Improved yields in the synthesis of spin-labeled fatty acids

Article abstract of DOI:10.1081/SCC-200032488

Paramagnetic amide side products (6a-g) have been isolated from the reaction mixture in the synthesis of spin-labeled fatty acids of the doxyl type. After their hydrolysis to the corresponding acid, 7, the overall yield of spin-labeled fatty acids is significantly increased compared with published procedures.

Full text of DOI:10.1081/SCC-200032488

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Improved Yields in the
Synthesis of Spin‐Labeled
Fatty Acids
Janez Mravljak a & Slavko Pečar a b
a Faculty of Pharmacy , University of Ljubljana ,
Aškerčeva 7, SI‐1000, Ljubljana, Slovenia
b Institut Jožef Stefan , Jamova 39, SI‐1000,
Ljubljana, Slovenia
Published online: 19 Apr 2010.
To cite this article: Janez Mravljak & Slavko Pečar (2004) Improved Yields in the
Synthesis of Spin‐Labeled Fatty Acids, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 34:20, 3763-3771,
DOI: 10.1081/SCC-200032488
To link to this article: http://dx.doi.org/10.1081/SCC-200032488
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SYNTHETIC COMMUNICATIONSw
Vol. 34, No. 20, pp. 3763–3771, 2004
Improved Yields in the Synthesis of
Spin-Labeled Fatty Acids
1,2
Janez Mravljak1, and Slavko Pecar
*
ˇ
1
ˇ ˇ
Faculty of Pharmacy, University of Ljubljana, Askerceva 7,
SI-1000 Ljubljana, Slovenia
Institut Jozef Stefan, Jamova 39, SI-1000 Ljubljana, Slovenia
2
ˇ
ABSTRACT
Paramagnetic amide side products (6a–g) have been isolated from the
reaction mixture in the synthesis of spin-labeled fatty acids of the doxyl
type. After their hydrolysis to the corresponding acid, 7, the overall
yield of spin-labeled fatty acids is significantly increased compared
with published procedures.
Key Words: Amide hydrolysis; Fatty acid spin probe.
*
Correspondence: Janez Mravljak, Faculty of Pharmacy, University of Ljubljana,
ˇ ˇ
Askerceva 7, SI-1000 Ljubljana, Slovenia; E-mail: janez.mravljak@ffa.uni-lj.si.
3763
DOI: 10.1081/SCC-200032488
0039-7911 (Print); 1532-2432 (Online)
www.dekker.com
Copyright # 2004 by Marcel Dekker, Inc.

ˇ
Mravljak and Pecar
3764
INTRODUCTION
Fatty acid methyl esters and, especially, the corresponding acids contain-
ing the N-oxyl-4,4-dimethyl-oxazolidine (doxyl) group in the alkyl chain, are
useful molecular tools in electron spin resonance (ESR) spectroscopy as
lipophilic and amphiphilic spin probes for studying the structure and dynamics
of complex systems, especially phospholipid bilayers and biological mem-
branes.[1–4] The interaction of fatty acid spin labels with membrane com-
ponents reflects the structural and dynamic properties of the local
environment, accounting for their wide use as spin probes. They are also
used for preparing spin-labeled phospholipids[4–7] and biologically interesting
amphiphilic molecules.[8]
The doxyl group is usually prepared by oxidation of an oxazolidine com-
pound (2, Fig. 1) using 3-chloroperoxybenzoic acid.[4,9] The 2 is obtained by
condensation of a fatty acid ketoester (1) with 2-amino-2-methylpropanol.
After isolation and purification, doxyl fatty acid ester (5) is hydrolyzed
under standard conditions[1,5] to spin-labeled acid (7).[1,5,6] The condensation
of 1 with amino alcohol is normally carried out in boiling benzene, toluene, or
xylene in the presence of para-toluenesulfonic acid as a catalyst (Fig. 1).[1,4,5]
Although the water is continuously removed by a Dean–Stark water separator
and a large excess of amino alcohol is usually used, the formation of 2 is rather
slow, and after 7–9 days of heating (reflux), considerable amounts of the start-
ing 1 are still present. According to the published procedures,[1,5] 5 is isolated
.
Figure 1. a) 2-amino-2-methylpropanol, TsOH H2O, toluene, reflux, and water
separation by Dean–Stark separator; b) 3-chloroperoxybenzoic acid, ether; c) 1 M
NaOH, dioxane.

Improved Yields in the Synthesis of Spin-Labeled Fatty Acids
3765
in 15–30% overall yield. Ester (5) is then practically quantitatively hydro-
lyzed to acid 7. Thus, any increase in the overall yield of preparation of 7
will be acceptable, especially if 7 is used as a starting material for the synthesis
of more complex spin probes.
RESULTS AND DISCUSSION
During the synthesis of many different spin-labeled fatty acids (7),
we observed that after oxidation there are at least two paramagnetically
active compounds present in considerable amounts in the reaction mixture.
The fast-moving compound eluted with petroleum ether–ether (3 : 1 v/v)
from a silica gel column (5) is already known,[1,5] while a second paramagne-
tically active compound, under the stated conditions, stays bound to the silica
gel. Because of the low overall yield of doxyl acids (7) and because only the
isolation of doxyl esters (5) is described in published papers, we have studied
the reaction mixture resulting from the oxidation step more carefully. We iso-
lated and characterized two additional compounds from the silica gel column.
We found that, under the published conditions,[1,5] oxoester (1) reacts with
amino alcohol to give (Fig. 2) oxazolidine esters 2a–g, oxoamides 3a–g
and oxazolidine amides 4a–g.
.
Figure 2. a) 2-amino-2-methylpropanol, TsOH H2O, toluene, reflux, and water
separation by Dean–Stark separator.

ˇ
Mravljak and Pecar
3766
Because the isolation and purification of 2 from the mixture is complicated,
the published procedures use the mixture directly in the oxidation step. After
separation on the silica gel column, 5 is isolated with 15–30% overall yield,
while other side products, 6 and 3, have been ignored (Fig. 3).[1,5] To
improve the relatively low yield of 7, we purified and characterized the other
two side products that are formed in the condensation step in considerable
amounts. After elution of 7, the yellow residue at the top of the column was
eluted with a more polar eluant, ether. The corresponding yellow band was col-
lected and found to be composed of two compounds, one yellow and paramag-
netic 6a–g, and the other nonparamagnetic oxoamide 3a–g. Both compounds
have similar retention factors. On thin-layer chromatography (TLC), 6 is
readily seen under ultraviolet (UV), while 3 is visualized by spraying with a
reagent for ketones, 2,4-dinitrophenylhydrazine. The mixture of 3 and 6 was
further resolved by size exclusion chromatography (Sephadex LH-20,
ethanol as eluant). The analytically pure doxyl amide 6a–g was isolated
as an orange-yellow viscous oil or solid, in yields listed in Table 1. To prove
the structure of 6a–g, a sample of amide (6d) was hydrolyzed (Fig. 3). The
isolated spin-labeled fatty acid (7) was shown to be identical to a sample
synthesized from 5 according to Ref.[6]
.
EXPERIMENTAL
General
Chemicals were obtained (Acros, Aldrich, Fluka, Merck, Jannsen, and
Sigma) and used without further purification. Solvents were used without
Figure 3. b) 3-chloroperoxybenzoic acid, ether; c) 1 M NaOH, dioxane, 608C.

Improved Yields in the Synthesis of Spin-Labeled Fatty Acids
3767
Table 1. Yields of spin-labeled compounds.
Theoretical
overall yield
of 7 (%)
Yielda of 6b
(%)
Yielda of 5
(%)
Compound (m,n)
a(4,5)
25.4
22.5
18.3
20.0
24.2
16.3
23.8
28.1
28.5
30.2
17.2
20.8
20.4
25.7
53
51
48
37
45
37
49
b(10,5)
c(5,10)
d(12,3)
e(3,12)
f(7,8)
g(6,5)
aOf analytically pure material.
bAll compounds 6a–g have aN (ethanol) ¼ 1.485 mT.
additional purification or drying. Ketoesters (1a–g) were prepared according to
known procedures.[1,5] Analytical TLC plates (Merck, silica gel 60 F254) were
used to monitor the reactions. Silica gel 60 (70–230 mesh, Merck) was used for
column chromatography. Size exclusion chromatography was carried out on
Sephadex LH-20 (Pharmacia). Mass spectra were obtained with a VG-Analyti-
ˇ
cal Autospec Q mass spectrometer with EI ionization (MS Centre, Jozef Stefan
Institute). Infrared (IR) spectra were recorded on a Perkin–Elmer FT-IR 1600
spectrometer. Electron paramagnetic resonance (EPR) spectra of nitroxide sol-
utions were measured on a BRUKER X-band continuous wave ESR (CW-ESR)
ˇ
spectrometer ESP 300 (EPR Centre, Jozef Stefan Institute) at room temperature
in a glass capillary (1 mm inner diameter) and at 10 mW microwave power.
[1]
General Procedure (Modified from Refs. and
[5]
)
To 250 mL of toluene were added methylketoester 1a–g (0.05 mol),
50 mL (0.5 mol) of 2-amino-2-methylpropanol, and 50 mg (0.26 mmol) of p-
toluenesulfonic acid monohydrate. The mixture was heated at reflux for 9
days using a Dean–Stark trap for water removal. The toluene phase was
then washed (4 ꢀ 150 mL) with saturated sodium bicarbonate solution and
with brine (2 ꢀ 150 mL), then dried (Na2SO4). After evaporating the
toluene under reduced pressure, the colorless, viscous liquid containing com-
pounds 1a–g, 2a–g, 3a–g, and 4a–g was dissolved in 300 mL of ether and
cooled in an ice bath. 50 mL ether containing 0.055 mol of 3-chloroperoxy-
benzoic acid (80–90%, Jannsen) was added over a period of 2 h. The
mixture was allowed to stand for 12 h, after which the ether phase was

ˇ
Mravljak and Pecar
3768
washed (8 ꢀ 150 mL) with saturated sodium bicarbonate, (2 ꢀ 100 mL) with
water, and with brine (2 ꢀ 100 mL), then dried (Na2SO4). After evaporating
the ether under reduced pressure, the yellow, viscous oil was chromatographed
on 500 g of silica gel, using ether/petrolether ¼ 1/3 (v/v) as eluent. The start-
ing compound 1a–g was eluted before the yellow colored zone. The center
portion of the first fast-moving yellow band was collected, yielding 0.0075
to 0.0150 mol of 5a–g as an orange oil. The yellow residue at the top of the
column was eluted with ether, and the main portion of the second moving
yellow band was collected. The mixture of compounds 3a–g and 6a–g was
further separated by size exclusion chromatography on Sephadex LH-20
using ethanol (96%) as eluent, yielding 0.0075 to 0.0125 mol of analytically
pure 6 as an orange oil (6a, 6b, 6c, 6e, 6f, and 6g) or yellow solid (6d).
N-(2-hydroxy-1,1-dimethylethyl)-6-(3-oxyl-4,4-dimethyl-2-penthyl-1,
3-oxazolidin-2-yl)hexanamide (6a)
IR (NaCl): n ¼ 3310, 2932, 2870, 1650, 1548, 1462, 1365, 1263, 1184,
1055 cm21; MS-EI m/e (relative intensity): 372 ((M þ 1)þ, 4), 353 (5), 340
(14), 301 (6), 286 (24), 228 (8), 212 (12), 186 (30), 151 (10), 129 (11), 95
(10), 90 (20), 72 (15), 58 (100); high-resolution mass spectra (HRMS) calcu-
lated for C20H40N2O4 (M þ 1)þ 372.2988, found 372.2998.
N-(2-hydroxy-1,1-dimethylethyl)-6-(3-oxyl-4,4-dimethyl-2-undecyl-1,
3-oxazolidin-2-yl)hexanamide (6b)
IR (NaCl): n ¼ 3307, 2926, 2854, 1650, 1548, 1462, 1366, 1263, 1184,
1056 cm21; MS-EI m/e (relative intensity): 456 ((M þ 1)þ, 14), 437 (6),
424 (11), 370 (33), 301 (15), 283 (6), 270 (30), 229 (10), 212 (10), 129
(10), 90 (23), 81 (6), 72 (15), 58 (100); HRMS calculated for C26H52N2O4
(M þ 1)þ 456.3927, found 456.3905.
N-(2-hydroxy-1,1-dimethylethyl)-11-(2-hexyl-3-oxyl-4,4-dimethyl-1,
3-oxazolidin-2-yl)undecanamide (6c)
IR (NaCl): n ¼ 3309, 2927, 2855, 1649, 1546, 1461, 1364, 1266,
1057 cm21; MS-EI m/e (relative intensity): 456 ((M þ 1)þ, 2), 437 (1), 424
(10), 370 (30), 352 (11), 339 (11), 282 (17), 242 (10), 200 (37), 129 (16),
113 (14), 90 (26), 72 (19), 58 (100); HRMS calculated for C26H51N2O4
(M)þ 455.3849, found 455.3862.

Improved Yields in the Synthesis of Spin-Labeled Fatty Acids
3769
N-(2-hydroxy-1,1-dimethylethyl)-4-(3-oxyl-4,4-dimethyl-2-tridecyl-1,
3-oxazolidin-2-yl)butanamide (6d)
IR (NaCl): n ¼ 3297, 2919, 2851, 1628, 1550, 1466, 1380, 1192,
1057 cm21; MS-EI m/e (relative intensity): 456 ((M þ 1)þ, 2), 438 (4), 424
(8), 370 (37), 352 (6), 298 (39), 281 (34), 273 (20), 253 (25), 239 (7), 214
(8), 201 (24), 184 (59), 126 (30), 113 (34), 97 (25), 90 (19), 83 (27), 69
(47), 58 (100); HRMS calculated for C26H52N2O4 (M þ 1)þ 456.3927,
found 456.3945.
N-(2-hydroxy-1,1-dimethylethyl)-13-(2-butyl-3-oxyl-4,4-dimethyl-1,
3-oxazolidin-2-yl)tridecanamide (6e)
IR (NaCl): n ¼ 3319, 2926, 2854, 1650, 1548, 1462, 1364, 1263,
1056 cm21; MS-EI m/e (relative intensity): 456 ((M þ 1)þ, 4), 440 (2), 425
(7), 399 (11), 370 (20), 352 (8), 310 (15), 270 (8), 228 (6), 172 (47), 156
(10), 129 (14), 113 (11), 97 (7), 90 (22), 72 (20), 58 (100); HRMS calculated
for C26H52N2O4 (M þ 1)þ 456.3927, found 456.3945.
N-(2-hydroxy-1,1-dimethylethyl)-9-(3-oxyl-4,4-dimethyl-2-octyl-1,
3-oxazolidin-2-yl)nonanamide (6f)
IR (NaCl): n ¼ 3308, 2927, 2856, 1645, 1543, 1463, 1364, 1263,
1057 cm21; MS-EI m/e (relative intensity): 456 ((M þ 1)þ, 3), 437 (1), 424
(16), 370 (45), 352 (11), 343 (20), 311 (15), 281 (10), 271 (13), 254 (20), 228
(55), 214 (16), 142 (14), 129 (23), 90 (38),72 (27), 58 (100); HRMS calculated
for C26H51N2O4 (M)þ 455.3849, found 455.3851.
N-(2-hydroxy-1,1-dimethylethyl)-6-(2-heptyl-3-oxyl-4,4-dimethyl-1,
3-oxazolidin-2-yl)hexanamide (6g)
IR (NaCl): n ¼ 3311, 2930, 2858, 1650, 1562, 1462, 1365, 1263,
1055 cm21; MS-EI m/e (relative intensity): 400 ((M þ 1)þ, 21), 381 (19),
368 (45), 314 (73), 301 (33), 283 (24), 269 (16), 242 (11), 229 (27), 214
(76), 198 (15), 179 (14), 142 (17), 129 (29), 107 (17), 90 (47), 81 (18), 72
(31), 58 (100); HRMS calculated for C22H44N2O4 (M þ 1)þ 400.3301,
found 400.3310.

ˇ
3770
Mravljak and Pecar
General Procedure for the Hydrolysis of Amides 6a–g
0.1 g of amide 6a–g was dissolved in 1.5 mL of dioxane, and 1 mL of 1 M
NaOH was added. The reaction mixture was stirred for 24 h at 608C. The chilled
solution was brought to pH 1 with 1 M HCl, 10 mL of water was added, and the
solution was extracted with ethyl acetate. The ethyl acetate phase was dried
(Na2SO4), and the solvent was removed under reduced pressure to yield the cor-
responding spin-labeled fatty acid 7a–g in almost quantitative yield.
4-(3-Oxyl-4,4-dimethyl-2-tridecyl-1,3-oxazolidin-2-yl)
butanoic acid (7d)
IR (NaCl): n ¼ 3496, 3298, 2919, 2850, 1713, 1470, 1429, 1216, 1142,
1044, 944, 782 cm21; MS-EI m/e (relative intensity): 384 ((M)þ, 3), 368
(2), 338 (3), 328 (12), 298 (46), 281 (100), 253 (51), 211 (11), 202 (72),
186 (16), 142 (21), 130 (50), 112 (32), 97 (22), 87 (45), 71 (25), 56 (63);
HRMS calculated for C22H42NO4 (M)þ 384.3114, found 384.3125.
6-(2-Heptyl-3-oxyl-4,4-dimethyl-1,3-oxazolidin-2-yl)
hexanoic acid (7g)
IR (NaCl): n ¼ 2930, 2858, 1711, 1464, 1261, 1052 cm21; MS-EI m/e
(relative intensity): 328 ((M)þ, 10), 312 (2), 282 (4), 272 (36), 243 (19),
230 (61), 225 (93) 214 (69), 207 (30), 198 (14), 179 (44), 158 (39), 142
(51), 129 (88), 109 (40), 97 (46), 86 (25), 81 (31), 69 (55), 56 (100);
HRMS calculated for C18H34NO4 (M)þ 328.2488, found 328.2490.
N-(2-hydroxy-1,1-dimethylethyl)-10-oxooctadecanamide (3f) as a
representative compound of oxoamides 3a–g
IR (NaCl): n ¼ 3165, 2968, 2718, 2027 1627, 1552, 1466, 1365,
1098 cm21; MS-EI m/e (relative intensity): 370 ((M þ 1)þ, 11), 352 (3),
338 (31), 327 (6), 298 (8), 281 (9), 256 (7), 228 (24), 214 (29), 184 (6), 144
(12), 131 (21), 113 (6), 97 (7), 90 (12), 83 (6), 72 (17), 58 (100); HRMS
calculated for C22H44NO3 (M þ 1)þ 370.3321, found 370.3330.
ACKNOWLEDGMENTS
This research work was financially supported by the Ministry of
Education, Science and Sports of the Republic of Slovenia. The authors
ˇ
thank the EPR Centre, Jozef Stefan Institute, for EPR spectra; the MS

Improved Yields in the Synthesis of Spin-Labeled Fatty Acids
3771
ˇ
Centre, Jozef Stefan Institute, for mass spectra; and Prof. Roger Pain for his
critical reading of the manuscript.
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Received in the UK March 30, 2004