An Improved, Versatile, and Easily Scalable Synthesis of Sphingomyelins: Application to Stable Isotope Labeling

Article abstract of DOI:10.1055/s-0039-1690863

With a view to make conveniently labeled mass spectrometry standards available, a set of deuterated sphingomyelins were prepared by a new expedient, flexible, robust, scalable, and high-yielding synthetic scheme starting from 2-azido-3- O -benzoylsphingos

Full text of DOI:10.1055/s-0039-1690863

SYNTHESIS
0
0
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9
-
7
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8
1
1
4
3
7
-
2
1
0
X
©
Georg Thieme Verlag Stuttgart · New York
2020, 52, A–E
paper
A
en
Synthesis
N. Philippe et al.
Paper
An Improved, Versatile, and Easily Scalable Synthesis of Sphingo-
myelins: Application to Stable Isotope Labeling
O
O
Nicolas Philippe*
Serge Pérard*
Franck Le Strat
Jörg Blankenstein
Sébastien Roy
0
0
0
0-0
0
0
3-
0
1
6
2-1
6
4
5
N3
N3
1
)
P
O–
P
Me(CH2)12
OH
Cl
Me(CH2)12
O
O
O
N+
TMEDA, MeCN
O
Ph
O
Ph
O
2
) NMe3, THF
O
(79% )
O
(48–60% on 3 steps)
-
Efficient synthesis
Sanofi-Aventis R & D, Integrated Drug Discovery,
Isotope Chemistry Department, 13 quai Jules
Guesde, 94403 Vitry-sur-Seine, France
nicolas.philippe-ICMS@sanofi.com
- Reproducible and scalable process
- Versatile methodology
O
CD2
CD3
CD2
CD2
(
)n
NH
serge.perard@sanofi.com
O–
CH3(CH2)12
O
O
P
N+
OH
O
[D9]-Sphingomyelin
C16:0 n = 9 or C24:0 n = 17
Received: 19.11.2019
ceramide
polar head
Accepted after revision: 06.03.2020
Published online: 24.03.2020
DOI: 10.1055/s-0039-1690863; Art ID: ss-2019-z0644-op
fatty acyl chain
O
phosphocholine group
N
–
Abstract With a view to make conveniently labeled mass spectrome-
try standards available, a set of deuterated sphingomyelins were pre-
pared by a new expedient, flexible, robust, scalable, and high-yielding
synthetic scheme starting from 2-azido-3-O-benzoylsphingosine as the
key intermediate. Unlike previously published procedures, this work
emphasizes the benefit arising from the choice of the azido function as
a masking group for the reactive primary amine during the trouble-
some, though crucial, phosphorylation step.
O
O
O
N+
P
OH
O
sphingosine
Figure 1 Structure of sphingomyelin
Key words sphingomyelin, sphingosine, stable isotope, deuterium,
mass spectrometry standard, chemical synthesis
mount importance for any pharmacological study. The use
of stable isotope labeled (SIL) standards has been recog-
4
Sphingomyelins are the main components of mammali-
an sphingolipids. Indeed, they are found in all cell mem-
branes and especially in the central nervous system at the
level of myelin sheath. These complex sphingolipids are
made up of both hydrophobic and hydrophilic groups. The
nonpolar part comprises the sphingoid base, which is amid-
ified by a fatty acid whereas the polar head consists of a
phosphocholine moiety (Figure 1). This amphipathic ar-
rangement allows sphingomyelins to play also a role in var-
ious cellular events such as signal transduction and apopto-
nized as the method of choice for this purpose. Ideally, SIL
standards should hold the very structure of the molecule to
be analyzed by mass spectrometry (LC-MS/MS). The syn-
thesis of these isotopologues has been a major concern for
5
the pharmaceutical industry and was highlighted recently.
Indeed, the commercial availability of these standards is
rather scarce and several strategies have been developed to
allow a realistic access to the target molecules.6
,7
SIL sphingomyelins can be prepared using conventional
organic synthesis. To the best of our knowledge, only a few
preparations of deuterated sphingomyelins were spreaded
out over the literature. Byun and Bittman described the to-
tal synthesis of 3-deuterio-D-erythro-sphingomyelin, an
1
sis. Furthermore, an anomaly in sphingomyelin metabo-
8
lism triggers severe illnesses such as the rare hereditary
2,3
Niemann–Pick disease. This well-known example of lyso-
some storage disorder (LSD) is the result of a deficiency in
the hydrolytic enzyme acid sphingomyelinase (ASM), lead-
ing to the accumulation of sphingomyelin in vital organs,
including brain, and inflicting, among others, irreversible
neurological damages.
isotopomer failing to achieve the degree of labeling gener-
ally requested by bioanalysis scientists. Bartels et al. very
9
succinctly (neither analytical data nor yield given) men-
tioned the preparation of N-perdeuteriopalmitoyl-D-eryth-
ro-sphingomyelin (d31) by acylation of the corresponding
amine (lysosphingomyelin). Matsumori et al. claimed the
total synthesis of many kinds of deuterated (d to d3) site-
Therefore, it is easy to understand that specific quantifi-
cation of sphingomyelins in biological materials is of para-
©
2020. Thieme. All rights reserved. Synthesis 2020, 52, A–E
Georg Thieme Verlag KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

B
Synthesis
N. Philippe et al.
Paper
specific labeled stearoylsphingomyelins10 and 1-palmitoyl-
reduction of the azido group and N-acylation15 in good
yield.
11
2
-stearoyl-sn-glycero-3-phosphocholines specifically de-
2
12
According to the literature,16 sphingomyelin could be
obtained in moderate to good yield from ceramides display-
ing either a free or protected secondary alcohol function by
using different phosphorylation reagents such as 2-chloro-
2-oxo-1,3,2-dioxaphospholane or 2-chloro-1,3,2-dioxa-
phospholane. Unfortunately, our attempts to introduce the
phosphocholine moiety under these conditions were un-
successful as we faced deplorable low (at best 10%) and in-
consistent yields.
signed for H NMR biophysical studies. Mehnert et al.
touched upon the preparation of three analogous deriva-
tives (C2-d2-palmitoylsphingomyelin, C3-d2-palmitoyl-
sphingomyelin and d31-palmitoylsphingomyelin). Finally,
13
Cui et al. were brought to prepare 2-(d9-trimethylammo-
nio)ethylphosphate-stearyl-D-erythro-sphingomyelin, as a
probe for lipid rafts Raman imaging, by nucleophilic substi-
tution of a 2-bromoethylphosphate-sphingosine precursor.
We now describe a refined, efficient, and more versatile
methodology to provide an easy access to any labeled or un-
labeled sphingomyelin variant, starting from 2-azido-3-O-
benzoylsphingosine (1) and fatty acids, deuterated where
needed.
Another access to sphingomyelin consists first in per-
forming the delicate step, that is, the primary alcohol phos-
phorylation, leading to lyso-sphingomyelin before adding
the fatty acid chain. In the literature, sphingosin’s phos-
This key intermediate 1 was synthesized in 8 steps from
D-arabitol based on the method of Demchenko.14 After opti-
mization of the experimental conditions to secure the over-
all yield and isomeric purity, this compound was obtained
at several tens of gram scale.
Our first tested access to labeled sphingomyelin consist-
ed in synthesizing first the ceramide, composed of sphin-
gosine and a labeled fatty acid, before introducing the phos-
phocholine moiety. The ceramide was obtained in three
steps from 1, after deprotection of the secondary alcohol,
phorylation required to protect the amine as N-Boc deriva-
8,10,11b,13,17
tives,
whereas the secondary alcohol function
could be left free. However, this strategy suffered the dis-
advantage of involving additional synthetic steps.
Nevertheless, Cairo et al.18 demonstrated that the phos-
phorylation step can be carried out with an azide acting as
a protected amino group, enabling them to synthesize
sphingomyelin analogues with modified cholyl headgroups.
The latter were obtained using modified phosphocholines
as reagents. We choose to adapt this strategy to finally get
N3
O
O
O
N3
N3
O–
P
Me(CH2)12
OH
P
O
Me(CH2)12
CH3(CH2)12
O
O
O
N+
Cl
NMe3
P
O
Ph
O
Ph
O
O
Ph
O
O
TMEDA, MeCN, rt , 16 h
MeCN/THF, 50 °C
(79%)
O
O
O
1
2
3
MeONa
(96%)
MeOH/CH2Cl2, rt
NH2
O–
propanedithiol
N3
Me(CH2)12
O
O
O–
O
N+
Me(CH2)
12
O
P
P
N+
MeOH, NEt3, rt
73%)
OH
O
(
OH
O
lyso-sphingomyelin
5
4
O
N+
pyridine, CHCl3, rt
O–
(69–86%)
O
O
4-nitrophenol, DCI
C4D9
(
)
C4D9
n
OH
(
)n
DMAP, CH2Cl2, rt
(74–80%)
O
C16:0 n = 9 or C24:0 n = 17
C16:0 n = 9 or C24:0 n = 17
6
7
O
CD2
CD2
(
)n
CD3
CD2
NH
O–
CH3(CH2)12
O
O
P
N+
OH
O
[D9]-Sphingomyelin
C16:0 n = 9 ou C24:0 n = 17
8
9
Scheme 1
©
2020. Thieme. All rights reserved. Synthesis 2020, 52, A–E

C
Synthesis
N. Philippe et al.
Paper
O
O
CD2
CD3
CD2
CD2
CH3(CH2)12
(
)n
CD2
CD3
CD2
CD2
NH
OMe
O–
P
MeO
( )n
N
O
O
O
N+
O
O
N+
P
OH
D9]-Sphingomyelin 9 (n = 17)
O
p-TsOH
toluene/CHCl3 (3:1)
overnight reflux
O–
O
[
10
(CH2)12CH3
(22%)
Scheme 2
sphingomyelin itself in only three steps featuring a short,
versatile, scalable and robust synthetic pathway as depicted
in Scheme 1.
The bulk synthesis of 2-azido-3-O-benzoylsphingosine was done in
collaboration with SynCom (Groningen, The Netherlands).
2
[13,13,14,14,15,15,16,16,16- H
]Hexadecanoic
acid
and
9
2
[
21,21,22,22,23,23,24,24,24- H ]tetracosanoic acid were purchased
The phosphocholine moiety was introduced via a one-
pot reaction by reacting 1 and 2-chloro-2-oxo-1,3,2-dioxa-
phospholane in acetonitrile overnight following by addition
of trimethylamine in THF at 50 °C for 2 days to give the azi-
dophosphodiester 3 in 79% yield (Scheme 1). For this reac-
tion, as the dioxaphospholane intermediate 2 is very sensi-
9
from C/D/N Isotopes Inc. (Quebec, Canada). All reagents and solvents
were obtained from commercial suppliers and used without further
purification. The solution of 1.0 M Me N in THF from Thermo Fisher
3
TM
Scientific was dried by adding a Trap-Pak bag (medium) from Ap-
plied Biosystems. The 99.9% anhydrous MeCN solution was purchased
from Sigma-Aldrich.
16c
tive to moisture, it was important to work under an inert
atmosphere with carefully dried solvents and compounds
to ensure an excellent reproducibility. The O-benzoyl deriv-
ative 3 was deprotected by sodium methoxide in methanol
to give the secondary alcohol 4 nearly quantitatively. The
azide 4 was reduced with propanedithiol in the presence of
trimethylamine in methanol to afford the lyso-sphingomy-
elin 5 in 73% yield. In the last step, the amine 5 was reacted
with labeled p-nitrophenyl palmitate 6 or lignocerate 7 in
pyridine to provide the corresponding sphingomyelins 8
and 9 in greater than 70% yield after crystallization (Scheme
Air and moisture sensitive reactions were conducted under an inert
atmosphere of argon and were magnetically stirred. Reactions were
monitored by TLC performed on 60 F254 silica gel plates. To locate
spots, plates were sprayed with 10% phosphomolybdic acid in EtOH
followed by heating.
1
H NMR spectra were recorded on a Bruker Avance 500 spectrometer
31
and P NMR spectra on a Bruker Avance 400 spectrometer in the
stated deuterated solvents. Proton decoupled C NMR spectra were
recorded on a Bruker Avance 500 spectrometer equipped with a
13
13
C
selective cryoprobe so that several deuterium coupled carbons be-
13
31
13
2
came detectable. These C- P and C- H coupling constants are thus
reported. The chemical shift data for each signal are given in units of 
1
13
relative to CH OH ( = 3.49 for H NMR spectra and  = 50.41 for
C
1).
3
1
NMR spectra) or to CHCl ( = 7.26 for H NMR spectra and  = 77.00
for C NMR spectra) and to phosphoric acid ( = 0.00 for P NMR
spectra). Data for H NMR are reported as follows: chemical shift (,
3
The relative configuration (erythro or threo) of sphin-
13
31
gomelins 8 and 9 can be most conveniently deduced from
1
1
their H NMR spectra, on the basis of data published by
ppm), multiplicity (standard abbreviations), coupling constants (J,
Hz), and integration.
19
Bruzik. Nevertheless, to avoid possible fallacies owing to
solvent variation, temperature or concentration effects, we
decided to validate this point by conducting a nuclear
Overhauser enhancement NMR experiment on a cyclic
derivative 10 (oxazolidone) prepared from compound 9
according to Scheme 2. The results are in full agreement
with the desired erythro stereochemistry.
High-resolution mass spectra were recorded on a Shimadzu hybrid
Ion Trap/Time of Flight spectrometer (IT-TOF). The molecular formula
determination was performed using Shimadzu’s Formula Predictor
software.
Melting points were determined on a Büchi B-545 apparatus, optical
rotations on a PerkinElmer 341 polarimeter at 589 nm, and IR spectra
were acquired with a Thermo Scientific (Nicolet) iS50 FT-IR spectro-
photometer.
We have described a hitherto unpublished and straight-
forward process leading to any sphingomyelin derivative in
four optimized steps with a mean overall yield of 40%, start-
ing from 2-azido-3-O-benzoylsphingosine, readily available
at a multi-gram scale. We demonstrated that this azido de-
rivative can be easily transformed into lyso-sphingomyelin,
skipping the usual protection/deprotection cycle of the pri-
mary amine. Afterwards, a simple acylation of lyso-sphin-
gomyelin by various stable isotope labeled or unlabeled fat-
ty acids will provide any sphingomyelin analogue. Alterna-
tively, this strategy could be applied to the labeling of the
choline moiety giving access to molecules such as 2-(d9-
trimethylammonio)ethylphosphate-sphingomyelin13 or the
corresponding lyso-sphingosine.
(E,2S,3R)-2-Azido-3-(benzoyloxy)octadec-4-en-1-yl 2-(Trimeth-
ylammonio)ethyl Phosphate (3)
In a 25 mL flask fitted with a rubber septum under argon, a stirred
solution of (2S,3R,E)-2-azido-1-hydroxyoctadec-4-en-3-yl benzoate
(
1; 1.0 g, 2.3 mmol) in anhyd MeCN (10 mL) was treated with a solu-
tion of TMEDA (0.3 mL, 2.0 mmol) in anhyd MeCN (0.5 mL) at 0 °C. A
solution of 2-chloro-1,3,2-dioxaphospholane-2-oxide (0.32 mL, 3.5
mmol) in anhyd MeCN (0.5 mL) was slowly added at 0 °C and the mix-
ture was stirred at rt overnight. The reaction was monitored by TLC
(
SiO , CHCl ). A solution of 1.0 M anhyd Me N in THF (20 mL, 25
2
3
3
mmol) was added dropwise at rt. The mixture was heated at 50 °C for
days. The reaction was monitored by TLC (SiO2, CHCl3 and
2
©
2020. Thieme. All rights reserved. Synthesis 2020, 52, A–E

D
Synthesis
N. Philippe et al.
Paper
1
CHCl /MeOH/H O, 70:30:4). After cooling to rt, the mixture was con-
H NMR (500 MHz, CDCl + CD OD 50:50):  = 5.91 (dt, J = 15.0, 6.9 Hz,
3
2
3
3
centrated under vacuum. The crude product was purified by flash
chromatography on silica gel (first elution with CHCl /MeOH/H O,
1 H), 5.63 (ddt, J = 15.3, 7.6, 1.4 Hz, 1 H), 4.42 (br m, 2 H), 4.12 (m, 3
H), 3.77 (br m, 2 H), 3.37 (s, 9 H), 2.95 (br m, 2 H), 2.23 (qd, J = 6.9, 1.1
Hz, 2 H), 1.56 (m, 2 H), 1.51–1.38 (br m, 20 H), 1.04 (t, J = 6.8 Hz, 3 H).
3
2
9
0:10:1 then with CHCl /MeOH/H O, 70:30:4) to afford 3 as a white
3 2
amorphous solid; yield: 1.1 g (79%); R = 0.3 (SiO , CHCl /MeOH/H O,
13C NMR (125 MHz, CDCl3 + CD OD 50:50):  = 137.01, 131.39, 75.3,
f
2
3
2
3
70:30:4).
68.69 (d, J = 5.8 Hz), 68.51 (m), 61.09 (d, J = 4.9 Hz), 57.77, 55.94,
1
H NMR (500 MHz, CDCl + CD OD 50:50):  = 8.21 (dd, J = 8.52, 1.30
55.91, 55.88, 34.44, 33.95, 31.69, 31.66, 31.65, 31.54, 31.37, 31.34,
31.28, 24.66, 15.73.
3
3
Hz, 2 H), 7.76 (tt, J = 7.40, 1.27 Hz, 1 H), 7.63 (tt, J = 7.8, 1.6 Hz, 2 H),
6
4
.11 (dt, J = 14.5, 6.8 Hz, 1 H), 5.74 (ddt, J = 14.5, 7.8, 1.30 Hz, 1 H),
.44 (br m, 2 H), 4.21 (dd, J = 6.2, 4.4 Hz, 1 H), 4.19 (dt, J = 10, 4.5 Hz, 1
31
P NMR (162 MHz, CDCl + CD OD 50:50):  = 0.059.
3
3
HRMS (ESI+): m/z [M + H]+ calcd for C23H49N O P: 465.3452; found:
2
5
H), 4.08 (m, 1 H), 3.76 (m, 2 H), 3.36 (s, 9 H), 2.24 (q, J = 7.1 Hz, 2 H),
.55 (br q, J = 7 Hz, 2 H), 1.49–1.37 (br m, 20 H), 1.03 (t, J = 7.1 Hz, 3
H).
465.3438.
1
2
4-Nitrophenyl [13,13,14,14,15,15,16,16,16- H ]Hexadecanoate (6)
13
9
C NMR (125 MHz, CDCl + CD OD 50:50):  = 167.68, 140.78, 135.44,
3
3
2
[
13,13,14,14,15,15,16,16,16- H ]Hexadecanoic acid (1.4 g, 5.2 mmol)
131.76, 131.66, 130.53, 124.86, 76.68, 68.41 (m), 66.55, 66.54 (d, J =
13 Hz), 61.15 (d, J = 5 Hz), 55.98, 55.95, 55.92, 34.27, 33.86, 31.57 (br),
31.54, 31.49, 31.32, 31.27, 31.04, 30.61, 24.58, 15.74.
9
was dissolved in CH Cl (10 mL) under argon. The solution was cooled
2
2
at 0 °C before 4-nitrophenol (440 mg, 3.2 mmol), DMAP (3 mg), and
N,N′-diisopropylcarbodiimide (0.535 mL, 5.2 mmol) were added. The
mixture was stirred at rt overnight and filtered through a pad of
Celite and washed with CH Cl . The filtrate was concentrated under
31
P NMR (162 MHz, CDCl + CD OD 50:50):  = –0.698.
3
3
HRMS (ESI+): m/z [M + H]+ calcd for C30H51N O P: 595.3619; found:
4
6
2
2
595.3591.
vacuum and the residue purified by chromatography using a silica gel
column eluted with CH Cl /n-pentane (7:3, v/v) to afford 6 as a white
2
2
(E,2S,3R)-2-Azido-3-hydroxyoctadec-4-en-1-yl 2-(Trimethylam-
solid; yield: 1.6 g (80%).
monio)ethyl Phosphate (4)
1
H NMR (500 MHz, CDCl :  = 8.27 (d, J = 9.1 Hz, 2 H), 7.27 (d, J = 9.1
3
Compound 3 (1.0 g, 1.7 mmol) was dissolved in a mixture of CH Cl2
Hz, 2 H),, 2.59 (t, J = 7.5 Hz), 1.76 (quint, J = 7.4 Hz, 2 H), 1.41 (m, 2 H),
2
(
(
30 mL) and MeOH (7 mL) under argon. After the addition of MeONa
150 mg, 2.6 mmol), the mixture was stirred at rt overnight. The reac-
1.38–1.22 (m, 16 H).
13
C NMR (500 MHz, CDCl ):  = 171.32, 155.54, 145.26, 125.19,
3
tion was monitored by TLC (SiO , CHCl /MeOH/H O, 70:30:4). Amber-
lite IR-120(H) resin (900 mg) was added and the mixture was stirred
for 5 min and filtered. The resin was washed with CH Cl and the fil-
trate concentrated under vacuum. The residue was purified by flash
chromatography on silica gel (first elution with CHCl /MeOH/H O,
9
2
3
2
122.42, 34.35, 30.59 (quint, J = 16 Hz), 29.69, 29.67, 29.65, 29.64,
29.58, 29.42, 29.21, 29.04, 28.28 (quint, J = 19 Hz), 24.75, 21.37 (quint,
2
2
J = 18 Hz), 12.94 (hept, J = 18.8 Hz).
3
2
2
4-Nitrophenyl [21,21,22,22,23,23,24,24,24- H ]Tetracosanoate (7)
9
0:10:1 and then with CHCl /MeOH/H O, 70:30:4) to give 4 as a
3 2
2
[
21,21,22,22,23,23,24,24,24- H ]Tetracosanoic acid (1.0 g, 2.7 mmol)
9
white amorphous solid; yield: 790 mg (96%); Rf
CHCl /MeOH/H O, 70:30:4).
=
0.2 (SiO2,
was dissolved in CH Cl (15 mL) under argon. The solution was cooled
2
2
3
2
at 0 °C, then 4-nitrophenol (440 mg, 3.2 mmol), DMAP (3 mg), and
N,N′-diisopropylcarbodiimide (0.535 mL, 5.2 mmol) were added. The
mixture was stirred at rt overnight, filtered through a pad of Celite,
and washed with CH Cl . The filtrate was concentrated under vacuum
1
H NMR (500 MHz, CDCl + CD OD 50:50):  = 5.94 (dtd, J = 15.4, 6.7,
3
3
0
.6 Hz, 1 H), 5.67 (ddt, J = 15.4, 7.5, 1.4 Hz, 1 H), 4.45 (br m, 2 H), 4.28
(t, J = 6.9 Hz, 1 H), 4.23 (ddd, J = 11.1, 5.8, 3.2 Hz, 1 H), 4.11 (m, 1 H),
2
2
3
1
H).
1
.78 (m, 2 H), 3.64 (td, J = 6.7, 3.30 Hz), 3.38 (s, 9 H), 2.22 (qd, J = 6.9,
.1 Hz, 2 H), 1.55 (m, 2 H), 1.51–1.37 (br m, 20 H), 1.04 (t, J = 6.9 Hz, 3
and the residue purified by chromatography using a silica gel column
eluting with CH Cl /n-pentane (7:3, v/v) to afford 7 as a white solid;
2
2
yield: 980 mg (74%).
3
C NMR (125 MHz, CDCl + CD OD 50:50):  = 170.80, 137.00, 130.31,
1
3
3
H NMR (500 MHz, CDCl ):  = 8.27 (d, J = 9.1 Hz, 2 H), 7.27 (d, J = 9.1
3
7
5
3
3.48, 68.43 (m), 68.35 (d, J = 7.3 Hz), 67.20 (d, J = 5.5 Hz), 61.15 (d, J =
Hz), 55.94, 55.91, 55.88, 33.35, 33.89, 31.62 (br), 31.60, 31.57, 31.46,
1.31, 31.19, 31.02, 24.60, 15.71.
Hz, 2 H), 2.59 (t, J = 7.5 Hz, 2 H), 1.76 (quint, J = 7.4 Hz, 2 H), 1.41 (m, 2
H), 1.37–1.22 (m, 32 H).
13
C NMR (500 MHz, CDCl ):  = 171.31, 155.54, 145.26, 125.18,
3
3
1
P NMR (162 MHz, CDCl + CD OD 50:50):  = –0.498.
3
3
122.42, 34.35, 30.60 (quint, J = 16 Hz), 29.70, 29.68, 29.64, 29.58,
HRMS (ESI+): m/z [M + H]+ calcd for C23H47N O P: 491.3357; found:
4
4
5
29.49, 29.43, 29.41, 29.28, 29.21, 29.05, 28.29 (quint, J = 19 Hz), 24.75,
21.37 (quint, J = 18 Hz), 13.08 (hept, J = 19 Hz).
91.3348.
2
(E,2S,3R)-2-Amino-3-hydroxyoctadec-4-en-1-yl 2-(Trimethyl-
(E,2S,3R)-2-[13,13,14,14,15,15,16,16,16- H ]Hexadecanoylamino-
9
ammonio)ethyl Phosphate (5)
3-hydroxytetradec-4-enyl 2-(Trimethylammonio)ethyl Phosphate
(8)
A stirred solution of 4 (780 mg, 1.6 mmol) in MeOH (40 mL) was
treated with 1,3-propanedithiol (4.0 mL, 39.5 mmol) and Et N (7.5
A stirred solution of 5 (520 mg, 1.1 mmol) in CHCl (20 mL) was treat-
3
3
mL, 53.5 mmol) under argon and the mixture was stirred at rt for 3
days. The reaction was monitored by TLC (SiO , CHCl /MeOH/H O,
ed with pyridine (5 mL) and ester 6 (550 mg, 1.4 mmol) under argon.
The mixture was stirred at rt for 2 days. The reaction was monitored
by TLC (SiO , CHCl and CHCl /MeOH/H O, 70:30:4) and after comple-
2
3
2
70:30:4) and after completion, the mixture was concentrated under
2
3
3
2
vacuum. The residue purified by flash chromatography using a silica
gel column (elution successively with CHCl /MeOH/H O, 90:10:1,
tion, the mixture was concentrated under vacuum. The residue was
taken up in a mixture of CHCl /MeOH/H O (90:10:1), then filtered,
3
2
3
2
7
0:30:4, 50:50:4, then with MeOH/H O, 100:4) to afford 5 as a white
and concentrated under vacuum. The crude material was purified by
flash chromatography using a silica gel column (first elution with
2
solid; yield: 540 mg (73%).
©
2020. Thieme. All rights reserved. Synthesis 2020, 52, A–E

E
Synthesis
N. Philippe et al.
Paper
CHCl /MeOH/H O, 90:10:1, then with CHCl /MeOH/H O, 70:30:4).
3
2
3
2
Supporting Information
The compound was dissolved in
a minimum amount of a
CHCl /MeOH mixture, then crystallized by addition of acetone to give
Supporting information for this article is available online at
3
8
as a white solid; yield: 690 mg (87%); mp 213 °C; R = 0.2 (SiO ,
https://doi.org/10.1055/s-0039-1690863.
S
u
p
p
orti
n
g Inform ati
o
n
S
u
p
p
orti
n
g Inform ati
o
n
f
2
25
CHCl /MeOH/H O, 70:30:4); [] +6.3 (c 0.44, CHCl /MeOH, 1:1).
3
2
D
3
IR (ATR): 3283 (br), 2916, 2849, 1644, 1547, 1467, 1378, 1226, 1085,
–1
References
1054, 986, 928, 875, 836, 753, 720, 504 cm (br).
1
H NMR (500 MHz, CDCl + CD OD 50:50):  = 5.86 (dt, J = 15.2, 6.6 Hz,
H), 5.60 (ddt, J = 15.3, 7.6, 1.3 Hz, 1 H), 4.40 (m, 2 H), 4.32 (m, 1 H),
.22 (t, J = 8.0 Hz, 1 H), 4.08 (m, 1 H), 4.05 (m, 1 H), 3.74 (br t, J = 4.6
(1) Rao, R. P.; Vaidyanathan, N.; Rengasamy, M.; Oommen, A. M.;
Somaiya, N.; Jagannath, M. R. J. Lipids 2013, 178910.
(2) Arenz, C. Future Med. Chem. 2017, 9, 1687.
(3) Wasserstein, M.; Dionisi-Vici, C.; Giugliani, R.; Hwu, W.-L.;
Lidove, O.; Lukacs, Z.; Mengel, E.; Mistry, P. K.; Schuchman, E.
H.; McGovern, M. Mol. Genet. Metab. 2019, 126, 98.
3
3
1
4
Hz, 2 H), 3.36 (s, 9 H), 2.32 (m, 2 H), 2.17 (br q, 2 H), 1.73 (m, 2 H), 1.58
(m, 2 H), 1.48–1.38 (m, 38 H), 1.04 (t, J = 6.8 Hz, 3 H).
13
C NMR (125 MHz, CDCl + CD OD 50:50):  = 176.59, 136.33, 131.36,
3
3
(
(
(
(
4) Nageswara, R. R. Mod. Appl. Pharm. Pharmacol. 2017, DOI:
0.31031/MAPP.2017.01.000508.
5) Atzrodt, J.; Derdau, V.; Kerr, W. J.; Reid, M. Angew. Chem. Int. Ed.
018, 57, 1758.
6) Atzrodt, J.; Derdau, V.; Kerr, W. J.; Reid, M. Angew. Chem. Int. Ed.
018, 57, 3022.
7) Allen, J.; Brasseur, D. M.; De Bruin, B.; Denoux, M.; Pérard, S.;
Philippe, N. J. Labelled Compd. Radiopharm. 2007, 50, 342.
8) Byun, H.-S.; Bittman, R. Chem. Phys. Lipids 2010, 163, 809.
9) Bartels, T.; Lankalapalli, R. S.; Bittman, R.; Beyer, K.; Brown, M. F.
J. Am. Chem. Soc. 2008, 130, 14521.
7
5
3
3
1
3.17, 68.41 (m), 66.57 (d, J = 5.8 Hz), 61.02 (d, J = 5.1 Hz), 55.98,
5.92, 55.89, 55.86, 38.41, 34.36, 33.88, 32.52 (quint, J = 19.3 Hz),
1.67, 31.65, 31.61, 32.56, 31.52, 31.45, 31.39, 31.35, 31.31, 31.25,
0.21 (quint, J = 19.7 Hz), 27.99, 24.59, 23.25 (quint, J = 20 Hz), 15.71,
4.55 (hept, J = 18.5 Hz).
1
2
2
31
P NMR (162 MHz, CDCl + CD OD 50:50):  = 0.244.
3
3
HRMS (ESI+): m/z [M + H]+ calcd for C39H702H N O P: 712.6313;
found: 712.6276.
9
2
6
(
(
2
(E,2S,3R)-2-[21,21,22,22,23,23,24,24,24- H ]Tetracosanoylamino-
9
3-hydroxytetradec-4-enyl 2-(Trimethylammonio)ethyl Phosphate
(10) Matsumori, N.; Yasuda, T.; Okazaki, H.; Suzuki, T.; Yamaguchi,
(9)
T.; Tsuchikawa, H.; Doi, M.; Oishi, T.; Murata, M. Biochemistry
2
012, 51, 8363.
A stirred solution of 5 (220 mg, 473 mol) in CHCl (15 mL) was treat-
3
(
11) (a) Yasuda, T.; Kinoshita, M.; Murata, M.; Matsumori, N. Bio-
phys. J. 2014, 106, 631. (b) Yo Yano, Y.; Hanashima, S.; Yasuda,
T.; Tsuchikawa, H.; Matsumori, N.; Kinoshita, M.; Al Sazzad, M.
A.; Slotte, J. P.; Murata, M. Biophys. J. 2018, 115, 1530.
ed with pyridine (3 mL) and ester 7 (400 mg, 802 mol) under argon.
The mixture was stirred at rt for 2 days. The reaction was monitored
by TLC (SiO , CHCl and CHCl /MeOH/H O, 70:30:4) and after comple-
2
3
3
2
tion, the mixture was concentrated under vacuum. The residue taken
up in a mixture of CHCl /MeOH/H O (90:10:1), filtered, and concen-
(c) Erratum: Yo Yano, Y.; Hanashima, S.; Yasuda, T.; Tsuchikawa,
3
2
H.; Matsumori, N.; Kinoshita, M.; Al Sazzad, M. A.; Slotte, J. P.;
Murata, M. Biophys J. 2019, 116, 1575.
12) Mehnert, T.; Jacob, K.; Bittman, R.; Beyer, K. Biophys. J. 2006, 90,
trated under vacuum. The crude material was purified by flash chro-
matography using silica gel column (first elution with
CHCl /MeOH/H O, 90:10:1, then with CHCl /MeOH/H O, 70:30:4).
a
(
3
2
3
2
939.
The compound was dissolved in
a minimum amount of a
(
13) Cui, J.; Matsuoka, S.; Kinoshita, M.; Matsumori, N.; Sato, F.;
Murata, M.; Ando, J.; Yamakoshi, H.; Dodo, K.; Sodeoka, M.
Bioorg. Med. Chem. 2015, 23, 2989.
14) Parameswar, A. R.; Hawkins, J. A.; Mydock, L. K.; Sands, M. S.;
Demchenko, A. V. Eur. J. Org. Chem. 2010, 3269.
CHCl /MeOH mixture and crystallized by addition of acetone to give 9
3
as a white solid; yield: 270 mg (69%); mp 211 °C; R = 0.3 (SiO ,
f
2
25
CHCl /MeOH/H O, 70:30:4); [] +7.9 (c 0.2, CHCl /MeOH, 1:1).
3
2
D
3
(
IR (ATR): 3282 (br), 2916, 2850, 1642, 1547, 1467, 1378, 1228, 1086,
–1
1
054, 968, 927, 875, 836, 720, 489 cm (br).
(
15) (a) Sonnenberger, S.; Lange, S.; Langner, A.; Reinhard, H. H.;
Neubert, R. H. H.; Dobner, B. J. Labelled Compd. Radiopharm.
1
H NMR (500 MHz, CDCl + CD OD 50:50):  = 5.86 (dt, J = 15.2, 6.6 Hz,
3
3
1
4
H), 5.60 (ddt, J = 15.3, 7.6, 1.3 Hz, 1 H), 4.40 (m, 2 H), 4.32 (m, 1 H),
.22 (t, J = 8.0 Hz, 1 H), 4.08 (m, 1 H), 4.05 (m, 1 H), 3.74 (br t, J = 4.6
2016, 59, 531. (b) Zimmermann, P.; Schmidt, R. R. Liebigs Ann.
Chem. 1988, 663.
Hz, 2 H), 3.36 (s, 9 H), 2.32 (m, 2 H), 2.17 (br q, 2 H), 1.73 (m, 2 H), 1.58
(
16) (a) Carmen, D.; Heckhoff, S.; Oswald, B.; Rebmann, P.; Peer, A.;
Gonzalez, M.; Sauter, P. Patent US 20140275590A1, 2014.
(b) Byun, H.-S.; Erukulla, R. K.; Bittman, R. J. Org. Chem. 1994,
59, 6495. (c) Dong, Z.; Butcher, J. A. Jr. Chem. Phys. Lipids 1993,
66, 41.
(17) Yamamoto, T.; Hasegawa, H.; Hakogi, T.; Katsumura, S. Org. Lett.
2006, 8, 5569.
(18) Sandbhor, M. S.; Key, J. A.; Strelkov, I. S.; Cairo, C. W. J. Org.
Chem. 2009, 74, 8669.
(m, 2 H), 1.48–1.38 (m, 54 H), 1.04 (t, J = 6.8 Hz, 3 H).
13
C NMR (125 MHz, CDCl + CD OD 50:50):  = 176.58, 136.34, 131.36,
3
3
73.18, 68.42 (m), 66.58 (d, J = 3.6 Hz), 61.03 (d, J = 5.1 Hz), 55.99,
55.93, 55.89, 55.86, 38.42, 34.38, 33.90, 32.59 (quint, J = 20.5 Hz),
31.69, 31.67, 31.65, 31.63, 31.58, 31.54, 31.46, 31.40, 31.34, 31.33,
31.27, 30.20 (quint, J = 19 Hz), 28.00, 24.58, 23.27 (quint, J = 18.4 Hz),
15.73, 14.55 (hept, J = 18.5 Hz).
31
P NMR (162 MHz, CDCl + CD OD 50:50):  = 0.122.
3
3
(
19) Bruzik, K. S. Biochim. Biophys. Acta 1988, 939, 2: 315.
HRMS (ESI+): m/z [M + H]+ calcd for C47H862H N O P: 824.7565;
9
2
6
found: 824.7557.
©
2020. Thieme. All rights reserved. Synthesis 2020, 52, A–E