Nitroxide-fluorophore double probes: A potential tool for studying membrane heterogeneity by ESR and fluorescence

Article abstract of DOI:10.1039/c0ob01173h

A serious drawback of ESR, particularly in its application to cells, is the lack of information on the location of spin probes in the system. In order to realize real time tracking, a spin probe was combined with a fluorophore in a new kind of nitroxide-fluorophore double probe which, in addition to information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to the water-lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on cells show preferential localization of both probes in the plasma membrane, with a relatively slow redistribution to other membranes of the cell. We believe that such double probes would be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.

Full text of DOI:10.1039/c0ob01173h

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PAPER
Nitroxide–fluorophore double probes: a potential tool for studying membrane
heterogeneity by ESR and fluorescence†
a
b
b
a,b
ˇ
Stane Pajk,* Maja Garvas, Janez Strancar and Slavko Pecˇar
Received 14th December 2010, Accepted 18th March 2011
DOI: 10.1039/c0ob01173h
A serious drawback of ESR, particularly in its application to cells, is the lack of information on the
location of spin probes in the system. In order to realize real time tracking, a spin probe was combined
with a fluorophore in a new kind of nitroxide–fluorophore double probe which, in addition to
information about lipid dynamics, enables visualization by fluorescence microscopy. The two sets of
probes synthesized are based on an amino-alkyne-functionalized sugar that serves as a central polar
group and as a linker between the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore and the
derivative of the spin labelled fatty acid. In this setting, the location of the fluorophore is restricted to
the water–lipid interface, while the nitroxide is located deep in the lipid bilayer. Preliminary tests on
cells show preferential localization of both probes in the plasma membrane, with a relatively slow
redistribution to other membranes of the cell. We believe that such double probes would be particularly
useful for studies of plasma membrane heterogeneity and associated cellular processes.
can be obtained.11 This methodology works well with simple mem-
Introduction
brane systems like liposomes, and can be applied to more complex
systems like cells, however, in the latter case specificity is a major
problem. Cells, unlike liposomes, comprise many membranes of
very different lipid and protein compositions that in turn yield
different spectra.12 Because spin probes introduced into the plasma
membrane of a cell rapidly undergo spontaneous or catalyzed flip-
flop, followed by redistribution to other intracellular membranes,
the resulting ESR spectra comprise the superimposition of spectra
of several membranes.13 This can be partially circumvented by
working with plasma membrane vesicles that lack cytosolic
organelles.14 Another way is to label the cells at lower temperatures,
where redistribution to other membranes is much slower, followed
by measurements at physiological temperature.13–15 However, the
redistribution increases with acquisition time, thus decreasing
the specificity of labelling. This lack of definition of spin probe
location in the system can be a serious drawback for the use of
ESR on cells.
One possible solution is to couple ESR with fluorescence
microscopy, a technique that provides real time visualization
together with high sensitivity. In order to realize this approach,
a double probe, containing nitroxide and fluorophore moieties in
the same molecule, is necessary. However, implementation of this
idea is hampered by the fact that nitroxides are strong quenchers of
fluorescence.16 This phenomenon is exploited by "profluorescent
nitroxides", which are double probes in which the nitroxide serves
as a ‘molecular switch’ that turns on fluorescence intensity by its
conversion to a diamagnetic moiety by reduction or oxidation.17
Profluorescent nitroxides have found application as sensors of
cationic metals,18 free radical generation in polymers,19–21 ROS
generation in cigarette smoke,22 cellular redox environment,23
Lipid-dependent plasma membrane heterogeneity is now widely
accepted as a requirement for the normal function of biological
membranes.1 Certain important cellular processes are associ-
ated with this heterogeneity including cell signalling, membrane
trafficking, pathogen invasion, neurodegenerative diseases and
angiogenesis.2–6 Microdomains of ordered lipids, the so-called
"lipid rafts", are believed to be platforms for performing these
biological functions.7 However, despite the need to determine the
exact molecular basis of these membrane organizations, progress
in this field is slow, mostly reflecting the technical and experimental
difficulties faced when studying lipid rafts in vivo.8
Electron spin resonance (ESR) is commonly employed to study
the membrane physical properties that reflect its lateral inhomo-
geneity. Owing to the unique time scale of ESR, which spans
almost all the motional range that occurs in membranes, ESR
spectroscopy is particularly useful for studying lipid dynamics.9
Since membranes do not possess paramagnetic moieties, ESR
investigation depends on spin probes (Fig. 1) inserted into the
membrane bilayer, that report on the properties of their immediate
surroundings.10 The resulting ESR spectra of model or biological
membranes are complex to analyze but, together with simulation
methods, much information on membrane dynamics and structure
aFaculty of Pharmacy, University of Ljubljana, Asˇkercˇeva 7, SI-1000,
Ljubljana, Slovenia. E-mail: stane.pajk@ffa.uni-lj.si; Fax: +386 1 425 80
31; Tel: +386 1 476 95 00
bLaboratory of Biophysics–EPR center, Institut Jozˇef Stefan, Jamova 39,
SI-1000, Ljubljana, Slovenia
† Electronic supplementary information (ESI) available: Absorption, flu-
orescence and NMR (1H and 13C) spectra. See DOI: 10.1039/c0ob01173h
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fluorophore was placed at position 2 or 6 of the hexose moiety,
depending on the position of an amino group on the hexose moiety.
An amino sugar was in both cases linked at position 1 to the spin
labelled fatty acid derivative by 1,2,3-triazol, employing copper(I)
catalyzed Huisgen cycloaddition.37 In addition to spin labelled
compounds, we made derivatives without a spin label, consisting
of a 14 C long alkyl chain, for comparison purposes.
Spin labelled azido derivatives were prepared from spin labelled
fatty acid methyl ester 1,38 which was first reduced to the alcohol
2 (Scheme 1.).39 Alcohol 2 was treated with mesylchloride to
obtain a mesylated derivative which yielded azide 3 after treatment
with NaN3 in DMF at elevated temperature.40 Similarly, azide 5
was obtained after treating 1-bromotetradecane 4 with NaN3 in
DMF.41
Fig. 1 Examples of a) fluorescent probe, b) spin probe, c) double
nitroxide–fluorophore probe.
nitric oxide,24,25 singlet oxygen,26,27 thiyl,28 superoxide29 and hy-
droxyl radicals,30 as well as various antioxidants.31–33 In a sim-
ilar manner, quantum dots have been utilized in the place
of the organic fluorophore, raising new possibilities for future
applications.34,35
Scheme 1 Reagents and conditions: (i) LiAlH4, diethyl ether, 0 ◦C, 73%;
(ii) MsCl, Et3N, DCM, 0 ◦C; (iii) NaN3, DMF, 55 ◦C (78% (over two steps)
for 3 and 94% for 5).
We present the design and synthesis of double nitroxide–
fluorophore probes that, after labelling the sample, enable detec-
tion by both ESR and fluorescence microscopy. Most importantly,
this does not require any modification of the nitroxide moiety,
as is the case with profluorescent nitroxides. To overcome the
problem of quenching we designed a class of probes in which
the fluorophore and nitroxide moieties are as far apart as
possible, since contact between the two is a precondition for
quenching.16 To minimize this interaction a hydrophilic sugar was
inserted between the lipophilic chain bearing the nitroxide and
the fluorophore. Additionally, the fluorophore was tightly bound
to the hydrophilic sugar moiety to prevent it from penetrating
through the water–lipid interface into the interior of the lipophilic
membrane and coming into contact with the spin labelled fatty
acid derivative located there. Also, quenching was reduced by
choosing a derivative of the spin labelled fatty acid in which the
nitroxide is located close to the end of the alkyl chain and near
the bilayer centre.36 In the set of double probes presented here,
a nitroxide reports on the lipid dynamics inside the membrane
bilayer, while the fluorophore indicates the distribution of the
probe among the membranes of the cell, as well as its lateral
distribution in the membrane. With proper design and a suitable
distance between the fluorophore and the nitroxide group we
have combined two powerful and independent techniques that
had previously been mutually exclusive. The described approach
could be a powerful new tool for research into membrane
heterogeneity. The synthesis of two double probes and their basic
characterization are described.
Synthesis of the glycosyl core for the first pair of compounds
that would bear the NBD fluorophore in position 2 (Scheme
2.) proceeded from readily obtainable D-glucosamine chloride
to compound 6 in four steps which are described elsewhere.42
Compound 6 was reacted with propargyl alcohol in the presence
of BF3·Et2O as an activator, to afford propargyl glycoside 7.43
Prolonged reaction times (3 days) were necessary, as alkyne 7
could not be separated from starting compound 6 and so complete
conversion was required, despite the fact that longer reaction
times lowered the yield. Nonetheless the reaction afforded the
propargyl moiety of alkyne 7 in b conformation only, showing
high stereoselectivity in this case.
Scheme 2 Reagents and conditions: (i) propargyl alcohol, BF3·Et2O,
DCM, 0 ◦C, 63%.
D-Galactose served as starting material for the synthesis of the
glycosyl core of the second pair of compounds that would bear
the NBD fluorophore in position 6 (Scheme 3.). To introduce the
precursor of the amino functionality, D-galactose was converted
to azide 8 in three previously described steps.44 Azide 8 was
next reduced to the amine by H2 in the presence of Pd/C,
followed by addition of trifluoroacetanhydride that afforded
Results and discussion
An amino-alkyne-functionalized sugar served as a central polar
group linking a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluo-
rophore and the derivative of the spin labelled fatty acid. The NBD
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compound 7 but, to our surprise, the reaction between compound
10 and propargyl alcohol in the presence of BF3·Et2O led to
an anomeric mixture of a and b propargyl glycoside 13, the b
anomer being the predominant one. To overcome this problem
a new approach to glycoside formation via trichloroacetimidate
was applied. In order to do so, the anomeric acetate group of
compound 10 was selectively removed with benzylamine to give
compound 11.46 The free anomeric hydroxyl group of compound
11 was coupled with trichloroacetonitrile in the presence of DBU
as a base, to yield trichloroacetimidate 12 in a conformation only.46
Glycoside formation from trichloroacetimidate 12 and propargyl
alcohol with TMSOTf as an activator went smoothly, affording
alkyne 13 in good yield and high stereoselectivity towards the b
product.47
Click reactions between azides 3 and 5 and alkynes 7 and
13 went smoothly, yielding compounds 14a, 14b and 17a, 17b
(Scheme 4).48 The best results were obtained when CuBr, in the
presence of N-methylmorpholine, was used as catalyst. Under
these conditions, the reaction proceeded to completion in just
30 min at room temperature. The trifluoroacetamide and acetate
protecting groups were removed in two consecutive steps, yielding
15a, 15b and 18a, 18b. In the first step, acetate groups were removed
in the presence of catalytic amounts of NaOMe in MeOH.49 To
our surprise, after addition of aqueous NaOH to the reaction
mixture, only partial cleavage of trifluoroacetamide was observed.
In contrast, complete cleavage of trifluoroacetamide was observed
if the solvent was removed prior to the addition of aqueous
NaOH.50 Finally, amines 15a, 15b and 18a, 18b were reacted with
fluorogenic benzofurazan reagent (NBD-Cl) to afford a set of
double probes 16a, 19a and its derivates 16b, 19b without the spin
label.51
Scheme 3 Reagents and conditions: H2, Pd/C, THF, RT; (ii) (CF3CO)2O,
Et3N, THF, 0 ◦C, 90% (over two steps); (iii) TFA, AcOH, H2O, 50 ◦C;
(iv) Ac2O, Py, 0 ◦C, 55% (over two steps); (v) BnNH2, THF, RT, 72%;
(vi) CCl3CN, DBU, 0 ◦C, 57%, (vii) propargyl alcohol, TMSOTf, DCM,
-30 ◦C, 63%.
trifluoroacetamide 9, both steps being conducted in one pot.
The switch from acetonide to acetate protecting groups was
accomplished by treatment of compound 9 with aqueous trifluoric
acid, that released hydroxyl groups,45 and subsequent introduction
of acetates with acetic anhydride that afforded compound 10,
again both steps being conducted in one pot. Under conditions of
acid hydrolysis of acetonides, the trifluoroacetamide functionality
proved to be only moderately stable, the yield being considerably
lower in the case of longer reaction times. To form a glycosidic
bond a similar strategy was employed to that used previously for
Absorption and emission fluorescence spectra of fluorescent
and double probes were recorded in methanol (Fig. 2). When
the nitroxide moiety of the double probes was reduced, fluores-
cence intensity significantly increased. However, the increase of
Scheme 4 Reagents and conditions: (i) CuBr, NMM, EtOAc, RT, 63% for 14a, 78% for 14b; 63% for 17a, 90% for 17b (ii) NaOMe, MeOH, RT; (iii)
NaOH(aq), 77% for 15a, 78% for 15b, 31% for 18a, 53% for 18b (over two steps); (iv) NBD-Cl, K2CO3, THF, 0 ◦C, 59% for 16a, 43% for 16b, 13% for 19a,
24% for 19b.
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19a the lipophilic NBD fluorophore has more conformational
freedom and can easily move into the interior of the lipophilic
membrane. This can explain the higher degree of quenching
observed with 19a in comparison to more tightly bound NBD
fluorophore of double probe 16a.
Fluorescence and ESR properties of the probes were further
studied on MCF-7 cells. For fluorescence microscopy, the probes
were dissolved in DMSO for addition to the medium, since they
were not sufficiently soluble in buffer. Labelling of the membrane
was almost instantaneous, lag times arising mainly from the
preparation of the optical equipment after adding the probes.
Fluorescence microscopy revealed that all probes concentrated
specifically in the plasma membrane, with little redistribution to
intracellular membranes even after one hour of incubation (Chart
1.). The preferential localization in the plasma membrane should
make these probes particularly interesting for research of lipid
dynamics and changes that occur during certain cell events e. g.
cell signalling.
Fig. 2 Normalised absorption and fluorescence emission spectra shown
together with fluorescence emission spectrum after reduction of the nitrox-
ide of double probe 16a. Fluorescence emission spectra were recorded with
10-7 M solutions of 16a in methanol (lex = 450 nm). Absorption spectrum
was recorded with 5 ¥ 10-5 M solution of 16a in methanol. For absorption
and fluorescence emission spectra of other probes see ESI.†
fluorescence intensity is not as great as it was reported in the
literature for double probes, where nitroxide is in close proximity
of the fluorophore.19–21
The exact degree of quenching present when the probe is in
the membrane compared to the case when the probe is in the
solution, is hard to establish due to the sensitivity of the NBD
fluorescence to the environment and insolubility of the probes
in water. Nevertheless, the influence of the double probe’s design
on quenching of the fluorescence was studied by comparing the
fluorescence intensity of probes 16a and 19a incorporated into
the liposomes before and after reduction of nitroxide (Fig. 3).
An increase in fluorescence was observed after reduction of the
nitroxide group with probes 16a and 19a, which means that
some quenching persists even when the probe is incorporated into
the membrane. The increase of fluorescence after reduction of
nitroxide is more pronounced with 19a. In the case of double probe
Chart 1 Confocal fluorescence images (upper row) and corresponding
bright field images (lower row) of MCF-7 cells labelled with 16a (A), 16b
(B), 19a (C) and 19b (D). Images were taken 5 min after addition of probes
dissolved in DMSO.
ESR spectra of MCF-7 cells labelled with double probes 16a
and 19a were recorded (Fig. 4.). The ESR signal-to-noise ratio
was satisfactory up to 30 min after labelling. Afterwards the signal
was lost due to the reduction of the nitroxide group. In order to
assess the influence of the fluorophore and sugar moiety on the
mobility and the ESR signature of the probes, ESR spectra of
liposomes labelled with 1, 16a and 19a were recorded (see ESI†).
The recorded spectra are very similar, therefore we believe that
the fluorophore and the sugar moiety do not have any significant
Fig. 3 Fluorescence emission spectra of double probe 16a incorporated
into the liposomes before and after reduction of the nitroxide. Probe
concentration was 10-7 M (lex = 470 nm). For fluorescence spectra of
other probes see the ESI.†
Fig. 4 ESR spectra of MCF-7 cells labelled with double probes 16a and
19a.
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impact on the motion of the doxyl group on the fatty acid chain.
No other particular differences regarding fluorescence or ESR
properties were observed, either between double probes 16a and
19a or in comparison with their non-paramagnetic analogues 16b
and 19b.
mosphere unless otherwise stated. Analytical TLC was performed
on Merck silica gel (60 F254) plates (0.25 mm) and visualized
with ultraviolet light and detected with 20% sulfuric acid in
ethanol. Flash chromatography was performed on an Isolera
One flash purification system from Biotage using KP-Sil SNAP
cartridges. 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 Brucker AVANCE DPX300
spectrometer in CDCl3, DMSO-d6, MeOH-d4, acetone-d6 and
pyridine-d5 solution, with TMS or residual undeuterated solvent
as the internal standards. In the case of compounds 15b and 19b
13C NMR spectra were additionally recorded at 353 K, in order
to confirm assignation of signals, since some were broad when
recorded at 302 K (see ESI†). Optical rotation was measured
using a Perkin–Elmer 241 MC polarimeter at 589 nm (sodium
D-line) and the [a]D values are quoted in units 10-1 deg cm2 g-1.
Microanalysis was performed on a Perkin–Elmer C, H, N analyzer
240C. Mass spectra were recorded using a VG-Analytical Q-TOF
Premier mass spectrometer. Fluorescence spectra were measured
with Perkin–Elmer LS 55 fluorescence spectrophotometer with
a 10 mm cuvette. The excitation and emission wavelength band-
passes were both set at 10 nm. Absorption spectra were measured
with Varian Cary 50 UV-Vis spectrophotometer.
Conclusions
To summarize, we have succeeded in synthesizing new double
nitroxide–fluorophore probes 16a and 19a, based around a
hydrophilic amino sugar that connects an NBD fluorophore and
a spin labelled fatty acid derivative. Both probes and their non-
paramagnetic analogues were tested on the MCF-7 cell line by
ESR spectroscopy and fluorescence microscopy, providing, for
the first time, ESR spectra with corresponding information of
the probe’s distribution in the cells. Furthermore, early testing on
MFC-7 cells showed that all the probes concentrated specifically
in the plasma membrane with little redistribution to intracellular
membranes. Based on these encouraging results, we believe that
these double nitroxide–fluorophore probes will open up new
possibilities for studying plasma membrane heterogeneity.
Experimental
Fluorescence microscopy
2-(12-Hydroxydodecyl)-4,4-dimethyl-2-propyloxazolidin-3-oxyl
(2). Spin-labelled fatty acid methyl ester 1 (690 mg, 1.86 mmol)
was dissolved in dry diethyl ether (20 mL) and added dropwise to
a suspension of LiAlH4 (141 mg, 2 equiv, 3.72 mmol) in dry diethyl
ether (20 mL) cooled to 0 ◦C on an ice bath. The temperature
was allowed to reach ambient and stirring was continued for
2 h. A saturated solution of NaHCO3 (250 mL) was added to
the reaction mixture and stirring was continued for 15 min.
The reaction mixture was then filtered and the solvent removed
under reduced pressure. The crude product was purified by flash
chromatography (ethyl acetate–hexane, 1 : 3) to give the desired
product (461 mg, 1.35 mmol, 73%) as an orange oil. IR (KBr,
cm-1): 3448, 2927, 2854, 1465, 1363, 1260, 1054. HRMS (ESI),
m/z calcd for C20H42NO3 344.3165 (M–H2)+, found 344.3179.
Microanalysis calcd for C20H40NO3·0.17 H2O (%): C 69.52, H
11.77, N 4.05; found C 69.30, H 12.13, N 4.04.
Human breast adenocarcinoma cell line MCF-7 cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM) containing 10%
fetal calf serum (FSC) and antibiotics: 100 U mL-1 penicillin
and 100 mg mL-1 streptomycin (all from Gibco). The cells were
incubated at 37 ◦C in a humidified 5% CO2 atmosphere.
For fluorescence microscopy observation, cells were plated
on glass-bottom cell culture dishes (Lab-Tek(tm) Chambered
Coverglass) for one day. 0.25 mL 10-5 M of fluorescence probe
16a or 19a in DMSO was added to 30 000 cells/well at room
temperature and observed with an inverted Nikon TE-2000 E
fluorescence microscope, equipped with a confocal unit Carv II
(BD Biosciences) and a Rolera-MGi camera. The samples were
excited from 430 nm to 490 nm and emission was observed from
506 nm to 594 nm.
Electron spin resonance measurements
2-(12-Azidododecyl)-4,4-dimethyl-2-propyloxazolidin-3-oxyl
(3). Alcohol 2 (355 mg, 1.04 mmol) was dissolved in dry
dichloromethane (20 mL) and cooled to 0 ◦C on an ice bath.
Methanesulfonyl chloride (82 mL, 1.05 equiv, 1.09 mmol) was
added to the solution and stirring continued for 30 min. The
reaction mixture was washed with water (50 mL), saturated
solution of NaHCO3 (40 mL), 10% citronic acid (40 mL) and
brine (40 mL), dried with Na2SO4 and the solvent evaporated
under reduced pressure. To the oily residue were added sodium
azide (340 mg, 5 equiv, 5.2 mmol) and dimethylformamide (5 mL)
and the obtained suspension was stirred for 4 h at 55 ◦C. The
reaction mixture was diluted with ethyl acetate (50 mL) and
washed with water (3 ¥ 50 mL), brine (40 mL), dried with
Na2SO4 and the solvent evaporated under reduced pressure to
give the desired product (281 mg, 0.76 mmol, 78%) as an orange
oil. For analytical purposes azide 3 was purified using silica gel
chromatography (ethyl acetate–hexane, 1 : 4). IR (NaCl, cm-1):
2927, 2854, 2095, 1464, 1362, 1259, 1053. HRMS (ESI), m/z
For ESR measurements cells were spin labelled with double probes
16a and 19a. A thin film of the probe was prepared on the walls
of a glass tube by rotary evaporation of a methanol solution of
the probe (12 mL for 16a and 48 mL for 19a). 6 2 million cells (in
the case of 19a 8 2 million cells) were then mixed with 1.5 mL
of medium without serum, placed into the tube and vortexed
for 10 min at room temperature, then centrifuged for 2 min at
1500 rpm. Samples were transferred into a glass capillary for
ESR measurements which were performed on an X-band ESR
spectrometer Bruker ESP 300 at room temperature. Spectrometer
settings were: microwave power 10 mW, modulation amplitude
0.1 mT, frequency of modulation 100 kHz and 9 scans for each
spectrum.
General methods
Chemicals from Sigma-Aldrich and Acros were used without
further purification. All reactions were performed under argon at-
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calcd for C20H39N4O2Na 390.2971 (M–Na)+, found 390.2970.
Microanalysis calcd for C20H39N4O2·0.1 EtOAc + 0.1 hexane (%):
C 65.52, H 10.79, N 14.55; found C 65.52, H 10.76, N 14.50.
was filtered off and the solvent evaporated under reduced pressure.
The residue was dissolved in ethyl acetate and washed with water
(50 mL), saturated solution of NaHCO3 (50 mL), 1 M HCl (50 mL)
and brine (50 mL), dried with Na2SO4 and the solvent evaporated
under reduced pressure. The crude product was purified by flash
chromatography (ethyl acetate–hexane, 1 : 3 to 1 : 1) to give the
desired product (4.5 g, 12.5 mmol, 90%) as a colourless solid. Mp
93–95 ◦C. IR (KBr, cm-1): 3587, 3342, 2999, 1718, 1560, 1459, 1382,
1255, 1215, 1185, 1156, 1112, 1068, 1017, 925, 899, 878, 858, 767,
729, 685. [a]D 9.6 (c 0.20, MeOH). 1H NMR (CDCl3, 300 MHz):
d (ppm) 7.08 (bs, 1H, NH), 5.47 (d, 1H, J = 5.0 Hz, H-1), 4.59 (dd,
1H, J = 7.8, 2.4 Hz, H-3), 4.29 (dd, 1H, J = 5.0, 2.4 Hz, H-2), 4.20
(dd, 1H, J = 7.8, 1.8 Hz, H-4), 3.93–3.88 (m, 1H, H-5), 3.82–3.74
(m, 1H, H-6), 3.37–3.28 (m, 1H, H-6¢), 1.44 (s, 3H, CH3), 1.41 (s,
3H, CH3), 1.30 (s, 3H, CH3), 1.28 (s, 3H, CH3). 13C NMR (CDCl3,
75 MHz): d (ppm) 158.53, 158.04, 157.55, 157.06, 121.93, 118.12,
114.31, 110.50, 110.08, 109.34, 96.60, 72.37, 71.21, 70.85, 65.57,
41.04, 26.25, 26.16, 25.23, 24.53. HRMS (ESI), m/z calcd for
C14H21F3NO6 356.1312 (M + H)+, found 356.1315. Microanalysis
calcd for C14H20F3NO6·0.5EtOAc (%): C 48.12, H 6.06, N 3.51;
found C 48.35, H 6.13, N 3.61.
1-Azidotetradecane (5). 1-Bromotetradecane
4
(3 g,
10.8 mmol) and sodium azide (3.52 g, 5 equiv, 54.1 mmol)
were suspended in dimethylformamide (10 mL) and the obtained
suspension stirred for 6 h at 60 ◦C. The reaction mixture was
diluted with ethyl acetate (50 mL) and washed with water (3 ¥
50 mL), brine (40 mL), dried with Na2SO4 and the solvent
evaporated under reduced pressure to give the desired product
(2.44 g, 10.2 mmol, 94%) as a colourless liquid. IR (KBr, cm-1):
3370, 2922, 2857, 2094, 1877, 1595, 1460, 1258. 1H NMR (CDCl3,
300 MHz): d (ppm) 3.24 (t, 2H, J = 6.9 Hz, CH2) 1.59 (m, 2H,
CH2), 1.40–1.00 (m, 22H, 11 ¥ CH2), 0.88 (t, 3H, J = 6.9 Hz,
CH3). 13C NMR (CDCl3, 75 MHz): d (ppm) 51.44, 31.91, 29.67,
29.65, 29.62, 29.54, 29.48, 29.35, 29.15, 28.84, 26.71, 22.66, 14.02.
MS (ESI), m/z: 214.3 (MH-28)+.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-(prop-2-yn-1-yloxy)-5-
(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-3,4-diyl diacetate
(7). Compound 6 (2.5 g, 5.64 mmol) and propargyl alcohol
(1.64 mL, 5 equiv, 28.2 mmol) were dissolved in anhydrous
dichloromethane (5 mL) and the solution cooled to 0 ◦C on
an ice bath. BF3·Et2O (3.6 mL, 5 equiv, 28.2 mmol) was added
dropwise and the reaction mixture was allowed to reach room
temperature while stirring was continued for 72 h. The reaction
mixture was then diluted with ethyl acetate (50 mL) and poured
into saturated solution of NaHCO3 (50 mL). After gas stopped
evolving, the organic layer was separated, washed with brine and
dried with Na2SO4. The solvent was evaporated under reduced
pressure and the crude product purified by flash chromatography
(ethyl acetate–hexane, 1 : 2) to give the desired product (1.55 g,
3.53 mmol, 63%) as a colourless solid. Mp 175–177 ◦C. IR (KBr,
cm-1): 3315, 3270, 3112, 2975, 1749, 1714, 1562, 1379, 1233,
1181, 1075, 1048, 885, 680. [a]D -38.9 (c 0.23, MeOH). 1H NMR
(CDCl3, 300 MHz): d (ppm) 6.53 (d, 1H, J = 8.7 Hz, NH), 5.32
(dd, 1H, J = 10.5, 9.3 Hz, H-3), 5.12 (dd, 1H, J = 9.9, 9.3 Hz,
H-4), 4.88 (d, 1H, J = 8.4 Hz, H-1), 4.38 (d, 2H, J = 2.2 Hz,
CH2–C ), 4.29 (dd, 1H, J = 12.6, 4.8 Hz, Hab-6), 4.16 (dd, 1H,
J = 12.6, 2.7 Hz, Hab-6¢), 4.08–3.98 (m, 1H, H-2), 3.79–3.73 (m,
1H, H-5), 2.47 (t, 1H, J = 2.2 Hz, CH), 2.10 (s, 3H, CH3), 2.04
(s, 3H, CH3), 2.03 (s, 3H, CH3). 13C NMR (CDCl3, 75 MHz):
d (ppm) 171.14, 170.69, 169.21, 158.20, 157.70, 157.20, 156.70,
121.29, 171.45, 131.65, 101.13, 97.65, 78.02, 75.59, 72.01, 71.84,
68.47, 61.90, 56.00, 54.32, 20.64, 20.52, 20.33. HRMS (ESI), m/z
calcd for C17H20F3NO9Na 462.0988 (M + Na)+, found 462.0973.
Microanalysis calcd for C17H20F3NO9 (%): C 46.47, H 4.59, N
3.19; found C 46.67, H 4.46, N 3.16.
(3R,4S,5S,6R)-6-((2,2,2-Trifluoroacetamido)methyl)tetrahydro-
2H-pyran-2,3,4,5-tetrayl tetraacetate (10). Compound 9 (4.18 g,
11.7 mmol) was dissolved in methanol (20 mL). Trifluoroacetic
acid (5 mL) and water (10 mL) were added and the reaction
mixture was stirred at 50 ◦C for 15 h. Solvents were removed
under reduced pressure; removing traces of water from the residue
by toluene (2 ¥ 10 mL) co-evaporation under reduced pressure.
Residue was dissolved in pyridine (18 mL) and the solution was
cooled on an ice bath to 0 ◦C. Acetic anhydride (7.2 mL, 5.5
equiv, 65.2 mmol) was added dropwise to the solution and stirring
was continued for 24 h while the temperature was allowed to
reach room temperature. 10 mL of methanol was added to the
reaction mixture and, after 10 min, the solvent was evaporated
under reduced pressure. The residue was dissolved in ethyl acetate
and washed with water (50 mL), saturated solution of NaHCO3
(50 mL), 1 M HCl (50 mL) and brine (50 mL), dried with Na2SO4
and the solvent evaporated under reduced pressure. The crude
product was purified by flash chromatography (ethyl acetate–
hexane, 1 : 3 to 1 : 1) to give the desired product (2.6 g, 6.4 mmol,
55%) as a viscous oil. IR (KBr, cm-1): 3422, 1752, 1569, 1375, 1225,
1069, 932, 727, 600. HRMS (ESI), m/z calcd for C16H20F3NO10Na
466.0937 (M + Na)+, found 466.0940. Microanalysis calcd for
C16H20F3NO10·0.2EtOAc (%): C 43.78, H 4.72, N 3.04; found C
43.67, H 4.79, N 3.00.
(3R,4S,5S,6R) - 2 - Hydroxy - 6 - ((2 , 2 , 2 - trifluoroacetamido)-
methyl)tetrahydro-2H -pyran-3,4,5-triyl triacetate (11). Com-
pound 10 (733 mg, 1.65 mmol) was dissolved in tetrahydrofuran
(5 mL) followed by the addition of benzylamine (191 mL,
1.05 equiv, 1.73 mmol). Reaction mixture was stirred at room
temperature for 72 h. The solvent was evaporated under reduced
pressure and the residue dissolved in ethyl acetate (30 ml), washed
with saturated solution of 1 M HCl (2 ¥ 30 mL), NaHCO3 (30 mL),
and brine (50 mL). After drying with Na2SO4 the solvent was
evaporated under reduced pressure and the crude product purified
by flash chromatography (ethyl acetate–hexane, 1 : 1) to give the
desired product (480 mg, 1.2 mmol, 72%) as a white foam. Mp
2,2,2-Trifluoro-N-(((3aR,5R,5aS,8aS,8bR)-2,2,7,7-tetramethyl-
tetrahydro-3aH-bis([1,3]dioxolo)[4,5-b:4¢,5¢-d]pyran-5-yl)methyl)
acetamide (9). Azide 8 (4.0 g, 13.8 mmol) was dissolved in
anhydrous tetrahydrofuran (50 mL) and argon was bubbled
through the solution for 10 min. Pd/C (250 mg, 6.25%) was
added and the suspension stirred under hydrogen for 48 h. The
suspension was cooled to 0 ◦C on ice while argon was bubbled
through. Triethylamine (2.9 mL, 1.5 equiv, 20.7 mmol) was added
to the suspension followed by dropwise addition of trifluoroacetic
anhydride (2.1 ml, 1.1 equiv, 15.2 mmol). After 2 h of stirring Pd/C
◦
68–70 C. IR (KBr, cm-1): 3447, 1751, 1560, 1438, 1375, 1228,
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1158, 1062, 728, 600. HRMS (ESI), m/z calcd for C14H18F3NO9Na
424.0831 (M + Na)+, found 424.0834. Microanalysis calcd for
C14H18F3NO9 (%): C 41.90, H 4.52, N 3.49; found C 41.98, H 4.58,
N 3.36.
dissolved in ethyl acetate (5 mL). CuBr (7 mg, 1,1 equiv, mmol)
was added and the reaction mixture stirred for 30 min at room
temperature. The solvent was removed under reduced pressure
and the crude product was purified by flash chromatography.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-((1-(12-(3-oxyl-4,4-
dimethyl-2-propyloxazolidin-2-yl)dodecyl)-1H-1,2,3-triazol-4-yl)-
methoxy)-5-(2,2,2-trifluoroacetamido)tetrahydro-2H-pyran-3,4-
diyl diacetate (14a). The reaction between alkyne 7 and azide
3 was carried out according to the general procedure for click
reaction. Removal of the solvent under reduced pressure and
purification of the crude product by flash chromatography (ethyl
acetate–hexane, 2 : 3 to 3:2) yielded the desired product (63%) as an
orange viscous oil. IR (KBr, cm-1): 3448, 2929, 1751, 1643, 1567,
1466, 1378, 1233, 1182, 1047. [a]D -9.9 (c 0.15, MeOH). HRMS
(ESI), m/z calcd for C37H60F3N5O11 807.4241 (M + H)+, found
807.4252. Microanalysis calcd for C37H59F3N5O11 (%): C 55.08, H
7.37, N 8.68; found C 55.01, H 7.22, N 8.36.
(3R,4S,5S,6R)-2-(2,2,2-Trichloro-1-iminoethoxy)-6-((2,2,2-tri-
fluoroacetamido)methyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
(12). Compound 11 was dissolved in dry dichloromethane
(50 mL) and cooled to
0
◦C on an ice bath. 1,8-
Diazabicyclo[5.4.0]undec-7-ene (160 mL, 0.33 equiv, 1.1 mmol)
was added followed by trichloroacetonitrile (510 mL, 1.5 equiv,
5 mmol). The temperature was allowed to reach ambient temper-
ature and stirring was continued for 2 h. The reaction mixture was
adsorbed on silica gel, dried and purified by flash chromatography
(ethyl acetate–hexane, 1 : 10 to 1 : 2) to give the desired product
(1.0 g, 1.9 mmol, 57%) as a white foam. Mp 50–55 ◦C. IR (KBr,
cm-1): 3447, 2931, 1735, 1376, 1228, 1159, 1064, 833. [a]D 143 (c
0.26, CH2Cl2). 1H NMR (CDCl3, 300 MHz): d (ppm) 8.69 (s, 1H, =
NH), 6.72 (bs, 1H, NH), 6.55 (d, 1H, J = 3.4 Hz, H-1), 5.52 (dd,
1H, J = 3.0, 0.9 Hz, H-4), 5.42 (dd, 1H, J = 10.8, 3.0 Hz, H-3),
5.35 (dd, 1H, J = 10.8, 3.4 Hz, H-2), 4.33–4.29 (m, 1H, H-5), 3.65–
3.56 (m, 1H, H-6), 3.39–3.29 (m, 1H, H-6¢), 2.19 (s, 3H, CH3),
2.04 (s, 3H, CH3), 2.02 (s, 3H, CH3).13C NMR (CDCl3, 75 MHz):
d (ppm) 170.32, 170.10, 169.76, 161.02, 158.00, 157.53, 157.03,
156.54, 121.29, 117.51, 113.70, 109.22, 93.30, 30.57, 69.09, 68.32,
67.40, 66.81, 39.51, 20.58, 20.53, 20.50. HRMS (ESI), m/z calcd
for C16H18F3N2O9Cl3Na 566.9928 (M + Na)+, found 566.9930.
Microanalysis calcd for C16H18Cl3F3N2O9·0.33 H2O (%): C 34.83,
H 3.41, N 5.08; found C 34.76, H 3.39, N 4.93.
(2R,3S,4R,5R,6R)-2-(Acetoxymethyl)-6-((1-tetradecyl-1H-1,2,
3-triazol-4-yl)methoxy)-5-(2,2,2-trifluoroacetamido)tetrahydro-
2H-pyran-3,4-diyl diacetate (14b). The reaction between alkyne
7 and azide 5 was carried out according to general procedure
for click reaction. Removal of solvent under reduced pressure
and purification of the crude product by flash chromatography
(ethyl acetate–hexane, 1 : 1) yielded the desired product (78%) as
◦
a colourless solid. Mp 129–131 C. IR (KBr, cm-1): 3344, 2918,
2850, 1754, 1713, 1558, 1471, 1379, 1228, 1181, 1119, 1076, 1049,
884, 782, 664, 604. [a]D -13.5 (c 0.22, MeOH). 1H NMR (CDCl3,
300 MHz): d (ppm) 7.47 (s, 1H, H-Ar), 7.20 (d, 1H, J = 8.7 Hz,
NH), 5.24 (dd, 1H, J1,2 = 9.6 Hz, H-3), 5.13 (dd, 1H, J1,2 = 9.6
Hz, H-4), 4.95 (d, 1H, J = 8.4 Hz, H-1), 4.91 (d, 1H, J = 12.6
Hz, OCHab-Ar), 4.81 (d, 1H, J = 12.6 Hz, OCHab-Ar), 4.32 (dd,
1H, J = 10.0, 1.8 Hz, Hab-6), 4.29(t, 2H, J = 4.5 Hz, CH2–Ar),
4.17 (dd, 1H, J = 10.0, 2.4 Hz, Hab-6¢), 4.15–4.06 (m, 1H, H-2),
3.79–3.73 (m, 1H, H-5), 2.10 (s, 3H, CH3), 2.03 (s, 3H, CH3), 2.02
(s, 3H, CH3), 1.95–1.80 (m, 2H, CH2), 1.38–1.16 (m, 22H, 11 ¥
CH2), 0.88 (t, 3H, J = 6.6 Hz, CH3). 13C NMR (CDCl3, 75 MHz):
d (ppm) 171.07, 170.50, 169.23, 157.90, 157.41, 156.92, 156.42,
144.06, 122.74, 121.37, 117.55, 113.73, 109.91, 99.63, 72.25, 71.72,
68.35, 62.57, 61.91, 54.27, 50.33, 31.74, 30.02, 29.50, 29.43, 29.43,
29.38, 29.21, 29.17, 28.87, 26.34, 22.50, 20.52, 20.33, 20.24, 13.92.
HRMS (ESI), m/z calcd for C31H50F3N4O9 679.3530 (M + H)+,
found 679.3556. Microanalysis calcd for C31H49F3N4O9 (%): C
54.86, H 7.28, N 8.25; found C 54.93, H 7.39, N 8.18.
(2R,3R,4S,5S,6R)-2-(Prop-2-yn-1-yloxy)-6-((2,2,2-trifluo-
roacetamido)methyl)tetrahydro-2H-pyran-3,4,5-triyl
triacetate
(13). Trichloroacetimidate 12 (1.01 g, 1.85 mmol) and propargyl
alcohol (1.14 mL, 10 equiv, 19.5 mmol) were dissolved in dry
dichloromethane (10 mL). Molecular sieves (1 g) were added and
the solution cooled to -30 ◦C. Trimethylsilyl triflate (85 mL, 0.25
equiv, 0.47 mmol) was added to the solution, the temperature of
which rose to -15 ◦C in a period of 30 min. A saturated solution
of NaHCO3 (500 mL) was added to the reaction mixture and the
solvent removed under reduced pressure. The crude product was
purified by flash chromatography (ethyl acetate–hexane, 1 : 9 to
1 : 1) to give the desired product (510 mg, 1.16 mmol, 63%) as
◦
a white foam. Mp 40–42 C. IR (KBr, cm-1): 3362, 2926, 1750,
1559, 1372, 1223, 1156, 1073, 905, 727. [a]D 4.2 (c 0.29, MeOH).
1H NMR (CDCl3, 300 MHz): d (ppm) 6.85 (bs, 1H, NH), 5.33
(dd, 1H, J = 3.3 Hz, H-4), 5.26 (dd, 1H, J = 10.3, 8.0 Hz, H-2),
5.09 (dd, 1H, J = 10.3, 3.3 Hz, H-3), 4.76 (d, 1H, J = 8.0 Hz,
H-1), 4.40–4.39 (m, 2H, CH2–C ), 3.87 (dt, 1H, J = 6.9 Hz,
H-5), 3.65–3.56 (m, 1H, H-6), 3.49–3.40 (m, 1H, H-6¢), 2.50 (t,
1H, J = 2.1 Hz, CH), 2.09 (s, 3H, CH3), 2.06 (s, 3H, CH3), 2.03
(s, 3H, CH3). 13C NMR (CDCl3, 75 MHz): d (ppm) 170.64,
169.90, 169.55, 158.07, 157.58, 157.08, 156.59, 121.29, 117.47,
113.67, 109.86, 98.93, 77.99, 75.42, 70.61, 70.49, 68.42, 67.82,
56.13, 39.16, 20.52, 20.39, 20.29. HRMS (ESI), m/z calcd for
C17H19F3NO9 438.1012 (M - H)-, found 438.1020. Microanalysis
calcd for C17H20F3NO9·0.2 H2O (%): C 45.24, H 4.76, N 3.10;
found C 45.11, H 4.59, N 3.06.
General procedure for one pot removal of acetate and trifluo-
roacetamide protective groups. The product of the click reaction
(0.16 mmol) was dissolved in dry methanol (2 mL). Sodium
methoxide solution in methanol (20 mL, 30%) was added and
stirring continued for 1 h at room temperature. Solvent was
evaporated under reduced pressure and NaOH solution (2 mL,
1 M) was added to the residue. After approximately 1 h a white
precipitate was formed. A small volume of methanol (~2 mL) was
added to wash unreacted compound from the wall of the flask and
stirring was continued for 30 min.
(2R,3S,4R,5R,6R)-5-Amino-6-((1-(12-(3-oxyl-4,4-dimethyl-2-
propyloxazolidin-2-yl)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-2-
(hydroxymethyl)tetrahydro-2H-pyran-3,4-diol (15a). Reaction
was carried out according to the general procedure for one pot
General procedure for a click reaction. Alkyne (1 mmol),
azide (1 mmol) and N-methylmorpholine (103 mL, 1 mmol) were
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removal of acetate and trifluoroacetamide protective groups,
starting from compound 14a. After reaction proceeded to
completion, the reaction mixture was adsorbed on silica gel
followed by flash chromatography (dichloromethane–methanol–
ammonia(aq), 12 : 2 : 1), yielding the desired product (77%) as an
orange viscous oil. IR (KBr, cm-1): 3422, 2926, 2854, 1639, 1458,
1382, 1260, 1081, 1054, 618. [a]D -18.8 (c 0.19, MeOH). HRMS
(ESI), m/z calcd for C29H55N5O7 585.4101 (M + H)+, found
585.4113. Microanalysis calcd for C29H54N5O7·MeOH (%): C
58.42, H 9.48, N 11.35; found C 58.31, H 9.82, N 11.70.
(2R,3S,4R,5R,6R)-2-(Hydroxymethyl)-5-((7-nitrobenzo[c][1,2,
5]oxadiazol-4-yl)amino)-6-((1-tetradecyl-1H-1,2,3-triazol-4-yl)-
methoxy)tetrahydro-2H-pyran-3,4-diol (16b). Reaction was car-
ried out according to the general procedure for reaction
with NBD-Cl, starting from amine 15b. Flash chromatography
(dichloromethane–methanol, 96 : 4 to 92 : 8) of the crude product
yielded the desired compound (43%) as an orange solid. Mp 110–
112 ◦C. IR (KBr, cm-1): 3392, 2924, 2853, 1585, 1498, 1310, 1075,
1
1034, 902. [a]D 44.9 (c 0.21, MeOH). H NMR (acetone-d6, 300
MHz): d (ppm) 8.47 (d, 1H, J = 9.0 Hz, H-Ar), 8.04 (s, 1H, NH),
7.71 (s, 1H, H-Ar), 6.65 (d, 1H, J = 9.0 Hz, H-Ar), 4.92 (d, 1H,
J = 8.1 Hz, H-1), 4.91 (s, 1H, OH), 4.88 (d, 1H, J = 12.9 Hz,
OCHab-Ar), 4.73 (d, 1H, J = 12.9 Hz, OCHab-Ar), 4.59 (s, 1H,
OH), 4.27 - 4.22 (m, 2H, CH2–Ar), 4.10–3.85 (m, 4H, H-2, H-3,
Hab-6, OH), 3.78 (dd, 1H, J = 10.5, 5.4 Hz, Hab-6¢), 3.58 (dd, 1H,
J1,2 = 8.4 Hz, H-4), 3.51–3.45 (m, 1H, H-5), 1.79–1.73 (m, 2H,
CH2), 1.40–1.15 (m, 22H, 11 ¥ CH2), 0.88 (t, 3H, J = 6.6 Hz, CH3.
13C NMR (acetone-d6, 75 MHz): d (ppm) 147.66, 146.56, 145.97,
145.80, 138.65, 125.04, 124.38, 103.04, 102.56, 78.78, 77.18, 73.11,
64.36, 63.79, 62.59, 51.48, 33.60, 31.37, 31.31, 31.25, 31.13, 31.04,
30.62, 30.61, 28.06, 24.29, 15.33. HRMS (ESI), m/z calcd for
C29H46N7O8 620.3408 (M + H)+, found 620.3394. Microanalysis
calcd for C29H45N7O8·MeOH (%): C 55.29, H 7.58, N 15.04; found
C 55.03, H 7.32, N 15.16.
(2R, 3S , 4R, 5R,6R)-5-Amino-2-(hydroxymethyl)-6-((1-tetr-
adecyl-1H-1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4-
diol (15b). Reaction was carried out according to the general
procedure for one pot removal of acetate and trifluoroacetamide
protective groups, starting from compound 14b. After the reaction
proceeded to completion, the reaction mixture was adsorbed on
silica gel followed by flash chromatography (dichloromethane–
methanol–ammonia(aq), 12 : 2 : 1) that yielded the desired product
(78%) as a colourless solid. Mp 118–120 ◦C. IR (KBr, cm-1): 3360,
2920, 2849, 1609, 1466, 1376, 1224, 1054. [a]D 22.6 (c 0.24, MeOH).
1H NMR (Pyr-d5, 300 MHz): d (ppm) 7.60 (s, 1H, H-Ar), 5.35 (d,
1H, J = 12.3 Hz, OCHab-Ar), 5.12 (d, 1H, J = 12.3 Hz, OCHab-
Ar), 4.92 (d, 1H, J = 8.6 Hz, H-1), 4.54 (dd, 1H, J = 12.1, 2.4
Hz, Hab-6), 4.38 (dd, 1H, J = 12.1, 5.4 Hz, Hab-6¢), 4.34 (t, 2H,
J = 7.2 Hz, CH2–Ar), 4.21 (dd, 1H, J1,2 = 8.6 Hz, H-4), 4.02 (dd,
1H, J1,2 = 8.6 Hz, H-3), 3.96–3.90 (m, 1H, H-5), 3.32 (dd, 1H,
J1,2 = 8.6 Hz, H-2), 1.84–1.79 (m, 2H, CH2), 1.30–1.10 (m, 22H,
11 ¥ CH2), 0.88 (t, 3H, J = 6.3 Hz, CH3. 13C NMR (CD3OD,
75 MHz): d (ppm) 145.45, 125.35, 103.40, 78.32, 77.46, 71.90,
62.88, 58.28, 51.44, 33.09, 31.30, 30.80, 30.77, 30.74, 30.67, 30.57,
30.48, 30.12, 27.53, 23.75, 14.47. HRMS (ESI), m/z calcd for
C23H45N4O5 457.3390 (M + H)+, found 457.3399. Microanalysis
calcd for C23H44N4O5·0.33H2O (%): C 59.71, H 9.73, N 12.11;
found C 59.70, H 9.79, N 11.99.
(2R,3R,4S,5S,6R) - 2 - ((1 - (12 - (3 - Oxyl - 4,4 - dimethyl - 2-pro-
pyloxazolidin-2-yl)dodecyl)-1H-1,2,3-triazol-4-yl)methoxy)-6-((2,
2,2-trifluoroacetamido)methyl)tetrahydro-2H-pyran-3,4,5-triyl tri-
acetate (17a). The reaction between alkyne 13 and azide 3 was
carried out according to the general procedure for click reaction.
Removal of solvent under reduced pressure and purification of the
crude product by flash chromatography (ethyl acetate–hexane, 2 : 3
to 3 : 2) yielded the desired product (63%) as an orange viscous
oil. IR (KBr, cm-1): 3422, 2932, 2857, 1752, 1560, 1370, 1222,
1159, 1058, 905, 727. [a]D -6.8 (c 0.18, MeOH). HRMS (ESI),
m/z calcd for C37H60F3N5O11 807.4241 (M + H)+, found 807.4258.
Microanalysis calcd for C37H59F3N5O11 (%): C 55.08, H 7.37, N
8.68; found C 54.80, H 7.18, N 8.44.
General procedure for reaction with 4-chloro-7-nitrobenzofurazan
(NBD-Cl). Free amine (1.0 mmol) and K2CO3 (140 mg, 2.5 equiv,
2.5 mmol) were suspended in methanol (3 mL) and the reaction
mixture cooled to 0 ◦C on an ice bath. To the suspension was
added in small portions NBD-Cl (360 mg, 1.8 equiv, 1.8 mmol)
and the reaction mixture was allowed to reach room temperature
while stirring was continued for 12 h. Solvent was evaporated
under reduced pressure and the crude product was purified by
flash chromatography.
(2R,3R,4S,5S,6R)-2-((1-Tetradecyl-1H -1,2,3-triazol-4-yl)-
methoxy)-6-((2,2,2-trifluoroacetamido)methyl)tetrahydro-2H-py-
ran-3,4,5-triyl triacetate (17b). The reaction between alkyne 13
and azide 5 was carried out according to general procedure
for click reaction. Removal of solvent under reduced pressure
and purification of the crude product by flash chromatography
(ethyl acetate–hexane, 1 : 1) yielded the desired product (90%) as
a colourless viscous oil. IR (KBr, cm-1): 3422, 2927, 2856, 1752,
1568, 1458, 1375, 1223, 1159, 1074, 905, 726. [a]D 5.1 (c 0.24,
(2R,3S ,4R,5R,6R)-6-((1-(12-(3-Oxyl-4,4-dimethyl-2-pro-
pyloxazolidin-2-yl)dodecyl)-1H -1,2,3-triazol-4-yl)methoxy)-2-
(hydroxymethyl)-5-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)-
tetrahydro-2H-pyran-3,4-diol (16a). Reaction was carried out ac-
cording to the general procedure for reaction with NBD-Cl, start-
ing from amine 15a. Flash chromatography (dichloromethane–
methanol, 1 : 0 to 15 : 1) of the crude product yielded the desired
compound (59%) as an orange solid. Mp 71–73 ◦C. IR (KBr,
cm-1): 3849, 3741, 2926, 2849, 1699, 1649, 1539, 1075, 826. [a]D
41.8 (c 0.14, MeOH). HRMS (ESI), m/z calcd for C35H56N8O10
748.4114 (M + H)+, found 748.4127. Microanalysis calcd for
C35H55N8O10·MeOH (%): C 55.44, H 7.63, N 14.37; found C 55.12,
H 7.51, N 14.11.
1
MeOH). H NMR (CDCl3, 300 MHz): d (ppm) 7.94 (t, 1H, J =
5.1 Hz, NH) 7.52 (s, 1H, H-Ar), 5.33 (dd, 1H, J = 3.3, 0.6 Hz,
H-4), 5.23 (dd, 1H, J = 10.3, 8.0 Hz, H-2), 5.01 (dd, 1H, J = 10.3,
3.3 Hz, H-3), 4.90 (d, 1H, J = 12.9 Hz, OCHab-Ar), 4.82 (d, 1H, J =
12.9 Hz, OCHab-Ar), 4.58 (d, 1H, J = 8.0 Hz, H-1), 4.34 (t, 2H, J =
7.2 Hz, CH2–Ar), 3.84–3.79 (m, 1H, H-5), 3.63–3.54 (m, 1H, H-
6), 3.45–3.38 (m, 1H, H-6¢), 2.16 (s, 3H, CH3), 2.02 (s, 3H, CH3),
1.98 (s, 3H, CH3), 1.94–1.86 (m, 2H, CH2), 1.35–1.22 (m, 22H,
11 ¥ CH2), 0.88 (t, 3H, J = 6.6 Hz, CH3). 13C NMR (CDCl3, 75
MHz): d (ppm) 170.49, 769.87, 169.46, 158.40, 157.91, 157.41,
156.92, 143.51, 122.97, 121.48, 171.67, 113.86, 110.05, 100.26,
70.92, 70.677, 68.78, 67.98, 62.37, 50.37, 39.57, 31.80, 30.15, 29.55,
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29.52, 29.48, 29.41, 29.28, 29.22, 28.88, 26.40, 22.56, 20.61, 20.49,
20.40, 13.38. HRMS (ESI), m/z calcd for C31H50F3N4O9 679.3530
(M + H)+, found 679.3525.
(dichloromethane–methanol, 96 : 4 to 92 : 8) of the crude product
yielded the desired compound (24%) as an orange solid. Mp 99–
101 ◦C. IR (KBr, cm-1): 3392, 2926, 28.57, 2094, 1593, 1305, 1037,
824. [a]D 44.2 (c 0.095, MeOH).1H NMR (acetone-d6, 300 MHz):
d (ppm) 8.55 (d, 1H, J = 8.8 Hz, H-Ar), 7.88 (s, 1H, H-Ar), 6.61
(d, 1H, J = 8.8 Hz, H-Ar), 4.83 (d, 1H, J = 12.3 Hz, OCHab-
Ar), 4.67 (d, 1H, J = 12.3 Hz, OCHab-Ar), 4.40 (d, 1H, J = 7.2
Hz, H-1), 4.35 (t, 2H, J = 7.2 Hz, CH2–Ar), 4.05–3.97 (m, 3H,
H-5, H-6), 3.98 (dd, 1H, J = 2.9 Hz, H-4), 3.64–3.57 (m, 1H,
H-2), 3.56 (dd, 1H, J = 9.3, 2.9 Hz. H-3), 1.88–1.81 (m, 2H,
CH2), 1.40–1.00 (m, 22H, 11 ¥ CH2), 0.86 (t, 3H, J = 6.9 Hz,
CH3). 13C NMR (Acetone-d6, 75 MHz): d (ppm) 146.45, 146.11,
145.96, 138.92, 125.18, 124.17, 104.51, 101.27, 75.26, 74.54, 72.74,
71.34, 63.52, 51.63, 46.63, 33.54, 31.92, 31.30, 31.29, 31.26, 31.21,
30.97, 30.66, 28.07, 24.23, 15.29. HRMS (ESI), m/z calcd for
C29H46N7O8 620.3408 (M + H)+, found 620.3431. Microanalysis
calcd for C29H45N7O10·0.66MeOH (%): C 55.58, H 7.49, N 15.29;
found C 55.38, H 7.43, N 15.32.
(2R,3S ,4S ,5R,6R)-2-(aminomethyl)-6-((1-(12-(3-oxyl-4,4-
dimethyl-2-propyloxazolidin-2-yl)dodecyl)-1H-1,2,3-triazol-4-yl)-
methoxy)tetrahydro-2H-pyran-3,4,5-triol (18a). Reaction was
carried out according to the general procedure for one pot removal
of acetate and trifluoroacetamide protective groups starting
from compound 17a. After reaction proceeded to completion,
the reaction mixture was adsorbed on silica gel followed by
flash chromatography (dichloromethane–methanol–ammonia(aq)
,
8 : 2 : 1), yielding the desired product (31%) as an orange viscous
oil. IR (KBr, cm-1): 3431, 2927, 2854, 1672, 1456, 1378, 1204,
1135, 1062. [a]D 1.2 (c 0.14, MeOH). HRMS (ESI), m/z calcd for
C29H55N5O7 585.4101 (M + H)+, found 585.4108. Microanalysis
calcd for C29H54N5O7·2H2O (%): C 56.11, H 9.42, N 11.28; found
C 56.22, H 9.12, N 11.01.
(2R,3S,4S , 5R , 6R) - 2 - (Aminomethyl) - 6 - ((1 - tetradecyl - 1H-
1,2,3-triazol-4-yl)methoxy)tetrahydro-2H-pyran-3,4,5-triol (18b).
Reaction was carried out according to the general procedure
for one pot removal of acetate and trifluoroacetamide protective
groups starting from compound 17b. After reaction proceeded
to completion, the reaction mixture was adsorbed on silica gel
followed by flash chromatography (dichloromethane–methanol–
ammonia(aq), 8 : 2 : 1)◦, yielding the desired product (53%) as a white
solid. Mp 109–112 C. IR (KBr, cm-1): 3426, 2922, 2853, 1606,
Acknowledgements
We are grateful to the Slovenian research agency (ARRS) for
financial support (P1-0208) of our work, the Institute Jozˇef
Stefan for MS spectra and Dr R. Pain for critical reading of the
manuscript.
1
Notes and references
1340, 1064. [a]D -4.7 (c 0.28, MeOH). H NMR (Pyr-d5, 300
MHz): d (ppm) 8.12 (s, 1H, H-Ar), 5.39 (d, 1H, J = 12.0 Hz,
OCHab-Ar), 5.19 (d, 1H, J = 12.0 Hz, OCHab-Ar), 4.98 (d, 1H, J =
8.0 Hz, H-1), 4.49 (dd, 1H, J = 9.3, 8.0 Hz, H-2), 4.40 (dd, 1H,
J = 3.1 Hz, H-4), 4.32 (t, 2H, J = 7.2 Hz, CH2–Ar), 4.13 (dd, 1H,
J = 9.3, 3.1 Hz, H-3), 3.97–3.87 (m, 1H, H-5), 3.81–3.24 (m, 2H,
H-6), 1.89–1.71 (m, 2H, CH2), 1.40–1.07 (m, 22H, 11 ¥ CH2), 0.88
(t, 3H, J = 6.0 Hz, CH3). 13C NMR (Pyr-d5, 75 MHz): d (ppm)
146.90, 125.67, 106.04, 76.82, 73.89, 72.62, 64.56, 51.86, 44.93,
33.79, 32.21, 31.61, 31.58, 31.51, 31.35, 31.27, 30.94, 28.39, 24.60,
15.95. HRMS (ESI), m/z calcd for C23H45N4O5 457.3390 (M +
H)+, found 457.3382. Microanalysis calcd for C23H44N4O5·2H2O
(%): C 56.07, H 9.82, N 11.37; found C 56.09, H 9.80, N 11.16.
1 K. Jacobson, O. G. Mouritsen and R. G. W. Anderson, Nat. Cell Biol.,
2007, 9, 7–14.
2 K. Simons and D. Toomre, Nat. Rev. Mol. Cell Biol., 2000, 1, 31–39.
3 S. Manes, G. del Real and A. C. Martinez, Nat. Rev. Immunol., 2003,
3, 557–568.
4 J. S. Wadia, M. Schaller, R. A. Williamson and S. F. Dowdy, PLoS One,
2008, 3, e3314.
5 H. Cheng, K. S. Vetrivel, P. Gong, X. Meckler, A. Parent and G.
Thinakaran, Nat Clin Pract Neurol, 2007, 3, 374–382.
6 J. Oshikawa, N. Urao, H. W. Kim, N. Kaplan, M. Razvi, R. McKinney,
L. B. Poole, T. Fukai and M. Ushio-Fukai, PLoS One, 2010, 5, e10189.
7 D. Lingwood and K. Simons, Science, 2010, 327, 46–50.
8 H. T. He and D. Marguet, EMBO Rep., 2008, 9, 525–530.
9 B. Dzikovski, J. H. Freed and T. P. Begley, Membrane Fluidity, John
Wiley & Sons, Inc., 2007.
10 G. I. Likhtenshtein, J. Yamauchi, S. Nakatsuji, A. I. Smirnov and R.
Tamura, Nitroxide Spin Probes for Studies of Molecular Dynamics and
Microstructure, Wiley-VCH Verlag GmbH & Co. KGaA, 2008.
11 M. A. Hemminga and L. J. Berliner, ESR spectroscopy in membrane
biophysics, Springer, New York, 2007.
12 G. van Meer, D. R. Voelker and G. W. Feigenson, Nat. Rev. Mol. Cell
Biol., 2008, 9, 112–124.
(2R,3R,4S ,5R,6R)-2-((1-(12-(3-Oxyl-4,4-dimethyl-2-pro-
pyloxazolidin-2-yl)dodecyl)-1H -1,2,3-triazol-4-yl)methoxy)-6-
(((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)methyl)tetrahydro-
2H-pyran-3,4,5-triol (19a). Reaction was carried out according
to the general procedure for reaction with NBD-Cl, starting from
amine 18a. Flash chromatography (dichloromethane–methanol,
96 : 4 to 92 : 8) of the crude product yiel◦ded the desired compound
(13%) as an orange solid. Mp 80–82 C. IR (KBr, cm-1): 3422,
2931, 2857, 1752, 1560, 1458, 1370, 1222, 1159, 1058, 905, 827,
726. [a]D 28.4 (c 0.31, MeOH). HRMS (ESI), m/z calcd for
C35H56N8O10 748.4119 (M + H)+, found 748.4141. Microanalysis
calcd for C35H55N8O10 (%): C 56.21, H 7.41, N 14.98; found C
56.50, H 7.64, N 15.37.
13 T. Pomorski, P. Muller, B. Zimmermann, K. Burger, P. F. Devaux and
A. Herrmann, J. Cell Sci., 1996, 109, 687–698.
14 A. Gidwani, D. Holowka and B. Baird, Biochemistry, 2001, 40, 12422–
12429.
15 M. Ge, A. Gidwani, H. A. Brown, D. Holowka, B. Baird and J. H.
Freed, Biophys. J., 2003, 85, 1278–1288.
16 J. R. Lakowicz, Principles of fluorescence spectroscopy, 3rd edn,
Springer, New York, 2006, pp. 278–351.
17 N. V. Blough and D. J. Simpson, J. Am. Chem. Soc., 1988, 110, 1915–
1917.
18 V. Y. Nagy, I. y. M. Bystryak, A. I. Kotel’nikov, G. I. Likhtenshtein, O.
M. Petrukhin, Y. A. Zolotov and L. B. Volodarskii, Analyst, 1990, 115,
839–841.
(2R,3R,4S,5R,6R)-2-(((7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl)-
amino)methyl) - 6 - ((1 - tetradecyl - 1H - 1 , 2 , 3 - triazol - 4 - yl)me-
thoxy)tetrahydro-2H-pyran-3,4,5-triol (19b). Reaction was car-
ried out according to the general procedure for reaction
with NBD-Cl, starting from amine 18b. Flash chromatography
19 J. P. Blinco, J. C. McMurtrie and S. E. Bottle, Eur. J. Org. Chem., 2007,
28, 4638–4641.
20 C. Coenjarts, O. Garc´ıa, L. Llauger, J. Palfreyman, A. L. Vinette and
J. C. Scaiano, J. Am. Chem. Soc., 2002, 125, 620–621.
4158 | Org. Biomol. Chem., 2011, 9, 4150–4159
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
21 A. S. Micallef, J. P. Blinco, G. A. George, D. A. Reid, E. Rizzardo, S.
H. Thang and S. E. Bottle, Polym. Degrad. Stab., 2005, 89, 427–435.
22 B. Miljevic, K. E. Fairfull-Smith, S. E. Bottle and Z. D. Ristovski,
Atmos. Environ., 2010, 44, 2224–2230.
23 B. J. Morrow, D. J. Keddie, N. Gueven, M. F. Lavin and S. E. Bottle,
Free Radical Biol. Med., 2010, 49, 67–76.
36 J. Mravljak, J. Konc, M. Hodoscek, T. Solmajer and S. Pecar, J. Phys.
Chem. B, 2006, 110, 25559–25561.
37 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int. Ed.,
2001, 40, 2004–2021.
38 S. Pecar, B. Sorg, M. Schara and E. Hecker, Chem. Phys. Lipids, 1984,
35, 151–159.
24 P. Meineke, U. Rauen, H. de Groot, H. G. Korth and R. Sustmann,
Chem.–Eur. J., 1999, 5, 1738–1747.
39 J. Mravljak and S. Pecar, Synth. Commun., 2004, 34, 3763–
3771.
25 P. M. Du¨ppe, P. M. Talbierski, F. S. Hornig, U. Rauen, H. G. Korth, T.
Wille, R. Boese, T. Omlor, H. de Groot and R. Sustmann, Chem.–Eur.
J., 2010, 16, 11121–11132.
40 S. Amel Diab, A. Hienzch, C. Lebargy, S. Guillarme, E. Pfund and T.
Lequeux, Org. Biomol. Chem., 2009, 7, 4481–4490.
41 L. Yu, X. Li, J. Dong, X. Zhang, Z. Guo and X. Liang, Anal. Methods,
2010, 2, 1667–1670.
´
26 E. Hideg, T. Ka´lai, P. B. Ko´s, K. Asada and K. Hideg, Photochem.
Photobiol., 2006, 82, 1211–1218.
27 P. Bilski, K. Hideg, T. Ka´lai, M. A. Bilska and C. F. Chignell, Free
Radical Biol. Med., 2003, 34, 489–495.
28 G. G. Borisenko, I. Martin, Q. Zhao, A. A. Amoscato and V. E. Kagan,
J. Am. Chem. Soc., 2004, 126, 9221–9232.
29 N. Medvedeva, V. V. Martin, A. L. Weis and G. I. Likhtenshten, J.
Photochem. Photobiol., A, 2004, 163, 45–51.
30 X.-F. Yang and X.-Q. Guo, Anal. Chim. Acta, 2001, 434, 169–177.
31 C. Aliaga, J. M. Jua´rez-Ruiz, J. C. Scaiano and A. Aspe´e, Org. Lett.,
2008, 10, 2147–2150.
32 Y. Tang, F. He, M. Yu, S. Wang, Y. Li and D. Zhu, Chem. Mater., 2006,
18, 3605–3610.
42 H. Myszka, D. Bednarczyk, M. Najder and W. Kaca, Carbohydr. Res.,
2003, 338, 133–141.
43 H. N. Lin and C. T. Walsh, J. Am. Chem. Soc., 2004, 126, 13998–
14003.
44 V. L. Campo, I. Carvalho, C. H. T. P. Da Silva, S. Schenkman, L. Hill,
S. A. Nepogodiev and R. A. Field, Chem. Sci., 2010, 1, 507–514.
45 O. Monasson, M. Ginisty, J. Mravljak, G. Bertho, C. Gravier-Pelletier
and Y. Le Merrer, Tetrahedron: Asymmetry, 2009, 20, 2320–2330.
46 H.-S. Oh, R. Xuan and H.-Y. Kang, Org. Biomol. Chem., 2009, 7,
4458–4463.
47 R. Faure´, T. C. Shiao, D. Lagnoux, D. Gigue`re and R. Roy, Org. Biomol.
Chem., 2007, 5, 2704–2708.
33 E. Lozinsky, V. V. Martin, T. A. Berezina, A. I. Shames, A. L. Weis
and G. I. Likhtenshtein, J. Biochem. Biophys. Methods, 1999, 38, 29–
42.
34 C. Tansakul, E. Lilie, E. D. Walter, F. Rivera, A. Wolcott, J. Z. Zhang, G.
L. Millhauser and R. Braslau, J. Phys. Chem. C Nanomater Interfaces,
2010, 114, 7793–7805.
48 C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67,
3057–3064.
49 A. Babic, S. Gobec, C. Gravier-Pelletier, Y. Le Merrer and S. Pecar,
Tetrahedron, 2008, 64, 9093–9100.
50 A. Makino, K. Kurosaki, M. Ohmae and S. Kobayashi, Biomacro-
molecules, 2006, 7, 950–957.
35 W. Chen, X. Wang, X. Tu, D. Pei, Y. Zhao and X. Guo, Small, 2008, 4,
759–764.
51 R. A. Vishwakarma, S. Vehring, A. Mehta, A. Sinha, T. Pomorski, A.
Herrmann and A. K. Menon, Org. Biomol. Chem., 2005, 3, 1275–1283.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 4150–4159 | 4159
©