-8.28 (m, 2H, Ar-H), 7.56 (d, 7=2.4 Hz, 1H,
ether (2 x 50 ml) and bubbled with nitrogen gas CH=NO), 7.43-7.40 (m, 3H, Ar-H), 1.62 (d, 7=2.4
for 10 minutes. This solution contained the hydro-
Hz, 9H, C(CH3)3) ppm.
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Y.-K. Zhang • Synthesis of C-Phenyl N-tert-Buty\ [15N]nitrone (PBN-15N)
143
MS (rel. int.%) m/z 179 (M++l, 1), 178 (M+, 8),
123 (2), 122 (11), 121 (4), 105 (4), 89 (14), 77 (11),
66 (9), 65 (14), 63 (7), 57 (r-Bu+, 100), 41 (40),
39 (17).
experiments described in this paper also support
the correct structure of this compound.
Acknowledgment
The author would like to acknowledge encour-
agement and useful suggestions from Drs. Edward
G. Janzen and Yashige Kotake. Special thanks is
also expressed to the Department of Free Radical
Biology and Aging Research Program of Okla-
homa Medical Research Foundation for housing
OMRF Spin Trap Source, and the Oklahoma
Medical Research Foundation for financial sup-
port for this work.
C„H1515NO (178.2)
Calcd C 74.12 H 8.48 N 8.42%,
Found C 73.99 H 8.57 N8.36%.
These data are consistent with the desired
structure of the novel compound C-phenyl N-tert-
butyl[15N]nitrone (PBN-15N). The spin trapping
[1] a) A. H. Beth, S. D. Venkataramu, K. Balasubra-
manian, L. R. Dalton, B. H. Robinson, D. E. Pear-
son, C. R. Park, J. H. Park, Proc. Natl. Acad. Sei.
USA 78, 967 (1981);
d) D. D. Bono, W.-D. Yang, M. C. R. Symons, Free
Rad. Res. 20, 327 (1994);
e) M. G. Hartell, G. Borzone, T. L. Clanton, L. J.
Berliner, Free Rad. Biol. Med. 17, 467 (1994);
f) E. G. Janzen, R. A. Towner, P. H. Krygsman, E.
K. Lai, J. L. Poyer, G. Brueggemann, P. B. McCay,
Free Rad. Res. Commun. 9, 353 (1990);
g) P. B. McCay, E. K. Lai, J. L. Poyer, C. M. DuBose,
E. G. Janzen, J. Biol. Chem. 259, 2135 (1984);
h) B. Kalyanaraman, R. P. Mason, E. Perez-Reyes,
C. F. Chignell, C. R. Wolf, R. M. Philpot, Biochem.
Biophys. Res. Commun. 89, 1065 (1979);
i) J. L. Poyer, R. A. Floyd, P. B. McCay, E. G. Jan-
zen, E. R. Davis, Biochim. Biophys. Acta 539, 402
(1978).
b) J. F. W. Keana, L. Lex, J. S. Mann, J. M. May, J.
H. Park, S. P. Pou, V. S. Prabhu, G. M. Rosen, B. J.
Sweetman, Y. Wu, Pure Appl. Chem. 62, 201 (1990);
c) K. Makino, S. Nagahara, Y. Konishi, M. Mukae,
H. Ide, A. Murakami, Free Rad. Res. Comms. Sup-
plement 19 (SI), S109 (1993);
d) S. P. Pou, G. M. Rosen, Y. Wu, J. F. W. Keana, J.
Org. Chem. 55, 4438 (1990).
[2] At least six names for PBN are currently being used
in the literature: a-phenyl N-tert-b\ity\ nitrone, C-
phenyl N-tert-butyl nitrone, N-tert-butyl a-phenyl
nitrone, N-tert-butyl C-phenyl nitrone, ,/V-benzyli-
dene terr-butylamine N-oxide, and 2-methyl-7V-phe-
nylmethylene-2-propanamine 7V-oxide. Because
PBN-14N is 269-times more naturally abundant than
PBN-15N in normal PBN, PBN-14N will be used to
refer to normal PBN in the description of this work.
[3] a) E. G. Janzen, Acc. Chem. Res. 4, 31 (1971);
b) W. A. Pryor, Free Rad. Biol. Med. 17, 451 (1994);
c) L. A. Reinke, D. R. Moore, C. M. Hague, P. B.
McCay, Free Rad. Res. 21, 213 (1994);
[4] P. Kovacic, S. S. Chaudhary, Org. Syn. 48, 4 (1968).
[5] J. W. Strand, P. Kovacic, J. Am. Chem. Soc. 95,
2977 (1973).
[6]A. Calder, A. R. Forrester, S. P. Hepburn, Org. Syn.
52, 77 (1972).
[7] D. L. Haire, U. M. Oehler, P. H. Krygsman, E. G.
Janzen, J. Org. Chem. 53, 4535 (1988).
[8]CRC Handbook of Chemistry and Physics, the 70th
edition; CRC Press: Florida, p. B-229 (1989-1990).
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