Synthesis of novel amphiphilic spin probes with the paramagnetic doxyl group in the polar region

Article abstract of DOI:10.1016/j.tet.2008.11.016

The use of ESR and specially designed spin probes has led to major breakthroughs in understanding the complexity of biological membranes. Research has been focused mainly on molecular events within the?lipid bilayer, and few probes have been designed for studying events in the extracellular space near the membrane surface. We have prepared a series of amphiphilic spin probes in which an ethylene glycol type hydrophilic spacer was introduced between a hydrophobic anchor and the doxyl group, placing the latter above the membrane in the extracellular space. Furthermore, the 2pπ orbital, containing the unpaired electron of the nitroxide group, would be orientated perpendicular to the membrane surface, making it more useful for ESR investigations of structural and dynamic properties close to the membrane surface in different situations of the cell life.

Full text of DOI:10.1016/j.tet.2008.11.016

Tetrahedron 65 (2009) 659–665
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of novel amphiphilic spin probes with the paramagnetic
doxyl group in the polar region
,
a,b
Stane Pajk a , Slavko Pecar
*
ˇ
a
ˇ
ˇ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia
Institut Jozef Stefan, Jamova 39, 1000 Ljubljana, Slovenia
b
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 April 2008
Received in revised form 21 October 2008
Accepted 6 November 2008
Available online 12 November 2008
The use of ESR and specially designed spin probes has led to major breakthroughs in understanding the
complexity of biological membranes. Research has been focused mainly on molecular events within
the lipid bilayer, and few probes have been designed for studying events in the extracellular space near
the membrane surface. We have prepared a series of amphiphilic spin probes in which an ethylene glycol
type hydrophilic spacer was introduced between a hydrophobic anchor and the doxyl group, placing the
latter above the membrane in the extracellular space. Furthermore, the 2p
p orbital, containing the
unpaired electron of the nitroxide group, would be orientated perpendicular to the membrane surface,
making it more useful for ESR investigations of structural and dynamic properties close to the membrane
surface in different situations of the cell life.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
containing the nitroxide group (Fig. 2) was located mostly in a li-
pophilic part of the probe. Only in some cases was the nitroxide
Development of stable nitroxide free radicals has been intensive
in the last 50 years.1 Originally, they were used in electron spin
resonance (ESR) spectroscopy as spin probes and spin labels.2 Later
they became important in chemistry as co-oxidants and antioxi-
dants in materials science,3 as candidates for organic magnets,4 as
polymerization modulators,5 as pH sensitive probes6 and, in
biology and pharmacology, as biologically active ligands,7–10 etc.
Furthermore, the combination of two labels in the same molecule,
e.g., a paramagnetic nitroxide and a fluorescent dye opens up new
fields of use as dual probes.11
Nitroxides, with their unique stability of the radical, which is
resistant to many mild reaction conditions, and especially with
their paramagnetic anisotropy, are becoming more and more im-
portant as molecular tools, since their structure can be adapted to
particular requirements. The most popular and useful heterocyclic
compounds containing a nitroxide group are shown in Figure 1.
These compounds have contributed to the determination of the
structure and dynamics of biological membranes.12 More recently
they have been applied in ESR determination of membrane het-
erogeneity, aggregation of membrane components and membrane
domain formation. In the last 30 years, membrane investigations
have been focused mostly on molecular events within the lipid
bilayer. Consequently the paramagnetic oxazolidine (doxyl) ring
moiety (of piperidine or pyrrolidine type) located in the polar head
group of phospholipids.13 Due to the possibility of introducing the
doxyl group onto the fatty acid alkyl chain, such probes have a great
potential for investigating molecular events in different regions of
the membrane bilayer. Recently, molecular dynamics simulations of
spin labelled alkyl phosphates in model lipid bilayer have been
reported,14,15 showing that the position of the doxyl group along
the fatty acid alkyl chain has an influence on the overall spin probe–
membrane interaction. However, the small doxyl group shows only
a small perturbation effect on the surrounding alkyl chain motion
in a lipid bilayer.
While the structure and dynamics of the lipid part of the
membrane have been widely studied, molecular events in the ex-
tracellular space in the vicinity of the membrane surface remain to
be investigated. The structure of the extracellular matrix is much
more complex than that of the lipid bilayer. Also the expected dy-
namics of oligosaccharide units of glycolipids, glycopeptides and
other membrane constituents exposed to the outer membrane
space are likely to be important in cell–cell interactions. There is,
therefore, a need for spin probes reporting directly from the extra-
cellular membrane region. Recently, the first synthesis and appli-
cation16,17 of a spin probe to describe the dynamics close to
a biological membrane surface has been reported. The spin probe
has a piperidine type of nitroxide (Fig. 3) in the extracellular polar
region. However, due to the predominantly parallel orientation to
the membrane surface of the 2p
taining the unpaired electron of the nitroxide group, the spin probe
p orbital (along the z axis) con-
* Corresponding author. Tel.: þ386 1 4769 500; fax: þ386 1 4258 031.
E-mail address: stane.pajk@ffa.uni-lj.si (S. Pajk).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.016

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S. Pajk, S. Pecar / Tetrahedron 65 (2009) 659–665
Table 1
R
(R′)
R
R
R
Distances between the acyl carbonyl and nitrogen of the nitroxide group (Fig. 4)
N
O
Compounds
4, 5
15, 17
14, 16
22, 23
27, 28
R1
R2
N
O
N
O
N
O
N
O
N
O
Distance (d)/nm
1.7
1.3
0.9
0.9
1.3
a
b
c
d
e
Figure 1. Typical structures of stable nitroxides: pyrroline (a), pyrrolidine (b), imid-
azoline (c), oxazolidine (‘doxyl’, d) and piperidine (e). R marks the position where
different groups can be attached.
m = 12,14
X = O or NH
n = 1-3
O
H
N
N
O
X
O
m
O
n
O
d
Figure 4. Schematic structure of final compounds.
N O
COOR
n
R = H, CH3 or other
n, m - number of CH2 groups
O
H3C
m
2. Results and discussion
Figure 2. General structure of a fatty acid spin probe.
All paramagnetic compounds were characterized by NMR
spectroscopy, using in situ nitroxide reduction by phenylhydrazine
to the corresponding non-paramagnetic derivatives of hydroxyl-
amine.19 The final compounds (Scheme 1) have a tetradecanoyl (4,
14, 15, 22, 27) or palmitoyl (5, 16, 17, 23, 28) acyl residue as the li-
pophilic part of the molecule. Both acyl residues are natural an-
chors for proteins, which are bound to biological membranes.
was not optimal. A probe with predominantly perpendicular 2p
p
orbital orientation would be more useful.18
Here we present the synthesis of novel amphiphilic spin probes,
which (i) would be able to report from the extracellular region close
to membrane surface and in which, in its extended conformation,
(ii) the nitroxide 2p
p orbital would be perpendicular to the
O
HO
H
membrane surface. The design of these compounds is based on
three requirements: they should comprise (i) a lipophilic part act-
ing as an anchor, enabling its insertion into the lipid bilayer, (ii)
a polar spacer group of variable length unable to penetrate into the
lipid and (iii) at the opposite end of the polar spacer, a doxyl type of
nitroxide with minimal lipophilic character. The latter is of im-
portance due to the possibility that the nitroxide moiety might curl
back in the hydrophobic region of the membrane if a more hydro-
phobic nitroxide was used. According to our experiences from
synthesis of doxyl fatty acids the compounds with the doxyl group
are more polar than the parent carbonyl compounds. Moreover,
Mravljak et al.14 pointed out that more or less ordered membrane
surrounding tries to push the doxyl group out of the hydrophobic
region and only the length (i.e., large lipophilicity) of the alkyl
chains attached to the doxyl group force it to be located within the
lipid bilayer. In our case, the doxyl group is located at the very end of
the polar spacer and it has a little chance to penetrate into the hy-
drophobic membrane region. Furthermore, when the probe con-
n
n = 2,3
(e)
n = 3 (a)
O
m = 12,14
O
O
COOEt
O
H
n
HO
m
3
6 (94%), 7 (96%),
8 (60%), 9 (95%)
1 (56%)
H2N
O
N
O
(b,c)
(f)
2
O
O
O
O
COOH
O
HO
O
N
H
3
m
n-1
N
O
10 (30%), 11 (80%),
12 (20%), 13 (81%)
3 (37%)
m = 12,14 (d)
(g)
O
O
O
O
O
formation is extended, its 2p
p orbital should be perpendicular to
O
O
O
O
O
N
N
H
the membrane surface (Fig. 3). In the novel amphiphilic spin probes
presented, ethylene glycol and amino glycol types of spacer were
used. The distance between the nitroxide group and lipophilic part
of the probe would vary (Table 1, Fig. 4) from 0.9 nm to 1.7 nm.
m
m
N
n-1
N
3
H
O
O
14 (18%), 15 (25%),
16 (25%), 17 (16%)
4 (25%), 5 (42%)
Scheme 1. Reagents and conditions: (a) ethyl diazoacetate, BF3$Et2O, CH2Cl2, 0 ꢀC, 2 h;
(b) NaOH, dioxane/H2O¼2:1, rt, 2 h; (c) 2, DCC, CH2Cl2, 0 ꢀC, 3 h; (d) acyl chloride, Et3N,
CH2Cl2, 0 ꢀC, 0.5 h; (e) acyl chloride, Et3N, CH2Cl2, 0 ꢀC, 6 h; (f) CrO3 dissolved in 35%
H2SO4, acetone, 0 ꢀC/rt, 3 h; (g) 2, DCC, Et3N, acetone, 0 ꢀC/rt, 2 h.
R
polar spacer
O
N
z
z
O
N
R=
O
The central part of the probes is a polar spacer of variable length
(Table 1), for that role mono-, di- and triethylene glycols or the
corresponding amino ethylene glycols were chosen. Acyl residues
are bound to the polar spacer via an ester bond (4, 5, 14, 15, 16 and
17) (Scheme 1) or an amide bond (22, 23, 27 and 28) (Scheme 2).
Final compounds 4 and 5 have the longest spacer (Table 1), com-
posed of triethylene glycol residues. The synthetic strategy includes
the reaction of commercially available triethylene glycol with ethyl
lipophilic anchor
a
b
Figure 3. Amphiphilic spin probe after insertion of the lipophilic part into the mem-
brane bilayer has nitroxide group in water surroundings and its 2p orbital is per-
pendicular (a) or parallel (b) to the long molecular axis or parallel (a) and
perpendicular (b) to the membrane surface.
p

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S. Pajk, S. Pecar / Tetrahedron 65 (2009) 659–665
661
diazoacetate, hydrolysis of the resulting ester 1, followed by cou-
pling of the acid with amine 2.20 The free amine 2 was unstable
after removal of the phthalimido protecting group with methyl-
amine. It was necessary to use it immediately after deprotection
and without purification. The instability of free amine 2 was
probably the main reason for the relatively low yields of com-
pounds 3, 14, 15, 16, 17, 22, 23 and 26. Final esters 4 and 5 were
obtained by acylating the hydroxyl moiety of compound 3 with the
corresponding acid chloride (Scheme 1).
PBS buffer (a)
ethanol (b)
liposomes (c)
Cl
O
mT
O
OH
(d)
PhthN
O
O
O
H2N
OH
OH
24 (88%)
m=12,14
O
(a)
(b)
O
Figure 5. Recorded ESR spectra of 15 dissolved in (a) phosphate buffer saline (pH¼7.4),
O
(b) ethanol and (c) liposome suspension.21
PhthN
O
N
OH
O
OH
m
H
25 (86%)
(c)
18 (80%), 19 (92%)
In order to lengthen the spacer moiety by one oxyethyl segment
potassium phthalimide was alkylated with 2-(2-(2-chloroethoxy)-
ethoxy)ethanol to yield compound 24 (Scheme 2). Oxidation of
compound 24 with Jones reagent produced carboxylic acid 25,
which was coupled with amine 2 to yield phthalimide 26. TBTU was
once more used, since it gave better results than DCC as with the
synthesis of amides 22 and 23. After removal of the phthalimido
protection group of compound 26 with methylamine, the resulting
amine was directly acylated without purification to produce amides
27 and 28.
(b)
O
O
O
O
PhthN
O
O
N
H
O
N
H
OH
N
O
m
O
20 (62%), 21 (88%)
26 (37%)
(c)
m=12,14 (e,f)
O
O
O
O
O
O
O
N
N
N
H
N
N
N
m
m
H
H
2
H
O
O
By introducing polar spacer groups of different lengths between
the nitroxide moiety and the lipophilic anchor, information about
molecular dynamics of oligosaccharide chains of glycolipids and
glycoproteins at different distances from the membrane surface
should be obtainable. It is expected that in the case of ordered re-
gions of oligosaccharide chains in extracellular space the presented
spin probes will provide important and new information. Further-
more, a difference between compounds that have the lipophilic
anchor attached by an ester bond (e.g., 15) and by an amide bond
(e.g., 27) is anticipated. The amide functional group can establish
one more hydrogen bond and therefore should position itself
higher in the membrane, pushing the nitroxide reporting group
further away from the membrane. The ester functionality on the
other hand can penetrate deeper into the membrane, bringing the
nitroxide moiety closer to it. The ester group allows rotational
movement and is, in that sense, much less rigid than the amide.
From the ESR spectra signal intensity only slight solubility of
compound 15 in PBS buffer (a) is observed (Fig. 5). The increase of
the intensity of the ESR signal after the addition of liposome sus-
pension (c) is due to the incorporation of 15 into the liposome bi-
layer with its alkyl chain, while the doxyl group remains in water
surroundings. For comparison, ESR spectra of compound 15 in
ethanol (b) were recorded. In this solvent compound 15 is com-
pletely dissolved.
22 (24%), 23 (9%)
27 (21%), 28 (24%)
Scheme 2. Reagents and conditions: (a) acyl chloride, Et3N, CH2Cl2, 0 ꢀC, 4 h; (b) CrO3
dissolved in 35% H2SO4, acetone, 0 ꢀC/rt, 3 h; (c) 2, TBTU, Et3N, CH2Cl2, 0 ꢀC, 3 h; (d)
potassium phthalimide, KI, DMF, 110 ꢀC, 17 h; (e) NH2Me, MeOH, rt, 3 h; (f) acyl
chloride, Et3N, CH2Cl2, 0 ꢀC, 2 h.
For preparing probes with shorter spacer groups of one or two
ethylene glycol units, as in compounds 14–17, the same synthetic
pathway was tried. Analogous esters of compound 1 were prepared
from ethylene or diethylene glycol and ethyl diazoacetate, but hydro-
lysis of them under similar conditions did not yield expected
products. Therefore a different strategy was chosen. Firstly dieth-
ylene and triethylene glycol were acylated with the corresponding
acid chloride to produce monoesters 6–9. The monoesters were
then oxidized by Jones reagent on the terminal hydroxymethyl
group to yield the corresponding carboxylic acids 10–13. By coup-
ling them with amine 2 in the last stage, this approach has enabled
preparation of final compounds 14–17 (Scheme 1).
To make the spacer group more polar the ester moiety between
the acyl residue and spacer was replaced by an amide group in
compounds 22, 23, 27 and 28. Amides have the potential for an
additional hydrogen bond.
We expect that the novel spin probes presented here would be
a new potential tool for ESR study of extracellular space of cells.
Their application will give a new insight into structural and dy-
namic properties close to the membrane surface at different situ-
ations of the cell life, and information for further development of
spin probes for the extracellular space.
Synthesis of 22 and 23 (Scheme 2) started by selectively acyl-
ating the amino group of 2-aminoethoxyethanol to produce amides
18 and 19. Subsequent oxidation with Jones reagent afforded car-
boxylic acids 20 and 21. These products were much less susceptible
to oxidative shortening of the spacer moiety by one oxyethyl group,
in contrast to compounds 10–13 and 25, most probably because
carboxylic acids 20 and 21 were poorly soluble in acetone and
started to precipitate in the reaction mixture. During the final re-
action step TBTU was used instead of DCC as coupling reagent, since
it gave higher yields but most importantly it altered the byproduct
profile and made purification of amides 22 and 23 easier.
3. Conclusion
To summarize, we have succeeded in synthesizing new am-
phiphilic spin probes in which a polar spacer group is inserted

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S. Pajk, S. Pecar / Tetrahedron 65 (2009) 659–665
662
between the nitroxide and the lipophilic part. This will enable the
complex molecular motion of oligosaccharide chains and other
membrane constituents above the biological membrane surface to
be studied by ESR. Furthermore, by varying the spacer length and
type of bond that connects the spacer and lipophilic parts, we have
access to a versatile group of compounds from which to choose,
according to the experiment. Further work with synthesis and
testing on artificial and cellular membranes is in progress.
1H NMR (DMSO-d6, 300 MHz):
d (ppm) 1.10 (s, 3H, CH3), 1.11
(s, 3H, CH3), 1.16 (s, 3H, CH3), 3.03 (dd, 1H, J1¼13.2 Hz,
J2¼4.5 Hz, NHCHaHbC), 3.40–3.65 (m, 15H, 6ꢂOCH2, NHCHaHbC,
OCHaHbC(CH3)2), 3.90 (s, 2H, OCH2CONH), 4.27–4.31 (m, 1H,
CH2OH); 13C NMR (CDCl3, 75 MHz):
d (ppm) 20.4, 21.0, 27.8,
46.0, 61.4, 62.7, 69.8, 70.0, 70.2, 70.4, 70.6, 72.5, 75.0, 97.3,
170.1.
4.2.4. General procedure for acylation of compound 3
4. Experimental
4.1. General
Acyl chloride (0.24 mmol) dissolved in dichloromethane (2 mL)
was added dropwise to a stirred solution of 3 (55 mg, 0.16 mmol)
and Et3N (32 mg, 0.32 mmol) in dichloromethane (3 mL) at 0 ꢀC.
Reaction was continued for 0.5 h at room temperature. The reaction
mixture was washed with 0.1 M HCl (5 mL), saturated aqueous
solution of NaHCO3 (5 mL), brine (10 mL), dried with Na2SO4 and
the solvent evaporated under reduced pressure. The crude product
was purified using silica gel chromatography (dichloromethane/
hexane/methanol¼8:8:1).
Chemicals from Sigma–Aldrich, Acros, Avanti Polar Lipids and
Fluka were used without further purification. Solvents were used
without additional purification or drying. Analytical TLC was per-
formed on Merck silica gel (60 F 254) plates (0.25 mm) and com-
ponents visualized with ultraviolet light and dyed with 20% sulfuric
acid in ethanol, Rhodamine G6, ninhydrin and bromocresol green.
Silica gel 60 (0.04–0.063 mm, Merck) was used for column chro-
matography. Melting points were determined on a Reichert hot
stage microscope and are uncorrected. IR spectra were obtained on
a Perkin–Elmer FT-IR System Spectrum BX. 1H and 13C NMR spectra
were recorded on a Bruker AVANCE DPX300 spectrometer in CDCl3
and DMSO-d6 solution with TMS as internal standard. The nitroxyl
groups were reduced to the corresponding hydroxylamines with
2 equiv of phenylhydrazine dissolved in CDCl3, prior to NMR anal-
ysis. ESR spectra of ethanolic solutions were recorded at ambient
temperature in a glass capillary (1 mm i.d.) using a Bruker X-band
CW-ESR spectrometer ESR-300 at 10 mW microwave power; all aN
values of compounds 3–5, 14–17, 22, 23, 26–28 were approximately
1.45 mT. Microanalyses were performed on a 240 C Perkin–Elmer
CHN analyzer. Mass spectra were obtained using Micromass
AutospecQ.
4.2.5. 1-(3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)-3-oxo-5,8,11-
trioxa-2-azatridecan-13-yl tetradecanoate (4)
Yield 25%; orange oil; Rf¼0.75 (dichloromethane/methanol¼
15:1); IR (NaCl, cmꢁ1): 3352, 2923, 2853, 2360, 1736, 1687,
1530, 1464, 1367, 1248, 1111, 1049; MS (ESI) m/z: 560 (MH)þ;
HRMS (ESI) m/z: calcd for C29H56N2O8 m/z: 560.4037 (MH)þ,
found 560.4017; 1H NMR (CDCl3, 300 MHz):
d (ppm) 0.88 (t, 3H,
J¼6.3 Hz, CH3), 1.16 (s, 3H, CH3), 1.22 (s, 3H, CH3), 1.23–1.31 (m,
20H, CH2), 1.32 (s, 3H, CH3), 1.56–1.65 (m, 2H, CH2CH2COO), 2.31
(t, 2H, J¼7.5 Hz, CH2COO), 3.13 (dd, 1H, J1¼13.5 Hz, J2¼4.8 Hz,
NHCHaHbC), 3.57–3.70 (m, 13H, 5ꢂOCH2, OCH2CONH, NHCHaHbC),
3.92 (d, 1H, J¼15.6 Hz, OCHaHbC(CH3)2), 4.03 (d, 1H, J¼15.6 Hz,
OCHaHbC(CH3)2), 4.22 (t, 2H, J¼4.8 Hz, COOCH2); 13C NMR (CDCl3,
75 MHz):
d (ppm) 14.1, 20.1, 20.7, 22.6, 24.9, 25.4, 29.1, 29.2, 29.3,
29.4, 29.56, 29.60, 29.63, 31.9, 34.1, 46.3, 62.5, 63.1, 69.1, 70.37,
70.39, 70.44, 70.6, 71.1, 75.2, 97.1, 170.3, 173.8. Anal. Calcd for
C29H56N2O8 (%): C, 62.23; H, 9.90; N, 5.00. Found: C, 62.03; H,
10.23; N, 4.72.
4.2. Synthetic procedures
4.2.1. Ethyl 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy) acetate (1)
Compound 1 was synthesized according to Ref. 22. Spectral data
is in accordance with the literature.22
4.2.6. 1-(3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)-3-oxo-5,8,11-
trioxa-2-azatridecan-13-yl palmitate (5)
Yield 42%; orange oil; Rf¼0.75 (dichloromethane/methanol¼
15:1); IR (KBr, cmꢁ1): 3354, 2923, 2853, 1736, 1686, 1529, 1464,
1372, 1248, 1116, 1049, 948, 857, 720; MS (ESI) m/z: 588 (MH)þ;
HRMS (ESI) m/z: calcd for C31H60N2O8 m/z: 588.4350 (MH)þ, found
4.2.2. 2-(Aminomethyl)-2,4,4-trimethyloxazolidin-3-oxyl (2)
Compound 2 was synthesized according to Ref. 20. Spectral data
is in accordance with the literature.20
588.4370; 1H NMR (CDCl3, 300 MHz):
d
(ppm) 0.88 (t, 3H, J¼6.6 Hz,
4.2.3. Synthesis of N-((3-oxyl-2,4,4-trimethyloxazolidin-2-yl)
methyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)-acetamide (3)
To compound 1 (200 mg, 0.85 mmol) dissolved in a mixture of
dioxane (2 mL) and water (1 mL) was added powdered NaOH
(68 mg, 1.7 mmol). The reaction mixture was stirred at room tem-
perature for 2 h. HCl (2 M, 2 mL) was then added and the solvent
evaporated under reduced pressure. The residue was washed sev-
eral times with dichloromethane (100 mL), combined washings
were dried with Na2SO4 and the solvent evaporated under reduced
pressure. To the obtained colourless oil dissolved in ethyl acetate
(2 mL) and cooled to 0 ꢀC was added DCC (175 mg, 0.85 mmol).
After 10 min, compound 2 (135 mg, 0.85 mmol) dissolved in ethyl
acetate (1 mL) was added dropwise. Reaction was continued for 3 h
at room temperature. The reaction mixture was then filtered and
the solvent evaporated under reduced pressure. The crude product
was purified using silica gel chromatography (dichloromethane/
methanol¼20:1).
CH3),1.15 (s, 3H, CH3),1.22 (s, 3H, CH3),1.23–1.32 (m, 24H, CH2),1.33
(s, 3H, CH3), 1.55–1.66 (m, 2H, CH2CH2COO), 2.28–2.33 (m, 2H,
CH2COO), 3.13 (dd,1H, J1¼13.5 Hz, J2¼4.8 Hz, NHCHaHbC), 3.57–3.73
(m, 13H, 5ꢂOCH2, NHCHaHbC, OCH2CONH), 3.92 (d, 1H, J¼15.3 Hz,
OCHaHbC(CH3)2), 4.01 (d, 1H, J¼15.3 Hz, OCHaHbC(CH3)2), 4.28 (t,
2H, J¼4.3 Hz, COOCH2); 13C NMR (CDCl3, 75 MHz):
d (ppm) 14.1,
20.1, 20.7, 22.6, 24.9, 25.4, 29.1, 29.2, 29.3, 29.4, 29.60, 29.64, 31.9,
34.1, 46.3, 62.4, 63.1, 69.1, 70.36, 70.39, 70.43, 70.6, 71.0, 75.2, 97.1,
170.3, 173.8.
4.2.7. Synthesis of compounds 6–9
Compounds 6–9 were synthesized according to Ref. 23.
4.2.7.1. 2-(2-Hydroxyethoxy)ethyl tetradecanoate (6). Spectral data
is in accordance with the literature.23
4.2.7.2. 2-(2-Hydroxyethoxy)ethyl palmitate (7). Spectral data is in
Yield 37%; orange oil; Rf¼0.35 (dichloromethane/meth-
anol¼15:1); IR (NaCl, cmꢁ1): 3326, 2929, 1754, 1667, 1537, 1454,
1350, 1209, 1120, 938; MS (ESI) m/z: 350 (MH)þ; HRMS (ESI) m/
z: calcd for C15H30N2O7 m/z: 350.2066 (MH)þ, found 350.2061;
accordance with the literature.23
4.2.7.3. 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl tetradecanoate (8).
Spectral data is in accordance with the literature.23

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S. Pajk, S. Pecar / Tetrahedron 65 (2009) 659–665
663
4.2.7.4. 2-(2-(2-Hydroxyethoxy)ethoxy)ethyl palmitate (9). Spectral
0 ꢀC. After 10 min, 2 (0.52 mmol) dissolved in acetone (1 mL) was
added. The temperature of the reaction mixture was allowed to
reach room temperature and stirring was continued for 2 h. The
reaction mixture was filtered and the solvent evaporated under
reduced pressure. The residue was dissolved in ethyl acetate
(10 mL) and washed with 0.1 M HCl (10 mL), saturated aqueous
solution of NaHCO3 (10 mL), brine (30 mL), dried with Na2SO4 and
the solvent evaporated under reduced pressure. The crude product
was purified using silica gel chromatography; compounds 14, 16
(ethyl acetate/hexane¼1:1) and compounds 15, 17 (ethyl acetate/
hexane¼2:1).
data is in accordance with the literature.23
4.2.8. General procedure for oxidation of monoacylated derivatives
(10–13)
To a stirred solution of monoacylated derivative (5 mmol) in
acetone (60 mL) at 0 ꢀC, CrO3 (12 mmol) dissolved in 35% H2SO4
(25 mL) was added dropwise. The temperature of the reaction
mixture was then allowed to reach room temperature and stirring
was continued for 3 h. The reaction mixture was poured into water
(80 mL)and the productextractedwith ethylacetate(4ꢂ50 mL). The
combined organic phases were washed with brine (70 mL), dried
with Na2SO4 and the solvent evaporated under reduced pressure.
4.2.9.1. 2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methylamino)-
2-oxoethoxy)ethyl tetradecanoate (14). Yield 18%; orange oil; Rf¼0.6
(ethyl acetate); IR (NaCl, cmꢁ1): 3409, 2924, 2853, 1737, 1691, 1529,
1463, 1372, 1248, 1176, 1114, 1051, 720; MS (ESI) m/z: 472 (MH)þ,
473 (MH2)þ; HRMS (ESI) m/z: calcd for C25H49N2O6 m/z: 473.3591
4.2.8.1. 2-(2-(Tetradecanoyloxy)ethoxy)acetic acid (10). Yield 30%;
white solid; Rf¼0.3 (dichloromethane/methanol¼10:1); mp 43–
46 ꢀC; IR (KBr, cmꢁ1): 2917, 2849, 2367, 1733, 1701, 1473, 1420, 1280,
1253, 1228, 1202, 1182, 1149, 1064, 923, 715, 699; MS (ESI) m/z: 331
(MH)þ, 353 (MNa)þ; HRMS (ESI) m/z: calcd for C18H35O5 m/z:
331.2484 (MH)þ, found 331.2482; 1H NMR (CDCl3, 300 MHz):
(MH2)þ, found 473.3570; 1H NMR (CDCl3, 300 MHz):
d (ppm) 0.88
(t, 3H, J¼6.3 Hz, CH3), 1.16 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.25–1.31
(m, 20H, CH2), 1.33 (s, 3H, CH3), 1.56–1.68 (m, 2H, CH2CH2COO), 2.33
(t, 2H, J¼7.5 Hz, CH2COO), 3.19 (dd, 1H, J1¼13.5 Hz, J2¼5.1 Hz,
NHCHaHbC), 3.56 (dd, 1H, J1¼13.8 Hz, J2¼7.5 Hz, NHCHaHbC), 3.57
(d, 1H, J¼8.1 Hz, OCHaHbC(CH3)2), 3.67–3.73 (m, 2H, OCH2), 3.70 (d,
1H, J¼8.4 Hz, OCHaHbC(CH3)2), 4.00 (s, 2H, OCH2CONH), 4.27 (t, 2H,
d
(ppm) 0.88 (t, 3H, J¼6.6 Hz, CH3), 1.23–1.31 (m, 20H, CH2), 1.55–
1.70 (m, 2H, CH2CH2COO), 2.34 (t, 2H, J¼7.5 Hz, CH2COO), 3.80 (t,
2H, J¼4.2 Hz, OCH2) 4.18 (s, 2H, CH2COOH), 4.28 (t, 2H, J¼4.2 Hz,
COOCH2); 13C NMR (CDCl3, 75 MHz):
d (ppm) 14.0, 22.6, 24.8, 29.0,
29.2, 29.3, 29.4, 29.6, 31.9, 34.1, 63.1, 68.3, 69.6, 173.9, 174.7.
J¼4.5 Hz, COOCH2); 13C NMR (CDCl3, 75 MHz):
d (ppm) 14.1, 19.9,
21.0, 22.6, 24.9, 25.7, 29.1, 29.2, 29.3, 29.4, 29.57, 29.60, 29.64, 31.9,
34.2, 45.9, 62.3, 62.9, 69.5, 70.6, 75.4, 97.1, 169.9, 174.0.
4.2.8.2. 2-(2-(Palmitoyloxy)ethoxy)acetic acid (11). Yield 80%; white
solid; Rf¼0.3 (dichloromethane/methanol¼10:1); mp 68–69 ꢀC; IR
(KBr, cmꢁ1): 2914, 2849, 1731, 1700, 1472, 1420, 1287, 1266, 1244,
1220,1198,1181,1149,1064, 923, 715, 700; MS (ESI) m/z: 359 (MH)þ,
381 (MNa)þ; HRMS (ESI) m/z: calcd for C20H39O5 m/z: 359.2797
4.2.9.2. 2-(2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methylamino)-
2-oxoethoxy)ethoxy)ethyl tetradecanoate (15). Yield 25%; orange
oil; Rf¼0.45 (ethyl acetate); IR (NaCl, cmꢁ1): 3361, 2924, 2853, 1737,
1688, 1530, 1463, 1367, 1248, 1112, 1049, 516; MS (ESI) m/z: 515
(MH)þ; HRMS (ESI) m/z: calcd for C27H52N2O7 m/z: 516.3775 (MH)þ,
(MH)þ, found 359.2797; 1H NMR (CDCl3, 300 MHz):
d (ppm) 0.88 (t,
3H, J¼6.6 Hz, CH3), 1.20–1.34 (m, 24H, CH2), 1.56–1.70 (m, 2H,
CH2CH2COO), 2.34 (t, 2H, J¼7.5 Hz, CH2COO), 3.80 (t, 2H, J¼4.5 Hz,
OCH2) 4.17 (s, 2H, CH2COOH), 4.23 (t, 2H, J¼4.2 Hz, COOCH2); 13C
found 516.3765; 1H NMR (CDCl3, 300 MHz):
d (ppm) 0.88 (t, 3H,
J¼6.3 Hz, CH3), 1.15 (s, 3H, CH3), 1.23 (s, 3H, CH3), 1.24–1.31 (m, 20H,
CH2), 1.33 (s, 3H, CH3), 1.56–1.68 (m, 2H, CH2CH2COO), 2.32 (t, 2H,
J¼7.2 Hz, CH2COO), 3.14 (dd, 1H, J1¼13.5 Hz, J2¼5.1 Hz, NHCHaHbC),
3.56–3.74 (m, 9H, 3ꢂOCH2, NHCHaHbC, OCH2CONH), 3.92 (d,
1H, J¼15.6 Hz, OCHaHbC(CH3)2), 4.04 (d, 1H, J¼15.6 Hz,
OCHaHbC(CH3)2), 4.22 (t, 2H, J¼4.8 Hz, COOCH2); 13C NMR (CDCl3,
NMR (CDCl3, 75 MHz): d (ppm) 14.0, 22.6, 24.8, 29.1, 29.2, 29.3, 29.4,
29.55, 29.59, 29.6, 31.9, 34.1, 63.1, 68.0, 69.7, 173.9, 174.6.
4.2.8.3. 2-(2-(2-(Tetradecanoyloxy)ethoxy)ethoxy)acetic acid (12).
Yield 20%; white solid; mp 34–36 ꢀC; Rf¼0.3 (dichloromethane/
methanol¼10:1); IR (KBr, cmꢁ1): 2919, 2850,1729,1472,1249,1202,
1183, 1152, 1135, 1047, 954, 921, 716; MS (ESI) m/z: 375 (MH)þ, 397
(MNa)þ; HRMS (ESI) m/z: calcd for C20H38O6Na m/z: 397.2566
75 MHz):
d (ppm) 14.1, 20.0, 20.6, 22.6, 24.9, 25.5, 29.1, 29.2, 29.3,
29.4, 29.56, 29.60, 29.63, 31.9, 34.2, 46.3, 62.5, 62.9, 69.4, 70.3, 70.4,
70.9, 75.2, 97.1, 170.3, 173.9.
(MNa)þ, found 397.2565; 1H NMR (CDCl3, 300 MHz):
d (ppm) 0.89
(t, 3H, J¼6.9 Hz, CH3), 1.23–1.34 (m, 20H, CH2), 1.56–1.68 (m, 2H,
CH2CH2COO), 2.34 (t, 2H, J¼7.5 Hz, CH2COO), 3.69–3.78 (m, 6H,
OCH2), 4.18 (s, 2H, CH2COOH), 4.25 (t, 2H, J¼4.5 Hz, COOCH2); 13C
4.2.9.3. 2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methylamino)-
2-oxoethoxy)ethyl palmitate (16). Yield 25%; orange oil; Rf¼0.55
(ethyl acetate); IR (NaCl, cmꢁ1): 3411, 2923, 2853, 1737, 1692, 1529,
1464, 1371, 1248, 1176, 1137, 1051, 720; MS (ESI) m/z: 500 (MH)þ;
HRMS (ESI) m/z: calcd for C27H52N2O6 m/z: 500.3825 (MH)þ, found
NMR (CDCl3, 75 MHz): d (ppm) 14.0, 22.6, 24.8, 29.0, 29.1, 29.2, 29.3,
29.48, 29.52, 29.55, 31.8, 34.0, 63.0, 68.3, 69.1, 70.2, 70.9, 173.8,
173.9.
500.3839; 1H NMR (CDCl3, 300 MHz):
d
(ppm) 0.88 (t, 3H, J¼6.6 Hz,
CH3), 1.15 (s, 3H, CH3),1.24 (s, 3H, CH3), 1.25–1.31 (m, 24H, CH2),1.33
(s, 3H, CH3), 1.56–1.68 (m, 2H, CH2CH2COO), 2.32 (t, 2H, J¼7.5 Hz,
CH2COO), 3.17 (dd, 1H, J1¼13.8 Hz, J2¼5.1 Hz, NHCHaHbC), 3.56 (dd,
1H, J1¼13.8 Hz, J2¼7.5 Hz, NHCHaHbC), 3.58 (d, 1H, J¼8.1 Hz,
OCHaHbC(CH3)2), 3.68 (t, 2H, J¼4.8 Hz, OCH2), 3.70 (d, 1H,
J¼8.4 Hz, OCHaHbC(CH3)2), 3.96 (s, 2H, OCH2CONH), 4.25 (t, 2H,
4.2.8.4. 2-(2-(2-(Palmitoyloxy)ethoxy)ethoxy)acetic acid (13). Yield
81%; white solid; Rf¼0.3 (dichloromethane/methanol¼10:1); mp
38–40 ꢀC; IR (KBr, cmꢁ1): 2916, 2849, 1734, 1467, 1245, 1220, 1198,
1180, 1125, 954, 721; MS (ESI) m/z: 403 (MH)þ, 425 (MNa)þ; HRMS
(ESI) m/z: calcd for C22H42O6Na m/z: 425.2879 (MNa)þ, found
425.2885; 1H NMR (CDCl3, 300 MHz):
d
(ppm) 0.88 (t, 3H, J¼6.3 Hz,
J¼4.5 Hz, COOCH2); 13C NMR (CDCl3, 75 MHz):
d (ppm) 14.0, 19.7,
20.9, 22.6, 24.8, 25.6, 29.1, 29.2, 29.3, 29.4, 29.55, 29.58, 29.62, 31.9,
34.1, 45.9, 62.3, 62.6, 69.6, 70.5, 75.4, 97.1, 169.9, 173.9.
CH3), 1.20–1.36 (m, 24H, CH2), 1.56–1.68 (m, 2H, CH2CH2COO), 2.33
(t, 2H, J¼7.5 Hz, CH2COO), 3.68–3.74 (m, 6H, OCH2), 4.12 (s, 2H,
CH2COOH), 4.25 (t, 2H, J¼4.5 Hz, COOCH2); 13C NMR (DMSO-d6,
75 MHz):
d
(ppm) 13.7, 22.0, 24.4, 28.4, 28.7, 28.9, 29.1, 31.3, 33.3,
4.2.9.4. 2-(2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methylamino)-
2-oxoethoxy)ethoxy)ethyl palmitate (17). Yield 16%; orange oil;
Rf¼0.4 (ethyl acetate); IR (NaCl, cmꢁ1): 3364, 2923, 2853, 1737,
1688, 1530, 1464, 1367, 1249, 1113, 1051, 500; MS (ESI) m/z: 544
(MH)þ, 545 (MH2)þ; HRMS (ESI) m/z: calcd for C29H57N2O7 m/z:
545.4166 (MH2)þ, found 545.4180; 1H NMR (CDCl3, 300 MHz):
62.9, 67.5, 68.2, 69.7, 171.4, 172.6.
4.2.9. General procedure for synthesis of 14–17
To a solution of compounds 10–13 (1 mmol) and Et3N (100 mg,
1 mmol) in acetone (2 mL), was added DCC (160 mg, 0.77 mmol) at

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S. Pajk, S. Pecar / Tetrahedron 65 (2009) 659–665
664
d
(ppm) 0.88 (t, 3H, J¼6.6 Hz, CH3), 1.16 (s, 3H, CH3), 1.23 (s, 3H,
4.2.12.2. N-(2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methyl-
amino)-2-oxoethoxy)ethyl)palmitamide (23). Yield 9%; orange
solid; Rf¼0.5 (dichloromethane/methanol¼10:1); mp 40–42 ꢀC; IR
(KBr, cmꢁ1): 3315, 2917, 2848, 2370, 1636, 1559, 1458, 1374, 1249,
1127, 1049, 719; MS (ESI) m/z: 499 (MH)þ, 500 (MH2)þ; HRMS (ESI)
m/z: calcd for C27H54N3O5 m/z: 500.4063 (MH2)þ, found 500.4080;
CH3), 1.24–1.31 (m, 24H, CH2), 1.33 (s, 3H, CH3), 1.56–1.68 (m, 2H,
CH2CH2COO), 2.33 (t, 2H, J¼7.5 Hz, CH2COO), 3.14 (dd, 1H,
J1¼13.5 Hz, J2¼5.1 Hz, NHCHaHbC), 3.57–3.76 (m, 9H, 3ꢂOCH2,
NHCHaHbC, OCH2CONH), 3.93 (d, 1H, J¼15.6 Hz, OCHaHbC(CH3)2),
4.06 (d, 1H, J¼15.9 Hz, OCHaHbC(CH3)2), 4.27 (t, 2H, J¼4.8 Hz,
COOCH2); 13C NMR (CDCl3, 75 MHz):
d
(ppm) 14.0, 20.0, 20.6, 22.6,
1H NMR (CDCl3, 300 MHz):
d
(ppm) 0.88 (t, 3H, J¼6.3 Hz, CH3), 1.19
24.8, 25.4, 29.0, 29.17, 29.25, 29.4, 29.50, 29.55, 29.58, 31.8, 34.1,
46.2, 62.4, 62.9, 69.3, 70.2, 70.4, 70.8, 75.1, 97.1, 170.2, 173.8.
(s, 3H, CH3), 1.22–1.30 (m, 27H, CH3, 12ꢂCH2), 1.33 (s, 3H, CH3),
1.58–1.68 (m, 2H, CH2CH2COO), 2.18 (t, 2H, J¼7.2 Hz, CH2COO), 3.16
(dd, 1H, J1¼13.2 Hz, J2¼4.5 Hz, NHCHaHbC), 3.48–3.62 (m, 5H, OCH2,
CONHCH2, NHCHaHbC), 3.59 (d, 1H, J¼7.8 Hz, OCHaHbC(CH3)2), 3.70
(d, 1H, J¼8.1 Hz, OCHaHbC(CH3)2), 3.95 (s, 2H, OCH2CONH), 5.77–
4.2.10. Synthesis of compounds 18 and 19
Compounds 18 and 19 were synthesized according to Ref. 24.
5.84 (m, 1H, CONH); 13C NMR (CDCl3, 75 MHz):
d (ppm) 14.0, 20.1,
4.2.10.1. N-(2-(2-Hydroxyethoxy)ethyl)tetradecanamide (18). Spectral
21.1, 22.6, 25.4, 25.7, 29.24, 29.27, 29.3, 29.4, 29.57, 29.61, 31.8, 36.7,
39.1, 46.2, 62.7, 70.4, 70.6, 75.2, 97.3, 169.7, 174.1.
data is in accordance with the literature.24
4.2.10.2. N-(2-(2-Hydroxyethoxy)ethyl)palmitamide (19). Spectral
4.2.13. N-(2-(2-(2-Hydroxyethoxy)ethoxy)ethyl) phthalimide (24)
Compound 24 was synthesized according to Ref. 27. Spectral
data is in accordance with the literature.27
data is in accordance with the literature.25
4.2.11. Synthesis of compounds 20 and 21
Compounds 20 and 21 were prepared as described for com-
pounds 10–13.
4.2.14. 2-(2-(2-(1,3-Dioxoisoindolin-2-yl)ethoxy)ethoxy) acetic
acid (25)
Compound 25 was synthesized according to Ref. 28. Spectral
4.2.11.1. 2-(2-Tetradecanamidoethoxy)acetic acid (20). Yield 62%;
white crystals; Rf¼0.2 (dichloromethane/methanol¼10:1); mp 72–
74 ꢀC; IR (KBr, cmꢁ1): 3301, 2916, 2848, 2344,1718,1635,1559,1472,
1425, 1260, 1149, 883, 719; MS (ESI) m/z: 330 (MH)þ, 352 (MNa)þ;
HRMS (ESI) m/z: calcd for C18H36NO4 m/z: 330.2644 (MH)þ, found
data is in accordance with the literature.28
4.2.15. Synthesis of 2-(2-(2-phthalimido ethoxy)ethoxy)-N-((3-
oxyl-2,4,4-trimethyloxazolidin-2-yl)methyl) acetamide (26)
To a solution of 25 (150 mg, 0.5 mmol), Et3N (100 mg, 1 mmol)
and 2 (0.5 mmol) in dichloromethane (3 mL) was added TBTU
(240 mg, 0.75 mmol) at 0 ꢀC. The temperature of the reaction
mixture was allowed to reach room temperature and stirring was
continued for 2 h. The reaction mixture was filtered and solvent
evaporated under reduced pressure. The residue was dissolved in
ethyl acetate (10 mL) and washed with 0.1 M HCl (10 mL), saturated
aqueous solution of NaHCO3 (10 mL), brine (30 mL), dried with
Na2SO4 and the solvent evaporated under reduced pressure. The
crude product was then purified using silica gel chromatography
(ethyl acetate/hexane¼4:1).
330.2629; 1H NMR (CDCl3, 300 MHz):
d
(ppm) 0.88 (t, 3H, J¼6.3 Hz,
CH3), 1.22–1.32 (m, 20H, CH2), 1.58–1.68 (m, 2H, CH2CH2COO), 2.23
(t, 2H, J¼7.8 Hz, CH2COO), 3.46 (q, 2H, J¼5.1 Hz, NHCH2CH2), 3.65 (t,
2H, J¼5.1 Hz, OCH2), 4.12 (s, 2H, OCH2COOH), 6.60–6.80 (m, 1H,
CONH); 13C NMR (DMSO-d6, 75 MHz):
d (ppm) 13.8, 22.0, 25.2, 28.6,
28.7, 28.8, 28.9, 29.01, 29.04, 31.3, 35.3, 38.3, 67.4, 69.3, 171.6, 172.1.
4.2.11.2. 2-(2-Palmitamidoethoxy)acetic acid (21). Spectral data is
in accordance with the literature.26
4.2.12. General procedure for synthesis of 22 and 23
Yield 37%; orange oil; Rf¼0.75 (dichloromethane/meth-
anol¼10:1); IR (NaCl, cmꢁ1): 3372, 2931,1773,1712,1682,1530,1466,
1427, 1394, 1249, 1108, 1024, 721, 529; MS (ESI) m/z: 435 (MH)þ;
HRMS (ESI) m/z: calcd for C21H29N3O7 m/z: 435.2006 (MH)þ, found
To a solution of compound 20 or 21 (0.5 mmol), Et3N (100 mg,
1 mmol) and 2 (0.5 mmol) in dichloromethane (3 mL) was added
TBTU (240 mg, 0.75 mmol) at 0 ꢀC. The temperature of the reaction
mixture was then allowed to reach room temperature and stirring
was continued for 2 h. After that the reaction mixture was filtered
and solvent evaporated under reduced pressure. The residue was
dissolved in ethyl acetate (10 mL) and washed with 0.1 M HCl
(10 mL), saturated aqueous solution of NaHCO3 (10 mL), brine
(30 mL), dried with Na2SO4 and the solvent evaporated under re-
duced pressure. The crude product was purified using silica gel
chromatography (ethyl acetate/acetone¼5:1).
435.2019; 1H NMR (CDCl3, 300 MHz):
d (ppm) 1.14 (s, 2H, CH3), 1.17
(s, 2H, CH3), 1.29 (s, 2H, CH3), 3.15 (dd, 1H, J1¼13.5 Hz, J2¼5.1 Hz,
NHCHaHbC), 3.49–3.67 (m, 7H, 3ꢂOCH2, NHCHaHbC), 3.76 (t, 2H,
J¼5.4 Hz, NCH2), 3.76 (d, 1H, J¼15.9 Hz, OCHaHbC(CH3)2), 3.91 (s,
2H, OCH2CONH), 3.93 (d, 1H, J¼15.9 Hz, OCHaHbC(CH3)2), 7.69 (dd,
2H, J1¼5.4 Hz, J2¼3.3 Hz, ArH), 7.83 (dd, 2H, J1¼5.4 Hz, J2¼3.3 Hz,
ArH); 13C NMR (CDCl3, 75 MHz):
d (ppm) 20.0, 20.5, 25.3, 36.9, 46.0,
62.3, 68.0, 69.6, 70.3, 70.7, 75.1, 97.0, 123.2, 131.9, 133.9, 168.3, 170.0.
4.2.12.1. N-(2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methyl-
amino)-2-oxoethoxy)ethyl)tetradecanamide (22). Yield 24%; orange
oil; Rf¼0.5 (dichloromethane/methanol¼10:1); IR (KBr, cmꢁ1):
3310, 2924, 2853, 1653, 1541, 1456, 1374, 1249, 1141, 1052, 722; MS
(ESI) m/z: 471 (MH)þ; HRMS (ESI) m/z: calcd for C25H49N3O5 m/z:
471.3672 (MH)þ, found 471.3687; 1H NMR (CDCl3, 300 MHz):
4.2.16. General procedure for synthesis of compounds 27 and 28
Methylamine aqueous solution (0.5 mL) was added to a solution
of compound 26 (65 mg, 0.15 mmol) in methanol (1 mL) at room
temperature. After 3 h of stirring, the solvents were evaporated
under reduced pressure. To the residue dissolved in dichloro-
methane (3 mL) were added Et3N (26 mg, 0.25 mmol) and acylating
reagent (0.23 mmol) in dichloromethane (1 mL) at 0 ꢀC. The tem-
perature of the reaction mixture was then allowed to reach room
temperature and stirring was continued for 2 h. To the reaction
mixture was added dichloromethane (10 mL) and the obtained
solution was washed with 0.1 M HCl (10 mL), saturated aqueous
solution of NaHCO3 (10 mL), brine (50 mL), dried with Na2SO4 and
the solvent evaporated under reduced pressure. The crude product
was purified using silica gel chromatography (dichloromethane/
methanol¼20:1/15:1).
d
(ppm) 0.88 (t, 3H, J¼6.6 Hz, CH3), 1.18 (s, 3H, CH3), 1.22–1.30 (m,
23H,10ꢂCH2, CH3),1.33 (s, 3H, CH3),1.57–1.67 (m, 2H, CH2CH2COO),
2.18 (t, 2H, J¼7.2 Hz, CH2COO), 3.16 (dd, 1H, J1¼13.5 Hz, J2¼4.8 Hz,
NHCHaHbC), 3.47–3.62 (m, 5H, OCH2, CONHCH2, NHCHaHbC), 3.59
(d, 1H, J¼7.8 Hz, OCHaHbC(CH3)2), 3.70 (d, 1H, J¼8.1 Hz,
OCHaHbC(CH3)2), 3.94 (s, 2H, OCH2CONH), 5.78–5.84 (m, 1H,
CONH); 13C NMR (CDCl3, 75 MHz):
d (ppm) 14.8, 20.1, 21.1, 22.6,
25.5, 25.7, 29.25, 29.28, 29.31, 29.4, 29.58, 29.61, 31.85, 36.7, 39.1,
46.2, 62.7, 70.4, 70.6, 75.2, 97.3, 169.8, 174.1.

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665
4.2.16.1. N-(2-(2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methyl-
References and notes
amino)-2-oxoethoxy)ethoxy)ethyl) tetradecanamide (27). Yield
21%; orange oil; Rf¼0.55 (dichloromethane/methanol¼10:1); IR
(NaCl, cmꢁ1): 3317, 2923, 2853, 2359, 2340, 1651, 1538, 1463,
1371, 1248, 1112, 1050, 671; MS (ESI) m/z: 515 (MH)þ, 537
(MNa)þ; HRMS (ESI) m/z: calcd for C27H53N3O6 m/z: 515.3934
1. Nakatsuji, S. In Nitroxides; Likhtenstein, G. I., Yamauchi, J., Nakatsuji, S., Smirnov,
A. I., Tamura, R., Eds.; Wiley-VCH: Weinheim, 2008; pp 161–204.
2. Likhtenshtein, G. I. In Nitroxides; Likhtenstein, G. I., Yamauchi, J., Nakatsuji, S.,
Smirnov, A. I., Tamura, R., Eds.; Wiley-VCH: Weinheim, 2008; pp 331–399.
3. Likhtenshtein, G. I. In Nitroxides; Likhtenstein, G. I., Yamauchi, J., Nakatsuji, S.,
Smirnov, A. I., Tamura, R., Eds.; Wiley-VCH: Weinheim, 2008; pp 239–268.
4. Yamauchi, J. In Nitroxides; Likhtenstein, G. I., Yamauchi, J., Nakatsuji, S., Smirnov,
A. I., Tamura, R., Eds.; Wiley-VCH: Weinheim, 2008; pp 47–70.
5. Miller, W. G. In Spin Labeling; Berliner, L. J., Ed.; Academic: 1979; Vol. 2, pp 173–221.
6. Voinov, M. A.; Polienko, J. F.; Schading, T.; Bobko, A. A.; Khramtsov, V. V.; Ga-
tilov, Y. V.; Rybalova, T. V.; Smirnov, A. I.; Grigorjev, I. A. J. Org. Chem. 2005, 70,
9702–9711.
(MH)þ, found 515.3959; 1H NMR (CDCl3, 300 MHz):
d (ppm)
0.88 (t, 3H, J¼6.3 Hz, CH3), 1.16 (s, 3H, CH3), 1.22–1.30 (m, 23H,
10ꢂCH2, CH3) 1.34 (s, 3H, CH3), 1.54–1.68 (m, 2H, CH2CH2COO),
2.15 (t, 2H, J¼7.2 Hz, CH2COO), 3.17 (dd, 1H, J1¼13.8 Hz, J2¼5.1
Hz, NHCHaHbC), 3.44–3.72 (m, 11H, 3ꢂOCH2, CONHCH2, OCH2
-
7. Chignell, C. F. In Spin Labeling; Berliner, L. J., Ed.; Academic: 1979; Vol. 2,
pp 223–246.
CONH, NHCHaHbC), 3.92 (d, 1H, J¼15.9 Hz, OCHaHbC(CH3)2),
4.03 (d, 1H, J¼15.9 Hz, OCHaHbC(CH3)2); 13C NMR (CDCl3,
ˇ
ˇ
¨
8. Ilas, J.; Pecar, S.; Hockemeyer, J.; Euler, H.; Kirfel, A.; Muller, C. E. J. Med. Chem.
75 MHz):
d (ppm) 14.0, 19.9, 20.7, 22.6, 25.6, 25.7, 29.2, 29.3,
2005, 48, 2108–2114.
ˇ
9. Mravljak, J.; Zeisig, R.; Pecar, S. J. Med. Chem. 2005, 48, 6393–6399.
29.4, 29.5, 29.6, 31.8, 36.5, 39.0, 46.0, 62.1, 70.0, 70.3, 70.4, 70.9,
75.4, 97.1, 170.4, 173.5.
10. Soule, B. P.; Hyodo, F.; Matsumoto, K.; Somone, N. L.; Cook, J. A.; Krisha, M. C.;
Mitchell, J. B. Free Radical Biol. Med. 2007, 42, 1632–1650.
11. Vladimir, V.M. WO/1999/050244, 1999.
12. Likhtenshtein, G. I. In Nitroxides; Likhtenstein, G. I., Yamauchi, J., Nakatsuji, S.,
Smirnov, A. I., Tamura, R., Eds.; Wiley-VCH: Weinheim, 2008; pp 205–238.
13. Griffith, O. H.; Jost, P. C. In Spin Labeling; Berliner, L. J., Ed.; Academic: 1976;
Vol. 1, pp 453–523.
4.2.16.2. N-(2-(2-(2-((3-Oxyl-2,4,4-trimethyloxazolidin-2-yl)methyl-
amino)-2-oxoethoxy)ethoxy)ethyl) palmitamide (28). Yield 24%;
orange solid; Rf¼0.45 (dichloromethane/methanol¼10:1); mp
34–36 ꢀC; IR (NaCl, cmꢁ1): 3305, 2916, 2848, 1673, 1640, 1545,
1462, 1371, 1249, 1136, 1049, 719; MS (ESI) m/z: 543 (MH)þ, 565
(MNa)þ; HRMS (ESI) m/z: calcd for C29H57N3O6 m/z: 543.4247
ˇ
14. Mravljak, J.; Konc, J.; Hodosˇcˇek, M.; Solmajer, T.; Pecˇar, S. J. Phys. Chem. B 2006,
110, 25559–25561.
15. Stimson, L.; Dong, L.; Karttunen, M.; Wisniewska, A.; Dutka, M.; Rog, T. J. Chem.
Phys. B 2007, 111, 12447–12453.
(MH)þ, found 543.4226; 1H NMR (CDCl3, 300 MHz):
d (ppm)
ˇ
ˇ
16. Strancar, J.; Schara, M.; Pecar, S. J. Membr. Biol. 2003, 193, 15–22.
ˇ
ˇ
J.; Pecˇar, S. Pharmazie 2003, 58, 599–600.
17. Babic, A.; Ilas,
18. Hubbell, W. L.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1969, 64, 20–27.
0.88 (t, 3H, J¼6.3 Hz, CH3), 1.17 (s, 3H, CH3), 1.20–1.30 (m,
27H, 12ꢂCH2, CH3) 1.35 (s, 3H, CH3), 1.56–1.68 (m, 2H,
CH2CH2COO), 2.16 (t, 2H, J¼7.5 Hz, CH2COO), 3.17 (dd, 1H,
J1¼13.8 Hz, J2¼5.4 Hz NHCHaHbC), 3.45–3.73 (m, 11H, 3ꢂOCH2,
CONHCH2, OCH2CONH, NHCHaHbC), 3.94 (d, 1H, J¼15.9 Hz,
OCHaHbC(CH3)2), 4.06 (d, 1H, J¼15.9 Hz, OCHaHbC(CH3)2); 13C
ˇ
ˇ
19. Babic, A.; Pecar, S. Synlett 2008, 1155–1158.
20. Harjani, J. H.; Nara, S. J.; Naik, P. N.; Salunkhe, M. M. Tetrahedron Lett. 2004, 45,
179–182.
21. ESR recording: the final concentration of ester 15 was 3.3ꢂ10ꢁ4 M in all cases
and the temperature of the samples when recorded was 310 K. Liposomes were
prepared from DPPC and PBS buffer (50 mg/mL) by lipid film method. Labelling:
a stock solution of 15 in ethanol (10ꢁ3 M) was added into a test tube. Ethanol
was removed by a stream of nitrogen to obtain a film of 15 on the test tube wall.
Liposomes were added to the film and the test tube was shaken for 30 min.
22. Newkome, G. R.; Kotta, K. K.; Mishra, A.; Moorefield, C. N. Macromolecules 2004,
37, 8262–8268.
NMR (CDCl3, 75 MHz):
d (ppm) 14.0, 19.9, 20.7, 22.6, 25.6, 25.7,
29.2, 29.3, 29.4, 29.5, 29.6, 31.8, 36.5, 39.0, 46.0, 62.1, 70.0, 70.3,
70.4, 70.9, 75.4, 97.1, 170.5, 173.5.
23. Riggs-Sauthier J. A.; Ekwuribe N. N. U.S. Patent Application Publication, Pub. No.
U.S. 2,006,008,850, 2006.
ˇ
ˇ
24. Korosec, E.; Poljsak, D.; Urleb, U. Arch. Pharm. Chem. Life Sci. 1992, 325, 251–252.
25. Tamotsu F.; Hirotaka S.; Kyoken U. Pub. No. JP7101933, 1995.
26. Krause, H. J.; Schmadel, E. Ger. Offen. Pub. No. DE2644498, 1978.
27. Sato, H.; Hayashi, E.; Yamada, N.; Yatagai, M.; Takahara, Y. Bioconjugate Chem.
2001, 12, 701–710.
Acknowledgements
We are grateful to the Ministry of Science and Education for
financial support of our work, the Institute Jozef Stefan for MS and
ESR spectra and Dr. R. Pain for critical reading of the manuscript.
ꢀ
28. Dolle´, F.; Hinnen, F.; Valette, H.; Fuseau, C.; Duval, R.; Pe´glion, J.; Crouzel, C.
Bioorg. Med. Chem. 1997, 5, 759–764.