Synthesis of 3,4-disubstituted 2,5-dihydropyrrol-1-yloxyl spin label reagents

Article abstract of DOI:10.1055/s-1998-2177

4-Alkyl(or aryl)-3-ethoxycarbonyl-2,5-dihydro-1H-pyrrol-1-yloxyl radicals 5a-g were prepared in a multistep reaction via base-catalyzed conjugate addition of 2-nitropropane to β-aryl(or -alkyl)-α, β- unsaturated-α-ethoxycarbonyl ketones to give γ-nitro ketones, followed by a cyclization to 3,4-dihydro-2H-pyrrole 1-oxides, dehydrobromination to 2H- pyrrole 1-oxides and then Grignard reaction. The esters 5a-g are versatile synthons for obtaining highly SH-specific allylic iodide 7a,c-g and methylsulfonylthiomethyl 8a-g spin label reagents.

Full text of DOI:10.1055/s-1998-2177

SYNTHESIS
October 1998
1497
Synthesis of 3,4-Disubstituted 2,5-Dihydropyrrol-1-yloxyl Spin Label Reagents
Cecília P. Sár,a József Jekö,b Kálmán Hideg*a
a Institute of Organic and Medicinal Chemistry, University of Pécs, H-7643 Pécs, P.O. Box 99, Hungary
Fax +36(72)325731; E-mail: KHIDEG@main.pote.hu
b ICN Akaloida Co. Ltd., H-4440 Tiszavasvári, P.O. Box 1, Hungary
Received 18 December 1997; revised 12 March 1998
Abstract: 4-Alkyl(or aryl)-3-ethoxycarbonyl-2,5-dihydro-1H-
gate addition of 2-nitropropane to compounds 1a–f in a
phase transfer reaction with K2CO3 and tetrabutylammo-
nium hydrogen sulfate;15 the use of 1,8-diazabicyc-
lo[5.4.0]undec-7-ene (DBU) resulted mainly in the elimi-
nation of the NO2 group as previously reported.16 It was
unnecessary to isolate and purify the resulting γ -nitro ke-
tones, which can be directly converted in aqueous dioxane
with Zn/NH4Cl to 3,4-dihydro-2H-pyrrole 1-oxides 2a–f.
This nitrone could be selectively brominated at the highly
activated 3-carbon with N-bromosuccinimide,17 or even
with pyrrolidone hydrotribromide (PHT) which was pre-
viously applied for the synthesis of α-bromo ketones18
without involvement of the 2-methyl or the benzyl car-
bons. Only in the case of 2b was an over bromination with
NBS observed that could be avoided by using PHT.
pyrrol-1-yloxyl radicals 5a–g were prepared in a multistep reac-
tion via base-catalyzed conjugate addition of 2-nitropropane to β-
aryl(or -alkyl)-α,β-unsaturated-α-ethoxycarbonyl ketones to give
γ-nitro ketones, followed by a cyclization to 3,4-dihydro-2H-pyr-
role 1-oxides, dehydrobromination to 2H-pyrrole 1-oxides and
then Grignard reaction. The esters 5a–g are versatile synthons for
obtaining highly SH-specific allylic iodide 7a,c–g and methyl-
sulfonylthiomethyl 8a–g spin label reagents.
Key words: nitrones, 3,4-disubstituted 2,5-dihydropyrrole nitrox-
ides, SH-specific spin label reagents
Nitroxide free radicals containing reactive arms are useful
reporter groups in spin labelling studies of proteins and
nucleic acids.1, 2 For site selective spin labelling of pro-
teins, a general strategy has been developed that employs
site-directed mutagenesis to introduce a unique cysteine
residue into the sequence.3 Modification of the free sulf-
hydryl group with a selective nitroxide reagent then gen-
erates a nitroxide containing side chain. Among the SH-
labelling reagents, 3-halomethyl- and 3-methylsulfonyl-
thiomethyl reagents have proven most useful.4–7 For ex-
ample, it has been shown that the EPR spectra of the cor-
responding nitroxide side chains provides detailed infor-
mation on solvent accessibility, secondary and tertiary
structure, and protein dynamics.8–11
In most cases bromination was accompanied with dehy-
drobromination that could be completed with DBU to give
2H-pyrrole 1-oxides 3a–f (Scheme 1).
The introduction of a second substituent in the nitroxide
ring is expected to result in specific steric and/or electron-
ic interaction with the local environment in the macromol-
ecule that can be identified by analysis of the EPR spectral
line shape. The availability of such reagents is anticipated
to significantly extend the information content of the spin
labelling experiment applied to analysis of protein struc-
ture.
(i) 2-nitropropane (2 equiv), K2CO3, Bu4NHSO4 (cat), benzene,
40°C, 30 min, evaporation then Zn, NH4Cl, dioxane/H2O, 5°C to r.t.,
2 h (36–47%); (ii) NBS, benzoyl peroxide, CCl4, reflux, 5 min then
DBU, r.t., 30 min (62–76%); (iii) PHT, THF, r.t., 2 min, (82%); (iv)
MeMgI or PhMgI, Et2O, 0°C to r.t. then NH4Cl then O2, MnO2 (cat),
CHCl3, 30 min (40–65%)
In previous work the introduction of 4-alkyl substituents
was achieved in a multistep synthesis,12 but the route has
two limitations due to the presence of the paramagnetic
center: 4-aryl-2,5-dihydro-1H-pyrroles cannot be directly
prepared, and formation of the 3,4-double bond in the end
products was only possible when the paramagnetic group
was protected as a diamagnetic N-acetoxy function.13
Scheme 1
Both the saturated 2a and unsaturated 3a–f nitrones could be
converted selectively to pyrrolidine and dihydropyrrole ni-
troxide esters 4a and 5a–g without the involvement of the
ester function by treatment with organomagnesium halides in
diethyl ether or THF. In a previous synthesis of 3-methoxy-
carbonyl-2,2,5,5-tetramethyl-1-oxyl-4-phenyl-pyrrol-
idine, 3-methoxycarbonyl-2,2,5,5-tetramethyl-1-oxyl-2,5 di-
hydro-1H-pyrrole was treated with an excess of phenylorga-
nocuprate, the reaction accompanied by the formation of 3-
benzoyl-2,2,5,5-tetramethyl-1-oxyl-4-phenylpyrrolidine.19
The synthesis reported in the present communication can
be applied to the preparation of a great variety of 3-esters
of both 4-alkyl- or 4-aryl-substituted pyrrolidine and di-
hydropyrrole radicals.
The starting β-substituted-α,β-unsaturated-α-ethoxycar-
bonyl ketones 1a–f are readily available by Knoevenagel
type reactions of ethyl acetoacetate with aldehyde.14 The
γ-nitro ketones were obtained by base-catalyzed conju-

SYNTHESIS
Papers
1498
The ester group of compounds 5a–g was reduced to the In conclusion, a multistep route was used for the synthesis
alcohol in 6a–g with sodium bis(2-methoxyethoxy)alumi- of a variety of both 4-aryl- and 4-alkyl-substituted 3-
num hydride (SMEAH) without reduction of the double iodomethyl or methylsulfonylthiomethyl reagents useful
bond. The allylic alcohols were converted to mesylates for spin labelling of SH groups of biomolecules.
which were used without isolation in the reaction in dry
acetone with NaI to yield the allylic iodides 7a,c–g. The
highly reactive iodides readily reacted in aqueous EtOH
with sodium methanethiosulfonate to give the compounds
Mps were determined on a Boetius micro melting point apparatus and
are uncorrected. Elemental analyses (C, H, N, S) were performed on
EA 1110 Elemental Analyser apparatus or (Hal) were carried out tit-
containing the methylsulfonylthiomethyl groups 8a,c–g.
In the synthesis of the 4-pyridyl compound 8b, the less
reactive mesylate was converted directly to methylsulfo-
nylthiomethyl function to avoid quaternerization of the
pyridine nitrogen (Scheme 2).
rimetrically by Schöniger’s method. The IR spectra (Specord 85)
were in each case consistent with the assigned structure. MS were
recorded on a VG TRIO-2 instrument in the EI mode (70 eV, direct
inlet). ESR spectra were obtained from a 10–5 molar solution (CHCl3)
using a Bruker 300-E spectrometer.
Flash column chromatography was performed on Merck Kieselgel 60
(0.04–0.063 mm). The physical and spectral data of all new com-
pounds are listed in the Table. Compounds 1a–f were prepared ac-
cording to the Cope modification of the Knoevenagel reaction using
piperidine acetate as catalyst and benzene as solvent.14
Synthesis of 3,4-Dihydro-2H-pyrrole 1-Oxides 2a–f from α,â-Un-
saturated Oxo Esters 1a–f; General Procedure:
To a solution of unsaturated oxo ester 1a–f (10 mmol) in benzene
(20 mL) were added K2CO3 (1.38 g, 10 mmol), 2-nitropropane
(1.78 g, 20 mmol) and Bu4HSO4 (cat., 20 mg). The mixture was
stirred at 40°C for 30 min, allowed to cool to r.t., and water (20 mL)
added. The organic layer was extracted with water (2 × 20 mL), dried
(MgSO4) and evaporated in vacuo. The residues were used without
purification in the ring-closure reaction as follows:
The above product and NH4Cl (0.53 g, 10 mmol) were dissolved in
H2O/dioxane (1:2, 30 mL) and cooled to 5°C. Under vigorous stirring
Zn powder (2.60 g, 40 mmol) was added and after 30 min the mixture
was allowed to warm to r.t. After stirring for 1 h, the mixture was
filtered and the precipitated salt washed with MeOH (3 × 10 mL). The
liquid phase was evaporated to its half volume and extracted with
CHCl3 (3 × 15 mL), dried (MgSO4) and evaporated. The residue was
purified by flash column chromatography (CHCl3/Et2O/MeOH) to
give 4-aryl(alkyl)-3-ethoxycarbonyl nitrones; yield 2a: 1.16 g (42%);
2b: 1.05 g (38%); 2c: 1.07 g (36%); 2d: 1.58 g (47%); 2e: 1.39 g
(45%); 2f: 1.29 g (38%).
(v) SMEAH, THF, –78 °C, 10 min then r.t., 1 h then 10% NaOH (61–
77%); (vi) MsCl, Et3N, CH2Cl2, 0 °C to r.t., 30 min then NaI, acetone,
reflux, 30 min (58–81%); (vii) NaSSO2Me, EtOH/H2O, r.t., 30 min
(61–76%); (viii) MsCl, Et3N, CH2Cl2, 0 °C to r.t., 30 min then
NaSSO2Me, EtOH/H2O, r.t., 1 h (57%)
Scheme 2
3-Alkyl(or Aryl)-4-ethoxycarbonyl-2,2,5-trimethyl-2H-pyrrole
1-Oxides 3a,c–f; General Procedure:
Compound 5e was nitrated without destruction of the ni-
troxide function. Nitration took place in the meta position
because of the meta director (α,β-unsaturated ester) and
ortho director (chloro) substituents to yield 4-(4-chloro-3-
nitrophenyl)-3-ethoxycarbonyl-2,2,5,5-tetramethyl-2,5-di-
hydro-1H-pyrrol-1-yloxyl radical (9), while nitration of
compound 4a took place in the para position as described
earlier.19 The nitrated product offers a further possibility to
develop heterocycles or a second reactive arm (Scheme 3).
To a solution of nitrone 2a,c–f (10 mmol) in anhyd CCl4 (20 mL)
were added NBS (2.22 g, 12.5 mmol) and dibenzoyl peroxide (cat-
alytic amount, 10 mg). The mixture was stirred and refluxed for
5 min, cooled to r.t., and DBU (1 mL) was added. After stirring for
30 min at r.t. to complete the dehydrobromination reaction, CHCl3
(20 mL) was added and the mixture extracted with 5% H2SO4
(30 mL), water (30 mL), dried (MgSO4) and evaporated. The resi-
due was chromatographed (silica gel, CHCl3/Et2O/MeOH) and after
purification kept at –18°C. Yield 3a: 1.97 g (72%), mp 120–122°C;
3c: 1.90 g (68%); 3d: 2.50 g (75%); 3e: 1.91 g (62%), mp 100–102°C;
3f: 2.56 g (76%).
4-Ethoxycarbonyl-2,2,5-trimethyl-3-(4-pyridyl)-2H-pyrrole
1-Oxide (3b):
To a solution of nitrone 2b (2.76 g, 10 mmol) in anhyd THF (30 mL)
was added pyrrolidone hydrotribromide (5.35 g, 1.1 mmol). The reac-
tion was complete in 1–2 min, then the mixture was extracted with sat.
K2CO3 (20 mL), brine (2 × 20 mL), dried (MgSO4) and evaporated.
The residue was purified by flash column chromatography (CHCl3/
MeOH). Yield: 2.25 g (82%), mp 74–75°C.
(ix) H2SO4, HNO3, 0 °C then r.t., 2 h (64%)
Scheme 3

SYNTHESIS
October 1998
1499
Table. Physical and Spectroscopic Data of New Compounds 2–9a
Product
Yield (%)
mp (°C)
IR (Nujol)
MS
m/z (%)
ν (cm–1)
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4a
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
7a
7c
7d
7e
7f
7g
8a
8b
8c
8d
8e
8f
42
38
36
47
45
38
72
82
68
75
62
76
57
62
40
47
52
65
59
54
77
61
74
68
67
75
70
81
58
69
77
72
68
76
57
68
61
65
70
72
64
oil
oil
oil
oil
oil
oil
120–122
74–75
oil
1730 (C=O), 1590 (C=C)
1728 (C=O), 1594 (C=C)
1730 (C=O), 1590 (C=C)
1728 (C=O), 1600, 1590 (C=C)
1730 (C=O), 1590 (C=C)
1722 (C=O)
1710 (C=O), 1580, 1530 (C=C, C=N)
1714 (C=O), 1582, 1530 (C=C, C=N)
1710 (C=O), 1574, 1524 (C=C, C=N)
1710 (C=O), 1580, 1530 (C=C, C=N)
1705 (C=O), 1600, 1530 (C=C, C=N)
1710 (C=O), 1600, 1530 (C=C, C=N)
1600 (C=C)
1700 (C=O), 1630, 1590 (C=C)
1708 (C=O), 1635, 1590 (C=C)
1708 (C=O), 1628 (C=C)
1694 (C=O), 1635, 1600 (C=C)
1705 (C=O), 1630, 1590 (C=C)
1708 (C=O), 1624 (C=C)
1706 (C=O), 1635, 1590 (C=C)
3360 (OH), 1590, 1566 (C=C)
3250–3150 (OH), 1590 (C=C)
3400–3200 (OH), 1630 (C=C)
3500–3300 (OH), 1600, 1580 (C=C)
3450–3200 (OH), 1650, 1590 (C=C)
3370 (OH), 1650 (C=C)
3380 (OH), 1650 (C=C)
1630 (C=C)
275 (M+, 100), 260 (18), 202 (19), 56 (86)
276 (M+, 1), 180 (7), 136 (100), 108 (16)
281 (M+, 34), 208 (15), 137 (18), 56 (100)
335 (M+, 2), 192 (22), 150 (48), 43 (100)
309 (M+, 20), 236 (17), 220 (13), 56 (100)
339 (M+, 2), 282 (22), 141 (35), 43 (100)
273 (M+, 69), 257 (24), 184 (54), 42 (100)
274 (M+, 2), 85 (100), 56 (18), 42 (78)
279 (M+, 15), 99 (57), 56 (57), 41 (100)
333 (M+, 6), 309 (6), 85 (100), 42 (96)
307 (M+, 100), 292 (16), 262 (14), 218 (24)
337 (M+, 1), 73 (88), 60 (96), 43 (100)
290 (M+, 27), 274 (6), 132 (65), 56 (100)
288 (M+, 100), 273 (90), 258 (55), 128 (56)
289 (M+, 20), 275 (40), 259 (42), 43 (100)
294 (M+, 100), 279 (97), 264 (73), 190 (66)
348 (M+, 100), 334 (95), 318 (88), 229 (31)
322 (M+, 100), 307 (82), 292 (62), 218 (34)
353 (M+H)+b
oil
100–102
oil
oil
75–77
92–94
64–66
55–57
107–109
oil
oil
350 (M+, 2), 320 (16), 105 (100), 94 (87)
246 (M+, 100), 231 (74), 216 (64), 128 (65)
247 (M+, 35), 232 (21), 56 (100), 43 (65)
252 (M+, 94), 237 (100), 222 (46), 189 (55)
306 (M+, 100), 292 (59), 276 (37), 246 (32)
280 (M+, 100), 265 (89), 250 (37), 236 (40)
310 (M+, 55), 295 (76), 280 (65), 43 (100)
308 (M+, 15), 294 (33), 278 (29), 43 (100)
356 (M+, 17), 342 (16), 214 (53), 43 (100)
362 (M+, 5), 294 (100), 279 (82), 264 (58)
416 (M+, 100), 402 (46), 274 (97), 151 (57)
390 (M+, 37), 376 (16), 248 (100), 177 (44)
421 (M+H)+b
111–113
167–168
118–120
oil
111–112
oil
oil
86–88
oil
1640 (C=C)
1640, 1600 (C=C)
1590 (C=C)
1640 (C=C)
1600 (C=C)
1630 (C=C)
1635 (C=C)
1630 (C=C)
110–112
101–103
oil
oil
419 (M+H)+b
108–110
145–147
oil
116–118
83–85
oil
340 (M+, 19), 326 (23), 246 (52), 41 (100)
341 (M+, 1), 289 (43), 275 (74), 43 (100)
346 (M+, 8), 264 (81), 237 (51), 43 (100)
400 (M+, 91), 386 (49), 370 (49), 274 (93)
374 (M+, 100), 360 (48), 344 (31), 248 (69)
404 (M+, 37), 374 (61), 278 (67), 43 (100)
402 (M+, 2), 388 (7), 378 (6), 45 (100)
1600 (C=C)
1600 (C=C)
1640 (C=C)
1596 (C=C)
8g
9
123–125
105–107
1705 (C=O), 1630, 1595 (C=C), 1540 (NO2) 367 (M+, 81), 352 (100), 337 (62), 246 (67)
a
Satisfactory elemental analyses obtained.
b Thermospray (TSP) mass spectrum was registered.
Grignard Reactions of Nitrones; Synthesis of Pyrrolidin-1-yloxyl
and 2,5-Dihydro-1H-pyrrol-1-yloxyl Radicals 4a, 5a–g; General
Procedure:
dropwise at –78°C under argon. The mixture was stirred at –78°C for
10 min then at r.t. for 1 h. After the reaction was complete, the mixture
was cooled in an ice bath and decomposed by dropwise addition of
10% NaOH (10 mL). The organic phase was washed with brine, dried
(MgSO4) and evaporated in vacuo to dryness. The residue was puri-
fied by flash column chromatography (hexane/EtOAc) to yield 6a:
0.95 g (77%), mp 111–113°C; 6b: 0.75 g (61%), mp 167–168°C; 6c:
0.93 g (74%), mp 118–120°C; 6d: 1.04 g (68%); 6e: 0.94 g (67%),
mp 111–112°C; 6f: 1.16 g (75%); 6g: 1.08 g (70%).
To a solution of MeMgBr or PhMgBr (20 mmol) in anhyd Et2O
(20 mL) was added dropwise the nitrone 2a or 3a–f (10 mmol) in an-
hyd THF (20 mL) at 0°C. The mixture was stirred at r.t. for 1 h, then
sat. NH4Cl (40 mL) was added. The organic layer was dried (MgSO4)
and evaporated. The residue was dissolved in CHCl3, MnO2 (catalytic
amount, 50 mg) was added, and the mixture was bubbled with O2 for
30 min. After filtration, the mixture was evaporated and chromato-
graphed (silica gel, Et2O/hexane) to yield 4a: 1.65 g (57%); 5a: 1.78
g (62%), mp 75–77°C; 5b: 1.15 g (40%), mp 92–94°C; 5c: 1.38 g
(47%), mp 64–66°C; 5d: 1.81 g (52%), mp 55–57°C; 5e: 2.09 g
(65%), mp 107–109°C; 5f: 2.07 g (59%); 5g: 1.89 g (54%).
Allylic Iodides 7a,c–g from Alcohols 6a,c–g; General Procedure:
To a solution of Et3N (1.20 g, 12 mmol) and the alcohol 6a,c–g
(10 mmol) in anhyd CH2Cl2 (30 mL) was added MsCl (1.37 g,
12 mmol) at 0°C. The mixture was stirred for 30 min at r.t., washed
with 5% NaHCO3, 5% H2SO4, dried (MgSO4) and evaporated. The
residue was dissolved in anhyd acetone and NaI (2.25 g, 15 mmol)
was added. The mixture was refluxed for 30 min and the solvent was
evaporated. The residue was dissolved in water and extracted with
Et2O, dried (MgSO4) and evaporated. Flash column chromatography
Reduction of Nitroxide Esters to Alcohols 6a–g; General Proce-
dure:
To a solution of nitroxide ester 5a–g (5 mmol) in anhyd THF (15 mL)
was added 70% SMEAH in toluene (1.5 mL) in anhyd THF (10 mL)

SYNTHESIS
Papers
1500
(hexane/Et2O) provided the iodides; yield 7a: 2.88 g (81%), mp 86–
88°C; 7c: 2.10 g, (58%); 7d: 2.87 g (69%), mp 110–112°C; 7e: 3.01
g (77%), mp 101–103°C; 7f: 3.03 g (72%); 7g: 2.84 g (68%).
This work was supported by grants from the Hungarian National
Research Foundation (T 017842). The authors wish to express their
thanks to B. Rozsnyai for technical assistance and for microanalyses
and to Professor W.L. Hubbell (UCLA School of Medicine, Los Ange-
les) for helpful discussions.
Methylsulfonylthiomethyl Nitroxides 8a,c–g from Iodides 7a, c–
g; General Procedure:
To a solution of the iodide compound 7a,c–g (5 mmol) in EtOH
(20 mL) was added NaSSO2Me (1.01 g, 7.5 mmol) in water (5 mL)
and the mixture was refluxed for 30 min. EtOH was evaporated, the
residue was taken up in CHCl3 (20 mL), washed with brine, dried
(MgSO4), evaporated and purified by flash column chromatography
(silica gel, hexane/EtOAc); yield 8a: 1.29 g (76%), mp 108–110°C;
8c: 1.18 g (68%); 8d: 1.22 g (61%), mp 116–118°C; 8e: 1.22 g (65%),
mp 83–85°C; 8f: 1.44 g (70%); 8g: 1.39 g (72%), mp 123–125°C.
(1) Hideg, K.; Hankovszky, H. O. In Biological Magnetic Reso-
nance, Vol. 8.; Berliner, L. J.; Reuben, J. Ed.; Plenum: New
York, 1989; p 427.
(2) Hideg, K. Pure Appl. Chem. 1990, 62, 207.
(3) Cornish, V. W.; Benson, D. R.; Altenbach, C. A.; Hideg, K.;
Hubbell, W. L.; Schultz, P. G. Proc. Natl. Acad. Sci., USA 1994,
91, 2910.
(4) Berliner, L. J.; Grunwald, J.; Hankovszky, H. O.; Hideg, K.
Anal. Biochem. 1982, 119, 450.
(5) Hankovszky, H. O.; Hideg, K.; Lex, L. Synthesis 1980, 914.
(6) Hideg, K.; Sár, P. C.; Hankovszky, H. O.; Jerkovich, Gy. Syn-
thesis 1991, 616.
2,2,5,5-Tetramethyl-3-methylsulfonylthiomethyl-4-(4-pyridyl)-
2,5-dihydro-1H-pyrrol-1-yloxyl Radical (8b):
To a solution of Et3N (0.606 g, 6 mmol) and alcohol 6b (1.24 g,
5 mmol) in anhyd CH2Cl2 (15 mL) was added MsCl (0.68 g, 6 mmol)
at 0°C. The mixture was stirred for 30 min, washed with 5%
NaHCO3, dried (MgSO4) and evaporated. The oily residue was dis-
solved in EtOH (10 mL) and NaSSO2Me (1.01 g, 7.5 mmol) was add-
ed in water (3 mL) and the mixture was stirred at r.t. for 1 h. EtOH
was evaporated, the residue was taken up in CHCl3 (15 mL), washed
with brine, dried (MgSO4), evaporated and purified by flash column
chromatography (silica gel, CHCl3/Et2O); yield: 0.97 g (57%), mp
145–147°C.
(7) Bárácz, M. N.; Hankovszky, H. O.; Sár, P. C.; Jerkovich, Gy.;
Hideg, K. Synthesis 1996, 204.
(8) Esmann, M.; Sár, P. C.; Hideg, K.; Marsh, D. Anal. Biochem.
1993, 213, 336.
(9) Farahbakhsh, T. Z.; Hideg, K.; Hubbell, W. L. Science 1993,
262, 1416.
(10) Mchaourab, H.; Lietzow, M. A.; Hideg, K.; Hubbell, W. L. Bio-
chemistry 1996, 35, 7692.
(11) Oh, J. K.; Zhan, H.; Cui, C.; Hideg, K.; Collier, R. J.; Hubbell,
W. L. Science 1996, 273, 810.
(12) Kálai, T.; Bárácz, M. N.; Jerkovich, Gy.; Hankovszky, H. O.;
Hideg, K. Synthesis 1995, 1278.
4-(4-Chloro-3-nitrophenyl)-3-ethoxycarbonyl-2,2,5,5-tetrameth-
yl-2,5-dihydro-1H-pyrrol-1-yloxyl Radical (9):
(13) Kálai, T.; Rozsnyai, B.; Jerkovich, Gy.; Hideg, K. Synthesis
1994, 1079.
To a stirred mixture of 5e (3.23 g, 10 mmol) in concd H2SO4 (10 mL)
at 0°C was added dropwise a mixture of concd H2SO4 (8 mL) and
67% HNO3 (4 mL), keeping the temperature <5°C. The deep red mix-
ture turned yellow. It was stirred at 0–5°C for 30 min and then at r.t.
for 2 h. The mixture was poured onto vigorously stirred crushed ice
(~100 g) and neutralized with 40% NaOH. The resulting mixture was
extracted with CHCl3 (3 × 15 mL), dried (MgSO4) and evaporated.
The resulting yellow crystalline product was recrystallized from
Et2O; yield: 2.14 g (64%), mp 105–107°C.
(14) Jones, G. Org. React. 1967, 15, 264.
(15) Krysthal, G. V.; Kulganek, V. V.; Kucherov, V. F.; Yanovska-
ya, L. A. Synthesis 1979, 107.
(16) Ballini, R.; Bosica, G. Synlett 1996, 1115.
(17) Black, D. St. C.; Strauch, R. J. Aust. J. Chem. 1988, 41, 183.
(18) Hankovszky, H. O.; Hideg, K.; Sár, P. C.; Lovas, M. J.; Jerko-
vich, Gy. Synthesis 1990, 59.
(19) Hideg, K.; Hankovszky, H. O.; Halász, H. A.; Sohár, P.
J. Chem. Soc., Perkin 1. 1988, 2905.