Synthesis of Nontoxic Fluorous Sphingolipids as Molecular Probes of Exogenous Metabolic Studies for Rapid Enrichment by Fluorous Solid Phase Extraction

Article abstract of DOI:10.1002/ejoc.201601302

Fluorous solid-phase extraction (FSPE) is a useful technique for efficient selective enrichment of fluorous compounds from nonfluorous molecules. Sphingolipids and their metabolites, which are ubiquitous building blocks of eukaryotic and prokaryotic cell membranes, play crucial roles, for example, as signaling molecules. However, details of the functions and metabolic mechanisms of exogenous sphingolipids have remained unknown compared with those of their endogenous analogs. To better understand these unknown roles, chemical probes with appropriate biological and physicochemical properties are needed. In this study, we designed and synthesized new fluorous sphingolipids to reveal these roles. Furthermore, we confirmed that they could be efficiently and rapidly separated from normal sphingolipids by FSPE, and that they hardly showed any cytotoxic activity, similarly to normal sphingolipids at the same dose. We also showed that these fluorinated ceramides could act as metabolic substrates for sphingomyelin synthase 2 (SMS2). This demonstrates their potential for further biological studies.

Full text of DOI:10.1002/ejoc.201601302

A Journal of
Accepted Article
Title: Synthesis of Nontoxic Fluorous Sphingolipids as Molecular
Probes of Exogenous Metabolic Studies for Rapid Enrichment by
FSPE
Authors: Shota Saito, Yuta Murai, Seigo Usuki, Masafumi Yoshida,
Mostafa A.S. Hammam, Susumu Mitsutake, Kohei Yuyama,
Yasuyuki Igarashi, and Kenji Monde
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601302
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601302
Supported by

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
Synthesis of Nontoxic Fluorous Sphingolipids as Molecular
Probes of Exogenous Metabolic Studies for Rapid Enrichment by
FSPE
Shota Saito,[a][‡] Yuta Murai,[b][‡] Seigo Usuki,[b] Masafumi Yoshida,[a] Mostafa A.S. Hammam,[b] Susumu
Mitsutake,[b] Kohei Yuyama,[b] Yasuyuki Igarashi,[b] and Kenji Monde[b]*
Abstract: Fluorous solid phase extraction (FSPE) is one of the useful
techniques for efficient selective enrichment of fluorous compounds
from nonfluoros molecules. Sphingolipids and their metabolites, which
are ubiquitous building blocks of eukaryotic and prokaryotic cell
membranes, play crucial roles such as signaling molecules. However,
details of the functions and metabolic mechanisms of exogenous
sphingolipids have remained unknown compared with that of
endogenous. To better understand the veiled roles, chemical probes
with appropriate biological and physicochemical properties are
needed. In this study, we designed and synthesized novel fluorous
sphingolipids to reveal their roles. Furthermore, we confirmed that
they could be efficiently and rapidly separated from normal
sphingolipids by FSPE and that they scarcely exhibited cytotoxic
activities as what normal sphingolipids did at the same dose. Also, we
demonstrated that these fluorinated ceramides could be metabolic
substrates utilizing sphingomyelin synthase 2 (SMS2) showing their
potential for further biological studies.
that achievement for analysis of protein-sphingolipid interactions
by using photoreactive and clickable analog of sphingosine.[8]
The use of fluorine chemistry involving trifluoromethyl groups or
perfluoroalkyl chains (fluorous compounds) with biological
properties is a well-known new strategy for application to
medicinal chemistry.[9] In addition, substitution of hydrogen by
fluorine cause scarcely steric effects despite their different van der
Waals radii.[10] Recently, applications of fluorous molecules to
various biological techniques have been increasing. For instance,
it has been applied for fluorous metabolomics[11], fluorous
microarrays[12], fluorous delivery systems[13], and fluorous photo
cross-linker.[14] Moreover, fluorous compounds can be rapidly and
efficiently separated from non-fluorous compounds with selective
retention on fluorous silica gel.[15] Separation of fluorous
peptides[16], oligosaccharides[17], and oligonucleotides[18] from
non-fluorous counterparts was also performed by fluorous solid
phase extraction (FSPE).
Introduction
Sphingolipids (SLs), which have common backbone sphingoid
bases of sphingosine, sphinganine and phytosphingosine, are
ubiquitous membrane components of all eukaryotic and
prokaryotic cells.[1] SLs have been reported to they play crucial
roles in many physiological processes, and their metabolites are
well known as bioactive compounds called lipid mediators that
regulate cell growth[2], apoptosis[3], migration, angiogenesis and
immune responses.[4] In addition, it has recently reported that they
are expected to be new pharmaceutical targets against metabolic
syndrome and Alzheimer’s disease.[5] Although these
mechanisms of endogenous SLs have gradually been elucidated
by mutations or knockouts in genes that encode for enzymes
involved in a specific metabolic step, details of the metabolic fate
of exogenous SLs are scarce. Recently, there have been a few
studies in which the function and metabolic mechanism of
exogenous SLs were investigated by using chemical probes
bearing a fluorescence tag[6] or using radioisotope labeling.[7]
(Fig.1) Those strategies are useful but have some problems such
as loss of original biological activity and affinity for the target
biomolecules or the requirement of special equipment. On the
other hand, recently Haberkant and Schults et al. has reported
[a] Graduate School of Life Science, Hokkaido University, Kita 21 Nishi 11,
Sapporo 001-0021, Japan
[b] Faculty of Advanced Life Science, Hokkaido University, Kita 21 Nishi
11, Sapporo 001-0021, Japan
E-mail: kmonde@sci.hokudai.ac.jp.
URL: http://altair.sci.hokudai.ac.jp/infchb/
[‡] These authors contributed equally to this work.
Supporting information for this article are available on the WWW under
http://********************.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
Fig. 1. Structures of sphingolipids labeled with a fluorescent tag (2), (3) and a
radioisotope tag (4) in previous studies and labeled with a fluorous tag (5) in this
study.
Scheme 1. Synthesis of perfluoroalkyl aldehydes (15 and 16).
Therefore, fluorous SLs (5) are useful for investigating the
functions and metabolic mechanisms of exogenous SLs. Here,
we describe the efficient synthesis of enantiopure D-erythro
fluorous SLs and sphinganine as the upstream precursor of SLs.
Furthermore, we also show that they scarcely exhibited
cytotoxicity and that they were efficiently separated from mixed
nonfluorous SLs by FSPE.
Next, we applied compound (9) and compounds (15, 16) to the
synthesis of D-erythro fluorous C18-sphingosine, which is one of
the essential components of SLs. Trans-selective double bond
formation with -ketophosphonate (9) and perfluoroalkyl
aldehydes (15, 16) were performed by modified HWE olefination
with K2CO3 in THF/H2O to obtain oxazolidine-3-keto fluorous
sphingosines (17, 18) in good yield (80%). Subsequently,
compound (17, 18) were subjected to diastereoselective
reduction by using Zn(BH4)2 at -78 oC and then at 0 oC to obtain
oxazolidine D-erythro fluorous C18-sphingosines (19, 20) in over
90% d.e., due to the high chelating ability of the Zn cation.[22] The
desired D-erythro fluorous SLs (23, 24) were synthesized in high
yield (90%) by deprotecting the oxazolidine moiety in compounds
(21, 22) with 1 M HCl / THF under reflux followed by acylation of
the amino group with hexanoyl chloride.
Results and Discussion
An important point in our synthesis of fluorous SLs and
sphinganine is the installation of perfluorinated aliphatic chains
(fluorous) instead of alkyl chains. There has only been a report on
the synthesis of fluorous C11,12,13-sphingosine mimics[8e], and the
synthesis of fluorous C18-sphingosine, SLs, and sphinganine has
not been reported so far. Additionally, their synthesis using olefin
metathesis remains a challenge because of poor yield, small-
scale reaction, and requirement of precious catalysts. To solve
these problems, Horner-Wadsworth-Emmons (HWE) olefination
is the most useful method to construct a sphingoid base skeleton
with inexpensive reagents. D-Erythro fluorous SLs and
sphinganine were synthesized as reported by Inazu et al. and
Koskinen et al.[19] with slight modification.
D-erythro fluorous C18-sphinganine (25) was also obtained in
moderate yield (60%) from compound (19) by reduction of the
alkene moiety with Pd/C in the presence of H2 atmosphere
followed by deprotection of the oxazolidine group under an acidic
condition. (Scheme 2)
As shown in Scheme 1, our synthesis started with commercially
available L-serine, and this was sequentially protected by Me
ester for the carboxylic acid group and Boc for the amino group to
obtain Boc-(S)-Ser-OMe (7) in a quantitative yield. Subsequently,
the alcohol moiety and carbamate group reacted with 2,2-
dimethoxypropane under an acidic condition to afford oxazolidine
(8) in excellent yield (97%). Compound (8) was transformed to the
corresponding -ketophosphonate (9) by treatment with excess
lithium dimethyl methylphosphonate at -78 °C (75%).
On the other hand, perfluorinated aliphatic chains (fluorous)
were synthesized from commercially available homoallylic alcohol
(10). Compound (10) was first subjected to radical addition with
iodide perfluoroalkyls (11, 12) by the radical initiator AIBN under
a neat condition, and this was followed by reductive deiodination
[20]
by LiAlH4 instead of Bu3SnH under a mild condition to obtain
perfluoroalkyl alcohols (13, 14) in two sequential steps in high
yield (80%). Additionally, Ni-catalyzed reductive deiodination
[21]
reported by Hájek et al.
could be also useful for these
perfluoroalkyl alcohols. Finally, perfluoroalkyl aldehydes (15, 16)
were created by oxidation of those alcohols with IBX in
DMSO/THF at room temperature under a mild condition. (Scheme
1)
Scheme 2. Synthesis of D-erythro fluorous SLs (23, 24) and sphinganine (25).
Subsequently, we evaluated the cytotoxicity of D-erythro fluorous
SLs (23, 24) and sphinganine (25) towards NIH3T3 cells and B16
cells. Cell viability was measured by using a cell counting kit-8
(CCK-8).[23] As shown in Fig. 2, all of the fluorous compounds
exhibited comparable cytotoxicity towards the cells as what
normal SLs and sphinganine did at the same dose (positive
controls). These results indicate that fluorous SLs (23, 24) and
sphinganine (25) are expected to be useful as metabolic
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
substrates and can assure their potential for further biological
studies.
(4.3 min). In Fig. 3, these results indicated that rapid and easy
separation of fluorous SLs from non-fluorous SLs by using FSPE
as well as the possibility of separating fluorous SLs bearing
different fluorous tags of different lengths.
NIH3T3 cell
OH
OH
OH
140"
HO
(CH2)12CH3
(CH2)4Rf8
(CH2)6Rf6
HN
C5H11
HO
HO
120"
100"
(×102
)
normal SLs
(24)
HN
C5H11
HN
C5H11 (23)
O
O
O
normal SLs
80"
60"
40"
20"
0"
compound (23)
compound (24)
Fig. 3. Fluorous LC-MS condition: the mobile phase comprised solvent (A)
MeCN/H2O/formic acid (20:80:0.1) and solvent (B) MeCN/i-PrOH/formic acid
(20:80:0.1) was performed in linear gradient program: 50% (B) 0.0-1.2 min.,
50%→80% (B) 1.2-3.0 min., 80% (B) 3.0–4.2 min., 80→90% (B) 4.2-6.0 min.,
flow: 0.3 mL/min.) .
1"
5"
10"
30"
50"
Compound concentration (mM)
B16 cell
120"
100"
80"
60"
40"
20"
0"
Finally, we examined whether compound 24 is available as the
substrate of sphingomyelin synthase 2 (SMS2), which exists in
lipid microdomains and partially associates with the fatty acid
transporter CD36/FAT.[24] Sphingomyelin (SM) has particularly
important roles in the formation of microdomains of the plasma
membrane involved in many cellular processes. The utility as a
substrate of fluorous SLs were measured by cell-based method[26]
using synthetic fluorescent normal and fluorous SLs attached
NBD (26, 28). Since this method has been recently established
and it is quite facile and reliable to confirm whether compound 24
becomes the substrate of SMS2 or not, we adopted it. The
metabolite assay revealed that synthetic compound (28) could be
metabolized to fluorous sphingomyelin (29).
normal SLs
compound (23)
compound (24)
1"
5"
10"
30"
50"
Compound concentration (mM)
NIH3T3 cell
120"
100"
80"
60"
40"
20"
0"
normal
sphinganine
compound (25)
OH
OH
O
O
HO
(CH2)12CH3
N
P
O
O
(CH2)12CH3
HN
N
H
HN
1"
5"
10"
30"
50"
N
H
NO2
O
NO2
Compound concentration (mM)
O
N
N
N
O
N
O
NBD-SLs
(26)
NBD-SM
(27)
B16 cell
120"
100"
80"
60"
40"
20"
0"
1000
normal
sphinganine
compound (25)
0
1
2
3
Retention time [min]
1"
5"
10"
30"
50"
Compound concentration (mM)
Fig. 2. Viability assays of NIH3T3 cells and B16 cells exposed to fluorous
compounds (23), (24), (25) and control normal SLs and sphinganine.
Next, fluorous SLs (23, 24) were subjected to analytical
separation by using fluorous LC-MS as shown in Fig. 3.
Compounds (23, 24) and normal SLs (control) were mixed, and
the mixture was injected on a commercial fluofix-II 120E analytical
column.[24] Firstly, normal SLs (control) emerged at 1.4 min, and
then fluorous SLs were eluted smoothly in order of their fluorous
content from (23): Rf6 = C6F13 (3.9 min) through (24): Rf8 = C8F17
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
Treatment of 5-hexen-1-ol (1.10 g, 11.0 mmol) and
heptadecafluoro-1-iodooctane (6.0 g, 11.0 mmol) was carried out
as described above to afford 14 (4.52 g, 79 %) as a white
amorphous solid. Analytical data were identical to those reported
in the literature.[27]
OH
(CH2)4Rf8
HO
HN
N
H
NO2
O
(28)
N
N
O
OH
O
O
O
N
P
(CH2)4Rf8
9,9,10,10,11,11,12,12,13,13,14,14,14-
tridecafluorotetradecanal (15)
O
O
1000
HN
(29)
N
H
NO2
N
N
O
Perfluoroalkyl alcohol 13 (2.24 g, 5.00 mmol) and IBX (2.80 g,
10.0 mmol) in DMSO (20 mL) and THF (20 mL) were stirred at
room temperature for 3 h. The THF was removed on a rotary
evaporator, added Et2O/water, then the insoluble material was
removed by filteration. The filtrate was extracted with Et2O. The
organic phase was washed with saturated NaHCO3 and brine and
dried with MgSO4, and the solvents were evaporated. The residue
was purified with SiO2 column chromatography (n-hexane/EtOAc
= 20:1) to afford 15 as a clear liquid (1.72 g, 77 %). 1H NMR (500
MHz, CDCl3):  = 9.77 (t, J= 1.6 Hz, 1H, CHO), 2.44 (dt, J = 7.3,
1.6 Hz, 2H, CH2CHO), 2.10–2.00 (m, 2H, CF2CH2), 1.66–1.59 (m,
0
1
2
3
Retention time [min]
Fig. 4. LC-MS condition: YMC-Pack Diol-120-NP (4.0 x 50mm), 2-propanol/n-
hexane/H2O = 52:40:8, flow rate 1 mL/min, fluorescent detector set to excitation
and emission wavelength of 470nm and 530nm.
4H,
CF2CH2CH2,
CH2CH2CHO),
1.41–1.35
(m,
6H,
Conclusions
CH2CH2CH2CH2CH2CHO) ppm; 13C NMR (125 MHz, CDCl3)  =
2
202.54 (CHO), 43.81, 30.85 (t, JC,F= 22.4 Hz, CF2CH2), 29.16,
In summary, we have described an efficient pathway for the
synthesis of perfluoroalkyl (fluorous) C18-sphingosine, SLs, and
sphinganine based on Horner-Wadsworth-Emmons olefination. A
cytotoxicity activity test by the CCK-8 assay showed that the
prepared compounds scarcely exhibited cytotoxicity towards the
cells as did normal SLs and sphinganine. In addition, they could
be readily and rapidly separated from non-fluorous SLs by FSPE.
Furthermore, we proved that in our synthesis, the fluorous SLs
could be chemical probes to elucidate those physiological
processes.
29.14, 29.01, 21.92, 20.05 ppm. HRMS (EI): calcd. for [M+H]+
C14H17F13O 446.0915, found 446.0916.
7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,14-
heptadecafluorotetradecanal (16)
Treatment of alcohol 14 (2.60 g, 5.00 mmol) was carried out as
described above to afford 16 (2.36 g, 91 %) as a white amorphous
solid. 1H NMR (500 MHz, CDCl3):  = 9.78 (s, 1H. CHO), 2.48 (t,
J = 7.3 Hz, 2H, CH2CHO), 2.13–2.02 (m, 2H, CF2CH2), 1.71–1.61
(m, 4H, CH2CH2CHO, CF2CH2CH2), 1.46–1.40 (m, 2H,
CF2CH2CH2CH2) ppm. 13C NMR (125 MHz, CDCl3):  = 201.96,
43.52, 30.67 (t, 2JC,F = 22.3 Hz), 28.55, 21.60, 20.00 (t, 2JC,F = 3.7
Hz). HRMS (APCI): calcd. for C14H10F17O [M+H]+ 517.04657,
found 517.04742.
Experimental Section
9,9,10,10,11,11,12,12,13,13,14,14,14-tridecafluoro-1-
tetradecanol (13)
(S,E)-tert-butyl
2,2-dimethyl-4-
(11,11,12,12,13,13,14,14,15,15,16,16,16-
tridecafluorohexadec-2-enoyl)oxazolidine-3-carboxylate (17)
7-octen-1-ol (1.70 g, 13.3 mmol), tridecafluoro-1-iodon-hexane
(6.50 g, 14.6 mmol), and AIBN (218 mg, 1.33 mmol) were stirred
for 4 h at 95 °C. The reaction was cooled to room temperature
and dissolved in dry Et2O (50 mL). To the reaction mixture was
added LiAlH4 (1.00 g, 26.4 mmol) in dry Et2O (5 mL) at 0 °C. The
reaction mixture was allowed to warm to room temperature and
stirred for 24 h. The mixture was cooled to 0 °C again, then
quenched with water (1 mL), NaOH (15 % w/v; 1 mL). The mixture
was filtered through Celite® and concentrated. The residue was
purified by FluoroFlash® solid-phase extraction (MeOH/H2O, 3:1
to acetone) and SiO2 column chromatography (n-hexane/EtOAc
= 9:1 to 7:1 to 4:1) to afford 13 as a white amorphous solid (4.75
g, 80 %). 1H NMR (500 MHz, CDCl3):  = 3.64 (q, J = 5.9 Hz, 2 H,
CH2OH), 2.10-1.99 (m, 2 H, CF2CH2), 1.63–1.55 (m, 4 H,
CH2CH2OH, CF2CH2CH2), 1.46 (t, J = 5.3 Hz, 1 H, OH), 1.38–1.35
(m, 8 H,CF2CH2CH2CH2CH2CH2) ppm. 13C NMR (125 MHz,
CDCl3):  = 62.93, 32.69, 30.86 (t, 2JC,F = 22.4 Hz, CF2CH2), 29.16,
29.14, 29.01, 25.64, 20.06 ppm. HRMS (ESI): calcd. for
C14H17F13ONa [M+Na]+ 471.0970, found 471.0967.
Perfluoroalkyl aldehyde 15 (1.68 g, 3.76 mmol) and -
ketophosphonate (1.52 g, 4.33 mmol) were dissolved in THF (15
mL) and water (15 mL) and cooled to 0 °C. K2CO3 (1.82 g, 13.2
mmol) was added and the reaction mixture was allowed to warm
to room temperature and stirred for 72 h. The reaction mixture
was acidified with citric acid solution and extracted with Et2O. The
organic phase was washed with NaHCO3, brine, dried with
MgSO4, and solvents were evaporated. The crude product was
purified by SiO2 column chromatography (n-hexane/EtOAc = 30:1
to 10:1) to afford 17 as a white amorphous solid (2.01 g, 79 %).
1H NMR (500 MHz, CDCl3, mixture of rotamers):  = 7.00–6.93
(m, 1H, COCH=CH), 6.33–6.23 (m, 1H, COCH=CH), 4.70–4.68,
4.51–4.49 (m, 1H, CHN), 4.20–4.16, 3.97–3.90 (m, 2H,
CHNCH2O), 2.27–2.21 (m, 2H, CH=CHCH2), 2.07–2.01 (m, 2H,
CF2CH2), 1.71(s), 1.65 (s), 1.60–1.46 (m), 1.41–1.35 (m) ppm. 13
NMR (125 MHz, CDCl3, mixture of rotamers):  = 196.67, 149.34,
C
126.02, 125.25, 95.15, 94.48, 80.84, 80.51, 65.92, 64.25, 63.94,
2
32.65, 30.88 (t, JC,F= 22.6 Hz, CF2CH2), 29.04, 29.01, 28.97,
7,7,8,8,9,9,10,10,11,11,12,12,13,13,14,14,14-heptadecafluoro-
1-tetradecanol (14)
28.39, 28.26, 27.95, 27.85, 26.10, 25.28, 25.15, 24.13, 20.12 ppm.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
HRMS (ESI): calcd. for C26H34F13NO4Na [M+Na]+ 694.2178, found
694.2162.
29.13, 28.78, 27.15, 24.75, 20.71 ppm. HRMS (ESI): calcd. for
C26H32F17NO4Na [M+Na]+ 768.19521, found 768.19528.
(S,E)-tert-butyl
2,2-dimethyl-4-
(2S,3R,E)-2-amino-13,13,14,14,15,15,16,16,17,17,18,18,18-
(9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-
heptadecafluorohexadec-2-enoyl)oxazolidine-3-carboxylate
(18)
tridecafluorooctadec-4-ene-1,3-diol (21)
Compound 19 (2.10 g, 3.12 mmol) in THF (50 mL) and 1M HCl
(50 mL) was stirred at 80 °C for 18 h. The THF was removed on
a rotary evaporator, and then the residue was dissolved in EtOAc.
The solution was basified with 1 M NaOH, then it was extracted
with EtOAc. The organic phase was washed with brine, dried with
Na2SO4, and the solvent was evaporated. The crude product was
purified by SiO2 column chromatography (CHCl3/MeOH = 9:1,
CHCl3/MeOH/NH3 aq. = 135:25:4) to afford 21 as a white
amorphous solid (1.41 g, 85 %). 1H NMR (500 MHz, CD3OD):  =
5.79–5.68 (m, 1H, CH=CH), 5.51 (dd, J = 15.3, 7.4 Hz, 1H,
CH=CH), 3.98 (t, J = 6.5 Hz, 1H, CHOHCH=CH), 3.68 (dd, J =
10.8, 4.5 Hz, 1H, CH2OH), 3.50 (dd, J = 11.0, 6.9 Hz, 1H, CH2OH),
2.76 (td, J = 6.3, 4.5 Hz, 1H, CHNH2), 2.22–2.06 (m, 4H, CF2CH2,
CH=CHCH2), 1.61 (quin, J = 7.6 Hz, 2H, CF2CH2CH2), 1.49–1.40
(m, 4H), 1.40–1.32 (m, 4H) ppm. 13C NMR (125 MHz, CD3OD): 
= 135.26, 131.04, 75.19, 64.43, 58.18, 33.53, 31.89 (t, 2JC,F = 22.4
Hz, CF2CH2), 30.42, 30.40, 30.26, 30.24, 21.44 ppm. HRMS
(ESI): calcd. for C18H25F13NO2 [M+H]+ 534.16722, found
534.16730.
Treatment of perfluoroalkyl aldehyde 16 (2.85 g, 5.50 mmol) was
carried out as described above to afford 18 (3.23 g, 79 %) as a
white amorphous solid. 1H NMR (500 MHz, CDCl3, mixture of
rotamers):  = 7.01–6.90 (m, 1H, COCH=CH), 6.38–6.23 (m, 1H,
COCH=CH), 4.68–4.67, 4.50–4.49 (m, 1H, CHN), 4.20–4.14,
3.97–3.90 (m, 2 H, CHNCH2O), 2.28–2.26 (m, 2H, CH=CHCH2),
2.14–1.97 (m, 2H, CF2CH2), 1.71 (s), 1.65 (br. s), 1.63 (br. s), 1.56
(s), 1.52 (br. s), 1.50 (s), 1.43 (br. s), 1.37 (s) ppm. 13C NMR (125
MHz, CDCl3, mixture of rotamers):  = 196.54, 195.72, 165.02,
152.26, 151.40, 148.62, 126.11, 125.39, 95.11, 94.44, 80.81,
80.46, 65.83, 65.41, 64.24, 63.95, 32.31, 30.73 (t, 2JC,F = 22.4 Hz),
28.61, 28.30, 28.18, 27.65, 27.52, 26.05, 25.23, 25.06, 24.03,
19.95 ppm. HRMS (ESI): calcd. for C26H30F17NO4Na [M+Na]+
766.17956, found 766.17950.
(S)-tert-butyl
2,2-dimethyl-4-((R,E)-
11,11,12,12,13,13,14,14,15,15,16,16,16-tridecafluoro-1-
hydroxyhexadec-2-enyl)oxazolidine-3-carboxylate (19)
(2S,3R,E)-2-amino-
11,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-
heptadecafluorooctadec-4-ene-1,3-diol (22)
Oxazolidine-3-keto 17 (2.56 g, 3.81 mmol) was dissolved in dry
Et2O (40 mL) and cooled to -78 °C. Freshly prepared zinc
borohydride solution in dry Et2O (0.2 M, 20 mL) was added
dropwise. The reaction mixture was allowed to warm to -30 °C
and stirred for 1 h, then warm to 0 °C and stirred for 1 h. The
reaction was quenched with 1M HCl and extracted with Et2O. The
organic phase was washed with NaHCO3, brine, dried with
MgSO4, and solvents were evaporated. The crude product was
purified by SiO2 column chromatography (n-hexane/EtOAc = 9:1
to 4:1) to afford 19 as a clear oil (2.27 g, 88 %). 1H NMR (500 MHz,
C6D6, 75 °C):  = 5.79–5.73 (m, 1H, CHOHCH=CH), 5.55 (dd, J =
5.9, 15.45 Hz, 1H, CHOHCH=CH), 4.31-4.29 (m, 1H, CHOH),
3.96 (br, 1H, CHN), 3.81 (br, 1H, CHNCH2O), 3.72–3.69 (m, 1H,
CHNCH2O), 2.02–1.97 (m, 2H, CH=CHCH2), 1.85–1.75 (m, 2H,
CF2CH2), 1.62 (s), 1.47–1.40 (m), 1.40-1.38 (m), 1.32–1.30 (m),
1.20–1.17 (m), 1.12–1.05 (m) ppm. 13C NMR (125 MHz, C6D6,
75 °C):  = HRMS (ESI): calcd. for C26H34F13NO4Na [M+Na]+
696.2334, found 696.2326.
Treatment of compound 20 (1.67 g, 2.24 mmol) was carried out
as described above to afford 22 (1.19 g, 88 %) as a white
amorphous solid. 1H NMR (500 MHz, CD3OD):  = 5.79–5.69 (m.
1H, CHOHCH=CH), 5.52 (dd,
J = 15.4, 7.4 Hz, 1H,
CHOHCH=CH), 3.99 (t, J = 6.6 Hz, 1H, CHOH), 3.68 (dd, J = 10.6,
4.5 Hz, 1H, CH2OH), 3.50 (dd, J = 11.0, 6.8 Hz, 1H, CH2OH), 2.77
(td, J = 6.4, 4.5 Hz, 1H, CHNH2), 2.23–2.07 (m, 4H, CF2CH2,
CH=CHCH2), 1.67–1.57 (m, 2H, CF2CH2CH2), 1.52-1.38 (m, 4H)
ppm. 13C NMR (125 MHz, CD3OD):  = 134.88, 131.33, 75.18,
2
64.47, 58.17, 33.28, 31.87 (t, JC,F = 21.9 Hz, CF2CH2), 30.10,
29.80, 21.31 ppm. HRMS (ESI): calcd. for C18H21F17NO2 [M+H]+
606.12953, found 606.12981.
N-((2S,3R,E)-13,13,14,14,15,15,16,16,17,17,18,18,18-
tridecafluoro-1,3-dihydroxyoctadec-4-en-2-yl)hexanamide
(23)
(S)-tert-butyl
2,2-dimethyl-4-((R,E)-
9,9,10,10,11,11,12,12,13,13,14,14,15,15,16,16,16-
heptadecafluoro-1-hydroxyhexadec-2-enyl)oxazolidine-3-
carboxylate (20)
Compound 21 (500 mg, 0.937 mmol) and NaOAc (3.08 g, 37.5
mmol) were dissolved in THF (10 mL) and water (5 mL). Hexanoyl
chloride (0.20 mL, 1.46 mmol) was added. The reaction mixture
was stirred for 18 h at room temperature. The reaction mixture
was extracted with EtOAc, then the organic phase was washed
with NaOH (1 M), brine, dried with MgSO4, and solvents were
evaporated. The crude product was purified by SiO2 column
chromatography (n-hexane/EtOAc = 1:1 to 1:4) to afford 23 (532
mg, 90 %) as a white amorphous solid. 1H NMR (500 MHz,
CDCl3):  = 6.40 (d, J = 7.6 Hz, 1H, NH), 5.81–5.73 (m, 1H,
CHOHCH=CH), 5.53 (dd, J = 15.4, 6.4 Hz, 1H, CHOHCH=CH),
4.28 (d, J = 4.5 Hz, 1H, CHOH), 3.95–3.86 (m. 2H, CHNH,
CH2OH), 3.69 (td, J = 7.1, 3.4 Hz, 1H, CH2OH), 3.50 (d, J = 5.4
Hz, 1H, CHOH), 3.43 (dd, J = 6.7, 3.8 Hz, 1H, CH2OH), 2.22 (t, J
= 7.9 Hz, 2H, COCH2), 2.12–1.98 (m, 4H, CF2CH2, CH=CHCH2),
1.68–1.55 (m, 4H), 1.42–1.26 (m, 14 H), 0.90 (t, J = 6.9 Hz, 3H,
Treatment of Oxazolidine-3-keto 18 (3.01 g, 4.05 mmol) was
carried out as described above to afford 20 (2.66 g, 88 %) as a
white amorphous solid. 1H NMR (500 MHz, C6D6, 75 °C):  =
5.76–5.66 (m, 1H, CHOHCH=CH), 5.53 (dd, J = 15.2, 5.3 Hz, 1H,
CHOHCH=CH), 4.30 (br. s., 1H, CHOH), 3.97 (br. s., 1H), 3.78
(br. s., 1H), 3.73–3.64 (m, 1H), 1.92 (q, J = 7.0 Hz, 2H,
CH=CHCH2), 1.86–1.72 (m, 2H, CF2CH2), 1.62 (br. s), 1.45 (br.
s), 1.42–1.31 (m), 1.20 (quin, J = 7.4 Hz, 2H, CH=CHCH2CH2),
1.13–1.02 (m, 2H, CH=CHCH2CH2CH2) ppm. 13C NMR (125 MHz,
C6D6, 75 °C):  = 165.88, 132.25, 130.98, 95.03, 80.65, 74.24,
2
65.35, 63.16, 32.69, 31.63 (t, JC,F = 22.6 Hz, CF2CH2), 29.39,
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
CH3) ppm. 13C NMR (125 MHz, CDCl3):  = 174.12, 133.76,
was dosed in the presence of 0.5 % DMSO. After 24 hours, 10μl
of Cell Counting Kit-8 Solution (obtained from Dojindo Molecular
Technologies, Inc.) was added. After 4 hours, Absorbance of
visible light (wavelength: 450nm) was measured and cell viability
was calculated as following formula:
128.97, 74.22, 62.28, 54.62, 36.75, 32.21, 31.40, 30.63 (t, 2JC,F
=
22.3 Hz, CF2CH2), 29.07, 29.02, 28.95, 25.42, 22.35, 20.07, 13.85
ppm. HRMS (ESI): calcd. for C24H34F13NO3Na [M+Na]+ 654.22233,
found 654.22232.
A A
s
b
Viability (%) 
100
A A
N-((2S,3R,E)-
c
b
11,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-
heptadecafluoro-1,3-dihydroxyoctadec-4-en-2-
yl)hexanamide (24)
As: Absorbance of sample well, Ac: Absorbance of Negative control
well (concentration : 0 μM with cells), Ab: Absorbance of blank well
(No lipids and cells, only medium).
Treatment of compound 22 (500 mg, 0.826 mmol) was carried
out as described above to afford 24 (523 mg, 90 %) as a white
amorphous solid. 1H NMR (500 MHz, CDCl3): = 6.39 (d, J = 7.4
Hz, 1H, NH), 5.80–5.74 (m, 1H, CHOHCH=CH), 5.54 (dd, J = 15.4,
6.4 Hz, 1H, CHOHCH=CH), 4.29 (br. s., 1H, CHOH), 3.97–3.86
(m, 2H, CHNH, CH2OH), 3.69 (d, J = 9.1 Hz, 1H, CH2OH), 3.46
(br. s., 1H, CHOH), 3.34 (br. s., 1H, CH2OH), 2.22 (t, J = 7.6 Hz,
2H, COCH2), 2.14–1.97 (m, 4H, CF2CH2, CH=CHCH2), 1.62 (m,
4H), 1.37–1.49 (m, 4H), 1.25–1.37 (m, 4H), 0.90 (t, J = 6.9 Hz, 3H,
CH3) ppm. 13C NMR (125 MHz, CDCl3): = 174.11, 133.25,
Cell-based assay using SMS2
SMS2-expressing cell lysates were prepared according to
previous method. [25] Lysates (20 mM Tris-buffer, 100 L) and
compound 26 and 28 (10 M as total concentration) were
incubated for 3 hours at 37°C, then fluorescent lipids were
extracted from lysates by the Bligh-Dyer method, and directly
applied to HPLC.
129.34, 74.21, 62.27, 54.58, 36.76, 31.98, 31.40, 30.77 (t, 2JC,F
=
22.3 Hz, CF2CH2), 28.74, 28.62, 25.43, 22.36, 19.97, 13.84 ppm. Acknowledgements
HRMS (ESI): calcd. for C24H30F17NO3Na [M+Na]+ 726.18464,
found 726.18502.
This work was partially supported by the Innovation COE
Project for Future Medicine and Medicinal Research, and Grants
in Aid for Scientific Research (24310151 and 15H03111 to K.M.
and 15K16552 to Y.M.)
(2S,3R)-2-amino-13,13,14,14,15,15,16,16,17,17,18,18,18-
tridecafluorooctadecane-1,3-diol (25)
To a solution of compound 19 (654 mg, 0.971 mmol) in EtOH (5
mL) was added Pd/C (65 mg) and vigorously stirred under H2
atmosphere. After 3 h, the solution was filtered by Celite® and the
filtrate was evaporated. To this residue was added MeOH (10 mL)
and 2 M aqueous HCl solution (0.5 mL) and stirred under reflux.
After 2 h, the solvent was evaporated, and residue was purified
Keywords: perfluoroalkyl chain • fluorous • sphingolipid • lipid
mediator • fluorous solid phase extraction
[1]
[2]
A. H. Merrill, Jr., M. D. Wang, M. Park, M. C. Sullards, Trends in
Biochemical Sciences 2007, 32, 457–468.
by SiO2 column chromatography (CHCl3:MeOH:NH4OH
=
a) A. Schwarz, E. Rapaport, K. Hirschberg, A. H. Futerman, J. Biol. Chem.
1995, 270, 10990-10998.; b) O. Cuvillier, G. Pirianov, B. Kleuser, P. G.
Vanek, O. A. Coso, J. S. Gutkind, S. Spiegel, Nature 1996, 381, 800-
803.; c) A. Schwarz, A. H. Futerman, J. Neurosci. 1997, 17, 2929-2938.;
d) M. Maceyka, S. Milstien, S. Spiegel, J. Lipid Res. 2009, 50, S272-
S276.; e) H. Fyrst, J. D. Saba, Nat. Chem. Biol. 2010, 6, 489-497.
a) L. M. Obeid,C. M. Linardic, L. A. Karolak, Y. A. Hannun, Science 1993,
259, 1769-1771.; b) T. Coetzee, N. Fujita, J. Dupree, R. Shi, A. Blight, K.
Suzuki, K. Suzuki, B. Popko, Cell 1996, 86, 209-219.; c) B. J. Pettus, C.
E. Chalfant, Y. A. Hannun, Biochim. Biophys. Acta 2002, 1585, 114-125.;
d) T. A. Taha, T. D. Mullen, T. D., L. M. Obeid, Biochim. Biophys. Acta
2006, 1758, 2027-2036.; e) A. Jana, E. L. Hogan, E. L., K. Pahan, J.
Neurol. Sci. 2009, 278, 5-15.
60:10:1) to afford 25 (326 mg, 63 %) as a white amorphous solid.
1H NMR (500MHz, CD3OD)  = 3.73 (dd, J = 4.3, 10.8 Hz, 1H,
CH2OH), 3.53–3.51 (m, 1H, CHOH), 3.47 (dd, J = 7.6, 11.1 Hz,
1H, CH2OH), 2.73–2.70 (m, 1H, CHNH2), 2.19–2.08 (m, 2H,
CF2CH2), 1.63–1.59 (m, 2H, CF2CH2CH2), 1.59–1.54 (m, 2H,
CHOHCH2), 1.45–1.35 (m, 16H) ppm. 13C NMR (125 MHz,
CD3OD)  = 74.17, 64.50, 58.31, 34.54, 31.90 (t, 2JC,F = 22.2 Hz,
CF2CH2), 30.90, 30.83, 30.66, 30.54, 30.29, 27.18, 21.43 ppm.
HRMS (ESI): calcd. for C18H26F13NO2 [M+H]+ 536.1834, found
536.1829.
[3]
Construction of cell culture
[4]
[5]
T. Hla, Semin. Cell Dev. Biol. 2004, 15, 513-520.
Cells were cultured in Dulbecco’s Modified Eagle’s Medium
(high glucose, -D6429- with 4500 mg/L glucose, L-glutamine,
sodium pyruvate, and sodium bicarbonate, obtained from
SIGMA) with 10 % fetal bovine serum (obtained from Gibco),
50U/mL penicillin and 50 mg/mL streptomycin (obtained from
SIGMA). All cells except for cell viability assay were cultivated in
10-cm dishes with 10mL of medium at 37 °C under a 5% CO2
humidified atmosphere.
K. Yuyama, S. Mitsutake, Y. Igarashi, Biochim. Biophys. Acta 2014, 1841,
793-798.
[6]
a) P. Bandhuvula, H. Fyrst, J. D. Saba, J. Lipid Res. 2007, 48, 2769-
2778.; b) Y. M. Lee, C. Lim, H. S. Lee, Y. K. Shin, K.-O. Shin, Y.-M. Lee,
S. Kim, Bioconjugate Chem. 2013, 24, 1324-1331.
[7]
[8]
V. Chigorno, M. Sciannamblo, J. Mikulak, A. Prinetti, S. Sonnino,
Glycoconj. J. 2006, 23, 159-165.
P. Haberkant, F. Stein, D. Höglinger, M. J. Gerl, B. Brügger, P. P. van
Veldhoven, J. Krijgsveld, A-C. Gavin, C. Schultz, ACS Chem. Biol. 2016,
11, 222-230.
Cell viability assay (Cell Counting Kit-8 assay)
[9]
a) H.-J. Böhm, D. Banner, S. Bendels, M. Kansy, B. Kuhn, K. Müller, U.
Obst-Sander, M. Stahl, ChemBioChem 2004, 5, 637-643.; b) C. Isanbor,
D. O’Hagan, J. Fluor. Chem. 2006, 127, 303-319.; c) J.-P. Bégué, D.
Bonnet-Delpon, J. Fluor. Chem. 2006, 127, 992-1012.; d) K. L. Kirk, J.
Cell viability assay was conducted as follows; NIH3T3 cell and
B16 cell were basically plated and incubated for 24 hours on 96-
well plate, which number of cells were 5000. Then, each lipid
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
Fluor. Chem. 2006, 127, 1013-1029.; e) E. Prchalová, I. Votruba, M.
Kotora, J. Fluor. Chem. 2012, 141, 49-57.; f) E. Prchalová, O. Stepánek,
S. Smrcek, M. Kotora, Future Med. Chem. 2014, 6, 1201-1229.
[10] a) M. Schlosser, D. Michel, Tetrahedron 1996, 52, 99-108.; b) D.
O'Hagan, H. S. Rzepa, Chem. Commun. 1997, 645-652.; c) K. Park, N.
R. Kitteringham, P. M. O'Neill, Annu. Rev. Pharmacol. Toxicol. 2001, 41,
443-470.; d) B. E. Smart, J. Fluorine Chem. 2001, 109, 3-11.; e) J. A.
Olsen, D. W. Banner, P. Seiler, U. Obst-Sander, A. D’Arcy, M. Stihle, K.
Mꢀller, F. Diederich, Angew. Chem. Int. Ed. 2003, 42, 2507-2511.; f) R.
Paulini, K. Mꢀller, F. Diederich, Angew. Chem. Int. Ed. 2005, 44, 1788-
1805.
[17] M. Tojino, M. Mori, M. C. Z. Kasuya, K. Hatanaka, A. Kawaguchi, K.
Nagata, T. Shirai, M. Mizuno, Bioorg. Chem. Lett. 2012, 22, 1251-1254.
[18] W. H. Pearson, D. A. Berry, P. Stoy, K. Y. Jung, A. D. Sercel, J. Org.
Chem. 2005, 70, 7114-7122.
[19] a) T. Yamanoi, T. Akiyama, E. Ishida, H. Abe, M. Amemiya, T. Inazu,
Chem. Lett. 1989, 335-336.; b) P. M. Koskinen, A. R. P. Koskinen,
Methods Enzymol. 1999, 311, 458-479.
[20] G. Johansson, V. Percec, Chem. Mater. 1997, 9, 164-175.
[21] M. Kotora, M. Hácek, B. Ameduri, B. Boutevin, J. Fluorine Chem. 1994,
68, 49-56.
[22] J.-M. Lee, H.-S. Lim, S.-K. Chung, Tetrahedron: Asymmetry 2002, 13,
343-347.
[11] E. P. Go, W. Uritboonthai, J. V. Apon, S. A. Trauger, A. Nordstrom, G.
O’Maille, S. M. Brittain, E. C. Peters, G. Siuzdak, J. Proteome Res. 2007,
6, 1492-1499.
[23] M. Song, W. Zang, B. Zhang, J. Cao, G. Yang, J. Exp. Clin. Cancer Res.
2012, 31:23.
[12] a) K. S. Ko, F. A. Jaipuri, N. L. Pohl, J. Am. Chem. Soc. 2005, 127,
13162-13163.; b) A. J. Vegas, J. E. Bradner, W. Tang, O. M. McPherson,
E. F. Greenberg, A. N. Koehler, S. L. Schreiber, Angew. Chem., Int. Ed.
2007, 46, 7960-7964.; c) F. A. Jaipuri, B. Y. Collet, N. L. Pohl, Angew.
Chem., Int. Ed. 2008, 47, 1707-1710.
[24] D. P. Curran, Z. Luo, J. Am. Chem. Soc. 1999, 121, 9069-9072.
[25] S. Mitsutake, K. Zama, H. Yokota, T. Yoshida, M. Tanaka, M. Mitsui, M.
Ikawa, M. Okabe, Y. Tanaka, T. Yamashita, H. Takemoto, T. Okazaki, K.
Watanabe, Y. Igarashi, J. Biol. Chem. 2011, 286, 28544-28555.
[26] K. Zama, S. Mitsutake, K. Watanabe, T. Okazaki, Y. Igarashi, Chem.
Phys. Lipids. 2012, 165, 760-768.
[13] a) L. Dafik, V. Kalsani, A. K. Leung, K. Kumar, J. Am. Chem. Soc. 2009,
131, 12091-12093.; b) E. Klein, M. Ciobanu, J. Klein, V. Machi, C.
Leborgne, T. Vandamme, B. Frisch, F. Pons, A. Kichler, G. Zuber, L.
Lebeau, Bioconjugate Chem. 2010, 21, 360-371.
[27] S. P. Pujari, E. Spruijt, M. A. C. Stuart, C. J. M. van Rijn, J. M. J. Paulusse,
H. Zuilhof, Langmuir 2012, 28, 17690-17700.
[14] Z. Song, W. Huang, Q. Zhang, Chem. Commun, 2012, 48, 3339-3341.
[15] D. P. Curran, Angew. Chem. Int. Ed. 1998, 37, 1174-1196
[16] S. M. Brittain, S. B. Ficarro, A. Brock and E. C. Peters, Nat. Biotechnol.
2005, 23, 463-468.
This article is protected by copyright. All rights reserved.

10.1002/ejoc.201601302
European Journal of Organic Chemistry
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Shota Saito, Yuta Murai, Seigo Usuki,
Masafumi Yoshida, Mostafa A.S.
Hammam, Susumu Mitsutake, Kohei
Yuyama, Yasuyuki Igarashi, and Kenji
Monde*
F
F
F
F
F
F
F
F
F
F
F
F
F F F F F
F
FSPE
F
F F F F F F
F
F
Rapid and facile separation
F
F F F F F
F
Page No. – Page No.
Metabolites of sphingolipids (SLs) are known to act as signaling molecules called
lipid mediators to regulate cell behavior. First synthesis of SLs bearing
perfluoroalkyl (fluorous) was reported. These derivatives could be applicable to
functional analysis of their metabolic mechanisms by FSPE.
Title:
Synthesis of Nontoxic Fluorous
Sphingolipids as Molecular Probes of
Exogenous Metabolic Studies for
Rapid Enrichment by FSPE
Key Topic:
Fluorous, Sphingosine
This article is protected by copyright. All rights reserved.