Synthesis of 2,2- and 2,5-disubstituted 1-oxyl pyrrolidine radicals as new homobifunctional cross-linking spin labels

Article abstract of DOI:10.1055/s-2003-41053

The synthesis of 2,2- and 2,5-disubstituted pyrrolidine nitroxide radicals starting from readily available nitrones 1, 11 is described. The stable radicals are acylating (10, 20), alkylating (7, 17) and thiol-specific (8, 18) reagents capable of producing cross-links in proteins over distances in the range of 10-15 A.

Full text of DOI:10.1055/s-2003-41053

2084
PAPER
Synthesis of 2,2- and 2,5-Disubstituted 1-Oxyl Pyrrolidine Radicals as
New Homobifunctional Cross-Linking Spin Labels
1
-Oxyl Pyrrolid
a
ine Radical
m
s
as
N
ew
H
omobifu
á
nctional Cr
s
oss-Linking Spi
K
n Labels álai,a József Jekő,b Wayne L. Hubbell,c Kálmán Hideg*a
a
Institute of Organic and Medicinal Chemistry, University of Pécs, 7602 Pécs, P. O. Box 99, Hungary
Fax +36(72)536219; E-mail: kalman.hideg@aok.pte.hu
b
c
ICN Hungary Ltd., 4440 Tiszavasvári, P. O. Box 1, Hungary
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095-7008, USA
Received 28 May 2003
able)11,12 and thiol-specific homobifunctional cross-link-
ing reagents13,14 containing reactive substituents only in
the 3,4-positions of the pyrroline nitroxide ring. In this pa-
per, we present an extension of the above idea with the
synthesis of novel 2,2- and 2,5-disubstituted pyrrolidine
nitroxide homobifunctional reagents that are designed to
serve the purposes mentioned above.
Abstract: The synthesis of 2,2- and 2,5-disubstituted pyrrolidine
nitroxide radicals starting from readily available nitrones 1, 11 is
described. The stable radicals are acylating (10, 20), alkylating (7,
17) and thiol-specific (8, 18) reagents capable of producing cross-
links in proteins over distances in the range of 10–15 Å.
Key words: cross linking reagents, proteins, free radicals, Grignard
reactions, nitrones, lithium
Treatment of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)
(1) or 2,5-dimethyl-1-pyrroline N-oxide15 (11) with 4-
dimethoxymethylphenylmagnesium bromide yielded
trisubstituted nitrones16 2 and 12, respectively, after oxi-
dation of the hydroxylamines to the nitrones by activated
MnO2. Further treatment of nitrone 12 with 4-
dimethoxymethylphenylmagnesium bromide gave trans
diphenyl nitroxide 13 analogously to earlier cases.17 On
the other hand, similar treatment of nitrone 2 yielded only
the unsubstituted hydroxylamine. Apparently, the second
phenyl substituent cannot be easily introduced into this
sterically hindered position, as was observed earlier by
Iwahashi et al.18 Treatment of compound 2 with the more
reactive 4-dimethoxymethylphenyllithium obtained by
translithiation19 of 4-bromobenzaldehyde dimethyl acetal
gave the desired nitroxide 3 after oxidation with activated
MnO2.
Nitroxides have received considerable attention as co-ox-
idants,1 spin labels,2 MRI agents,3 antioxidants4 and dou-
ble sensor molecules.5 Among these uses, spin labeling is
the most important and wide-spread application. A major
advance in the field of protein spin labeling was the intro-
duction of site directed spin labeling (SDSL). SDSL is
proving to be a general strategy for determining the struc-
ture of both soluble and membrane proteins of any molec-
ular weight.6–8 Moreover, recent work indicates that
protein dynamics, an important determinant of protein
function, can be extracted from the motion of nitroxide
side chains.9,10 Homo-bifunctional nitroxide reagents,
particularly those with reactive functionality toward sulf-
hydryl groups, promise to significantly extend the capa-
bilities of SDSL. For example, bifunctional reagents that
cross-link a pair of cysteine residues spaced by one or two
turns along an a-helix are strongly immobilized with re-
spect to the protein backbone, as expected. In this case,
motion of the nitroxide group, reflected in the EPR spec-
trum, is dominated by rigid body motions of the helix.
Thus, the EPR spectra of spin labels with dual attachment
points may provide a direct and powerful measure of pro-
tein dynamics without the usual contributions from the in-
ternal motion of the side chain itself.10 Because the
orientation of the nitroxide ring is necessarily fixed by the
cross-link, the angular dependence of the EPR spectrum
in an oriented system (i.e., membranes) can be used to de-
duce the orientation of the helical axis with respect to the
director of the system. Finally, the rate of cross-link for-
mation, easily monitored by EPR in real time, will provide
direct measures of both cysteine proximity and protein
flexibility. In our laboratory we have synthesized several
heterobifunctional (thiol-specific and photoactivat-
Dimethylacetals 3 and 13 could be deprotected by treat-
ment with aqueous H2SO4 in THF to give aldehydes 4 and
14,20 which were in turn reduced with NaBH4 in ethanol
to give alcohols 5 and 15. The alcohols were converted to
the corresponding mesylates, which yielded the benzylic
bromides 6 and 16 upon treatment with LiBr in ace-
tone.21,22 To obtain the more reactive benzylic iodides for
use as alkylating agents,23 compounds 6 and 16 were treat-
ed with NaI to give compounds 7 and 17. The thiol-specif-
ic bismethanethiosulfonate reagents 8 and 18 were
obtained by treatment of bromides 6 and 16 with
NaSSO2CH3 in aqueous acetone.24 Reagents 8 and 18 are
capable of making cross-links between SH of cysteine res-
idues. Dialdehydes 4 and 14 were oxidized to dicarboxyl-
ic acids 9 and 19 by Ag2O in an aq NaOH (10%)–THF
mixture.25 The dicarboxylic acids were converted to dis-
uccinimidyl esters26 10 and 20 by treatment with N-hy-
droxysuccinimide and N,N¢-dicyclohexylcarbodiimide
(Schemes 1 and 2). The resulting disuccinimides are acy-
lating reagents for amino groups and capable of making
cross-link between lysine side chains.
Synthesis 2003, No. 13, Print: 18 09 2003. Web: 22 08 2003.
Art Id.1437-210X,E;2003,0,13,2084,2088,ftx,en;P04103SS.pdf.
DOI: 10.1055/s-2003-41053
© Georg Thieme Verlag Stuttgart · New York

PAPER
1-Oxyl Pyrrolidine Radicals as New Homobifunctional Cross-Linking Spin Labels
2085
Scheme 1 Reagents and conditions: (a) 4-dimethoxymethylphenylmagnesium bromide (1.2 equiv) THF, 0 °CÆreflux, 3 h, under N2, then
activated MnO2 (1.0 equiv), O2, 10 min, CHCl3, reflux, 1 h, 70%; (b) 4-dimethoxymethylphenyllithium (1.2 equiv), THF, –78 °C, 20 min, then
2, reflux 5 h, then activated MnO2, CHCl3, O2, 30 min, 65%; (c) THF, aq H2SO4 (5%), r.t., 8 h, 92%; (d) NaBH4 (2.5 equiv) EtOH, 40 °C, 30
min, 78%; (e) MsCl (2.2 equiv), Et3N (2.2 equiv), CH2Cl2, 0 °C Æ r.t., 1 h, then LiBr (4.0 equiv), acetone, reflux, 1 h, 55%; (f) NaI, 4.0 equiv,
reflux, 30 min, 68%; (g) NaSSO2CH3 (4.0 equiv), acetone–H2O, 30 min, reflux, 43%; (h) Ag2O (4.0 equiv), aq NaOH (10%)–THF, 60 °C, 40
min, then H+, 45%; (i) N-hydroxysuccinimide (2.0 equiv), DCC (2.0 equiv), EtOAc, 0 °C ∆ r.t., 12 h, 61%.
Mps were determined with a Boetius micro melting point apparatus
and are uncorrected. Elemental analyses (C, H, N, S) were per-
formed on Carlo Erba EA 1110 CHNS elemental analyzer. IR
(Specord 75) spectra were in each case consistent with the assigned
structure. Mass spectra were recorded on a VG TRIO-2 instrument
in the EI mode (70 eV, direct inlet). ESR spectra were obtained from
10–5 molar solutions (CHCl3), using a Bruker ECS-106 spectrome-
ter. All monoradicals exhibited three equally spaced lines with
aN = 15.1–15.5 G. Preparative flash column chromatography was
performed on Merck Kieselgel 60 (0.040–0.063 mm). Qualitative
TLC was carried out on commercially prepared plates
In summary, new 2,2- and 2,5-disubstituted stable pyrro-
lidine radicals were obtained with simple procedures
starting from readily available nitrones. These homobi-
functional amino-specific and thiol-specific reagents
serve as cross-linkers between lysine and cysteine resi-
dues, respectively. The general synthetic procedure is
readily extendable to include spacer groups other than
benzyl to vary the cross-linking distance. Applications of
the reagents will be reported separately.
Scheme 2 Reagents and conditions: (a) 4-dimethoxymethylphenylmagnesium bromide (1.2 equiv) THF, 0 °C Æreflux, 3 h, under N2, then
activated MnO2 (1.0 equiv), O2, 10 min, CHCl3, reflux, 1 h, 55%; (b) 4-dimethoxymethylphenylmagnesium bromide (1.2 equiv), THF, 0 °C,
then 12, reflux, 3 h, under N2, then activated MnO2, CHCl3, O2, 30 min, 60%; (c) THF, aq H2SO4 (5%), r.t., 8 h, 85%; (d) NaBH4 (2.5 equiv)
EtOH, 40 ºC, 30 min, 70%; (e) MsCl (2.2 equiv), Et3N (2.2 equiv), CH2Cl2, 0 °C Ær.t., 1 h, then LiBr (4.0 equiv), acetone, reflux, 1 h, 53%;
(f) NaI (4.0 equiv), acetone, reflux, 30 min, 77%; (g) NaSSO2CH3 (4.0 equiv), acetone–H2O, 30 min, reflux, 28%; (h) Ag2O (4.0 equiv), aq
NaOH (10%)–THF, 60 °C, 40 min, then H+, 40%; (i) N-hydroxysuccinimide (2.0 equiv), DCC (2.0 equiv), EtOAc, 0 ºC Ær.t., 12 h, 55%.
Synthesis 2003, No. 13, 2084–2088 © Thieme Stuttgart · New York

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T. Kálai et al.
PAPER
Table 1 New Compounds (2–10, 12–20) Prepareda
Compound
Yield (%)
Mp (ºC)
60–62
IR (cm–1) Neat or Nujol
1600, 1500 (C=C)
MS (m/z)
2
3
4
5
6
70
60
92
78
55
263 (M+, 27), 248 (5), 232 (100), 216 (20)
414 (M+, 2), 297 (22), 246 (83), 75 (100)
322 (M+, 8), 308 (35), 292 (23), 236 (100)
326 (M+, 10), 312 (23), 296 (15), 240 (100)
oil
1600, 1500 (C=C)
133–135
107–109
174–176
1680 (C=O), 1590 (C=C)
3300 (OH), 1500 (C=C)
1500 (C=C)
450/452/454 (M+, 3/6/3), 436/438/440 (8/16/8),
364/366/368 (6/12/6), 285/287 (100/100)
7
8
68
43
45
61
55
65
85
70
53
183–185
115–117
143–145
103–105
oil
1520 (C=C)
546 (M+, 16), 473 (6), 420 (6), 333 (100)
514 (M+, <1), 484 (2), 404 (2), 65 (100)
354 (M+, 9), 340 (43), 324 (55), 268 (100)
548 (M+, <1), 435 (1), 115 (35), 55 (100)
263 (M+, 38), 232 (76), 161 (77), 75 (100)
414 (M+, 3), 384 (18), 192 (13), 161 (100)
322 (M+, 5), 308 (16), 292 (23), 146 (100)
326 (M+, 4), 312 (9), 296 (14), 148 (100)
1510 (C=C)
9
1680 (C=O), 1600, 1560 (C=C)
1760, 1720 (C=O), 1550 (C=C)
1600, 1510 (C=C)
10
12
13
14
15
16
55–57
1600, 1500 (C=C)
181–183
163–165
161–162
1680 (C=O), 1590, 1560 (C=C)
3300 (OH), 1500 (C=C)
1500 (C=C)
450/452/454 (M+, 1/2/1), 420/422/424 (2/4/2),
210/212 (14/14), 131 (100)
17
18
19
20
77
28
40
55
155–157
138–139
242–244
222–224
1510 (C=C)
546 (M+, 2), 389 (4), 258 (8), 131 (100)
514 (M+, <1), 484 (1), 80 (55), 65 (100)
354 (M+, 2), 340 (6), 310 (20), 148 (100)
548 (M+, <1), 435 (1), 115 (35), 55 (100)
1500 (C=C)
1680 (C=O), 1590 (C=C)
1760, 1720 (C=O), 1550 (C=C)
a All compounds gave satisfactory microanalyses: C, H, N, S (where appropriate) 0.3.
(20 × 20 × 0.02 cm) coated with Merck Kieselgel GF254. All re-
agents and 1 were purchased from Sigma-Aldrich, Hungary. Com-
pound 11 was prepared according to published procedures.15 The
physical and spectral data of all new compounds are listed in
Table 1.
Nitrone 2
Yield: 9.20 g (70%); off-white solid; mp 60–62 ºC; Rf 0.28 (CHCl3–
Et2O, 2:1).
Nitrone 12
Yield: 7.23 g (55%); oil; Rf 0.28 (CHCl3–MeOH, 9:1).
5-(4-Dimethoxymethylphenyl)-2,2-dimethyl-1-pyrroline N-Ox-
ide (2) and 2-(4-Dimethoxymethylphenyl)-2,5-dimethyl-1-pyr-
roline N-Oxide (12)
2,2-Bis-(4-dimethoxymethylphenyl)-5,5-dimethylpyrrolidin-1-
yloxyl Radical (3)
To a stirred solution of 4-dimethoxymethylphenylmagnesium bro-
mide [made from Mg turnings (1.44 g, 60.0 mmol) and 4-bro-
mobenzaldehyde dimethyl acetal (13.86 g, 60.0 mmol)] in THF (40
mL), nitrone 1 or 11 (5.65 g, 50.0 mmol) was added dropwise at 0
°C and the mixture was stirred and refluxed for 3 h under N2. After
cooling, sat. aq NH4Cl solution (20 mL) was added, the organic
phase was separated, and the aq phase was extracted with CHCl3
(2 × 20 mL). The combined organic phases were dried (MgSO4),
filtered and evaporated. The residue was dissolved in CHCl3 (40
mL), activated MnO2 (4.35 g, 50.0 mmol) was added in one portion,
O2 was bubbled through for 10 min, and the solution was stirred and
refluxed for 1 h. The MnO2 was filtered off, the solvent was evapo-
rated under reduced pressure and the residue was purified by flash
column chromatography (CHCl3–Et2O) to give nitrone 2 or 12.
To a stirred solution of 4-dimethoxymethylphenyl bromide (6.93 g,
30.0 mmol) in THF (30 mL), BuLi (2.5 M in hexane; 14 mL, 35
mmol) was added dropwise at –78 °C and the mixture was stirred
for 20 min at this temperature. Nitrone 2 (6.57 g, 25.0 mmol) dis-
solved in THF (20 mL) was added dropwise and the mixture was
stirred and refluxed for 5 h under N2. After cooling, the reaction
mixture was quenched with sat. aq NH4Cl solution (20 mL), the or-
ganic phase was separated and the aq phase was extracted with
CHCl3 (2 × 20 mL). The combined organic phases were dried
(MgSO4), activated MnO2 (870 mg, 10.0 mmol) was added, O2 was
bubbled through for 30 min, and the mixture was filtered and evap-
orated. The residue was purified by flash column chromatography
(hexane–EtOAc) to give compound 3.
Yield: 6.72 g (65%); red oil; Rf 0.28 (hexane–EtOAc, 2:1).
Synthesis 2003, No. 13, 2084–2088 © Thieme Stuttgart · New York

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1-Oxyl Pyrrolidine Radicals as New Homobifunctional Cross-Linking Spin Labels
2087
2,5-trans-Bis-(4-dimethoxymethylphenyl)-2,5-dimethylpyrroli-
din-1-yloxyl Radical (13)
organic phase was separated, dried (MgSO4), filtered, and the sol-
vent evaporated. The residue was purified by flash column chroma-
To a stirred solution of 4-dimethoxymethylphenylmagnesium bro-
mide [made from Mg turnings (720 mg, 30.0 mmol) and 4-bro-
mobenzaldehyde dimethyl acetal (6.93 g, 30.0 mmol)] in THF (25
mL), nitrone 12 (6.57 g, 25.0 mmol) dissolved in THF (20 mL) was
added dropwise at 0 °C and the mixture was stirred and refluxed for
3 h under N2. After cooling, sat. aq NH4Cl solution (20 mL) was
added, the organic phase was separated, and the aq phase was ex-
tracted with CHCl3 (2 × 20 mL). The combined organic phases
were dried (MgSO4), activated MnO2 (870 mg, 10.0 mmol) was
added, O2 was bubbled through for 30 min, and the mixture was fil-
tered and evaporated. The residue was purified by flash column
chromatography (hexane–EtOAc) to give compound 13.
tography (hexane–EtOAc) to give compound 6 or 16.
Compound 6
Yield: 2.48 g (55%); mp 174–176 ºC; Rf 0.56 (hexane–EtOAc, 2:1).
Compound 16
Yield: 2.39 g (53%); mp 161–162 ºC; Rf 0.60 (hexane–EtOAc, 2:1).
2,2-Bis-(4-iodomethylphenyl)-5,5-dimethylpyrrolidin-1-yloxyl
Radical (7) and 2,5-trans-Bis-(4-iodomethylphenyl)-2,5-dimeth-
ylpyrrolidin-1-yloxyl Radical (17)
A solution of bis(benzylic bromide) 6 or 16 (904 mg, 2.0 mmol) and
NaI (1.20 g, 8.0 mmol) in acetone was stirred and refluxed for 30
min. The NaBr was filtered off, and the acetone was evaporated un-
der reduced pressure. The residue was dissolved in EtOAc (20 mL)
and washed with aq Na2S2O3 solution (5%; 10 mL). The organic
phase was separated, dried (MgSO4), filtered, evaporated under re-
duced pressure and the residue was purified by flash column chro-
matography (hexane–Et2O) to give compound 7 or 17.
Yield: 6.21 g (60%); orange solid; mp 55–57 ºC; Rf 0.35 (hexane–
EtOAc, 2:1).
2,2-Bis-(4-formylphenyl)-5,5-dimethylpyrrolidin-1-yloxyl Rad-
ical (4) and 2,5-trans-Bis-(4-formylphenyl)-2,5-dimethylpyrro-
lidin-1-yloxyl Radical (14)
To a stirred solution of bis(dimethyl acetal) 3 or 13 (8.28 g; 20.0
mmol) in THF (40 mL),
aq H2SO4 (5%; 10 mL) was added and the mixture was stirred for 8
h at r.t. Brine (30 mL) and Et2O (30 mL) were then added, the or-
ganic phase was separated, dried (MgSO4), filtered, evaporated and
the residue was purified by flash column chromatography (hexane–
EtOAc) to give the aldehyde 4 or 14.
Compound 7
Yield: 742 mg (68%); yellow solid; mp 183–185 ºC; Rf 0.58 (hex-
ane–EtOAc, 2:1).
Compound 17
Yield: 841 mg (77%); yellow solid; mp 155–157 ºC; Rf 0.65 (hex-
ane–EtOAc, 2:1).
Aldehyde 4
Yield: 5.92 g (92%); yellow solid; mp 133–135 ºC; Rf 0.16 (hex-
ane–EtOAc, 2:1).
2,2-Bis-(4-methanethiosulfonylmethylphenyl)-5,5-dimethyl-
pyrrolidin-1-yloxyl Radical (8) and 2,5-trans-Bis-(4-methane-
thiosulfonylmethylphenyl)-2,5-dimethylpyrrolidin-1-yloxyl
Radical (18)
To a solution of dibromo compound 6 or 16 (452 mg, 1.0 mmol) in
acetone (10 mL) and H2O (5 mL), NaSSO2CH3 (536 mg, 4.0 mmol)
was added and the mixture heated under reflux for 30 min. After
cooling, the acetone was evaporated, and the aq phase was extracted
with CHCl3 (2 × 10 mL). The organic phase was separated, dried
(MgSO4), filtered, evaporated and the residue was purified by flash
column chromatography (CHCl3–Et2O) to give compound 8 or 18.
Aldehyde 14
Yield: 5.47 g (85%); yellow solid; mp 181–183 ºC; Rf 0.19 (hex-
ane–EtOAc, 2:1).
2,2-Bis-(4-hydroxymethylphenyl)-5,5-dimethylpyrrolidin-1-
yloxyl Radical (5) and 2,5-trans-Bis-(4-hydroxymethylphenyl)-
2,5-dimethylpyrrolidin-1-yloxyl Radical (15)
To a stirred solution of aldehyde 4 or 14 (6.44 g, 20.0 mmol) in
EtOH (50 mL), NaBH4 (1.89 g, 50.0 mmol) was added and the mix-
ture was stirred for 30 min at 40 ºC. The solvents were evaporated,
the residue was dissolved in brine (30 mL), the aq phase was ex-
tracted with CHCl3 (2 × 20 mL), dried (MgSO4), filtered and the
solvent evaporated. The residue was purified by flash column chro-
matography (CHCl3–MeOH) to give compound 5 or 15.
Compound 8
Yield: 221 mg (43%); mp 115–117 ºC; Rf 0.46 (CHCl3–Et2O, 2:1).
Compound 18
Yield: 144 mg (28%); mp 138–139 ºC; Rf 0.47 (CHCl3–Et2O, 2:1).
Compound 5
2,2-Bis-(4-carboxyphenyl)-5,5-dimethylpyrrolidin-1-yloxyl
Radical (9) and 2,5-trans-Bis-(4-carboxyphenyl)-2,5-dimeth-
ylpyrrolidin-1-yloxyl Radical (19)
Yield: 5.08 g (78%); yellow solid; mp 107–109 ºC; Rf 0.26 (CHCl3–
MeOH, 9:1).
To a vigorously stirred suspension of freshly precipitated Ag2O
(9.26 g, 40.0 mmol) in aq NaOH solution (10%; 40 mL), aldehyde
9 or 19 (3.22 g, 10.0 mmol) in THF (15 mL) was added at 60 ºC and
the mixture was stirred and refluxed for 40 min. After cooling, the
solution was filtered through a Celite pad and washed with MeOH
(20 mL). The organic solvents were evaporated off, the aq phase
was acidified with aq H2SO4 (5%) to pH 2, and extracted with
CHCl3 (3 × 20 mL). The organic phase was dried (MgSO4), filtered
and evaporated. The residue was purified by flash column chroma-
tography (CHCl3–MeOH) to give dicarboxylic acid 9 or 19.
Compound 15
Yield: 4.56 g (70%); yellow solid; mp 163–165 ºC; Rf 0.33 (CHCl3–
MeOH, 9:1).
2,2-Bis-(4-bromomethylphenyl)-5,5-dimethylpyrrolidin-1-ylox-
yl Radical (6) and 2,5-trans-Bis-(4-bromomethylphenyl)-2,5-
dimethylpyrrolidin-1-yloxyl Radical (16)
To a solution of bis(benzylic alcohol) 5 or 15 (3.26 g, 10.0 mmol)
and Et3N (2.22 g, 22 mmol) in CH2Cl2 (40 mL), methanesulfonyl
chloride (2.52 g, 22.0 mmol) was added at 0 °C and the mixture was
stirred at r.t. for 1 h. The reaction mixture was then washed with
H2O (30 mL), dried (MgSO4), filtered, and the solvent evaporated.
The residue was dissolved in anhyd acetone (50 mL), LiBr (3.48 g,
40.0 mmol) was added, and the mixture was heated at reflux and
stirred for 1 h. After cooling, the acetone was evaporated, the resi-
due was dissolved in Et2O (40 mL), washed with H2O (20 mL), the
Dicarboxylic Acid 9
Yield: 1.59 g (45%); mp 143–145 ºC; Rf 0.13 (CHCl3–MeOH, 9:1).
Dicarboxylic Acid 19
Yield: 1.41 g (40%); mp 242–244 ºC; Rf 0.16 (CHCl3–MeOH, 9:1).
Synthesis 2003, No. 13, 2084–2088 © Thieme Stuttgart · New York

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T. Kálai et al.
PAPER
2,2-Bis-(4-carboxyphenyl)-5,5-dimethylpyrrolidin-1-yloxyl
Bis(succinimide Ester) Radical (10) and 2,5-trans-Bis-(4-car-
boxyphenyl)-2,5-dimethylpyrrolidin-1-yloxyl Bis(succinimide
Ester) Radical (20)
(5) Bilski, P.; Hideg, K.; Kálai, T.; Bilska, M. A.; Chignell, C.
F. Free Radical Biol. Med. 2003, 34, 489.
(6) Hubbell, W. L.; Mchaourab, H. S.; Altenbach, C.; Lietzow,
M. A. Structure 1996, 4, 779.
To a stirred solution of carboxylic acid 9 and 19 (354 mg, 1.0 mmol)
and N-hydroxysuccinimide (230 mg, 2.0 mmol) in EtOAc (10 mL),
DCC (412 mg, 2.0 mmol) dissolved in EtOAc (5 mL) was added
dropwise at 0 ºC. The mixture was stirred at r.t. overnight, the pre-
cipitated dicyclohexyl urea was filtered off, the filtrate was evapo-
rated and purified by flash column chromatography (CHCl3–Et2O)
to give active ester 10 or 20.
(7) Hubbell, W. L.; Gross, A.; Langen, R.; Lietzow, M. A. Curr.
Opin. Struct. Biol. 1998, 8, 649.
(8) Hubbell, W. L.; Cafiso, D. S.; Altenbach, C. Nat. Struct.
Biol. 2000, 7, 735.
(9) Columbus, L.; Kálai, T.; Jekő, J.; Hideg, K.; Hubbell, W. L.
Biochemistry 2001, 40, 3828.
(10) Columbus, L.; Hubbell, W. L. Trends Biochem. Sci. 2002,
27, 288.
Ester 10
(11) Kálai, T.; Rozsnyai, B.; Jerkovich, Gy.; Hideg, K. Synthesis
1994, 1079.
(12) Lösel, R. M.; Philipp, R.; Kálai, T.; Hideg, K.; Trommer, W.
E. Bioconjugate Chem. 1999, 10, 578.
Yield: 335 mg (61%); mp 103–105 ºC; Rf 0.39 (CHCl3–MeOH,
9:1).
Ester 20
(13) Kálai, T.; Balog, M.; Jekő, J.; Hideg, K. Synthesis 1999, 973.
(14) Kálai, T.; Balog, M.; Jekő, J.; Hubbell, W. L.; Hideg, K.
Synthesis 2002, 2365.
Yield: 301 mg (55%); mp 222–224 ºC; Rf 0.35 (CHCl3–MeOH,
9:1).
(15) Keana, J. F. W. In Spin Labeling II, Theory and
Applications; Berliner, L. J., Ed.; Academic Press: New
York, 1979, 115.
Acknowledgments
(16) Sár, P. C.; Jekő, J.; Fajer, P.; Hideg, K. Synthesis 1999, 1039.
(17) Rockenbauer, A.; Korecz, L.; Hideg, K. J. Chem. Soc.,
Perkin Trans. 2 1993, 2149.
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This work was supported by grant from Hungarian National Rese-
arch Foundations (OTKA T34307). A Bolyai fellowship for T. K.
from the Hungarian Academy of Sciences is gratefully acknowled-
ged. The authors thank to Mária Balog for technical assistance and
Viola H. Csokona for elemental analysis and Mária Szabó for mass
spectral measurements (ICN, Hungary).
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Synthesis 2003, No. 13, 2084–2088 © Thieme Stuttgart · New York