A study of synthetic approaches to 2-acyl DHA lysophosphatidic acid

Article abstract of DOI:10.1039/c7ob01771e

Lysophosphatidic acid (LPA) is a chemical mediator with a very simple glycerophospholipid structure. 1-Acyl LPA and 2-acyl LPA are biosynthesized in vivo. Unlike 1-acyl LPA, the biological function of 2-acyl LPA has been hardly elucidated and even organic synthesis of 2-acyl LPA had not been established. We suppressed acyl migration by formation of a salt with a phosphate group in order to synthesize 2-acyl LPA condensed with docosahexaenoic acid.

Full text of DOI:10.1039/c7ob01771e

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A study of synthetic approaches to 2-acyl DHA lysophosphatidic
acid†
Yoshinori Yamamoto, Toshimasa Itoh, Keiko Yamamoto*
Received 00th January 20xx,
Accepted 00th January 20xx
Lysophosphatidic acid (LPA) is a chemical mediator with a very simple glycerophospholipid structure. 1-acyl LPA and 2-acyl
LPA are biosynthesized in vivo. Unlike 1-acyl LPA, the biological function of 2-acyl LPA has been hardly elucidated and even
organic synthesis of 2-acyl LPA had not been established. We suppressed acyl migration by formation of a salt with a
phosphate group in order to synthesize 2-acyl LPA condensed with docosahexaenoic acid.
DOI: 10.1039/x0xx00000x
www.rsc.org/
co-workers synthesized analogue 2, in which the sn-1 hydroxyl group
is protected by a hydroxy ethyl ether8. Tamaruya et al. designed and
synthesized 3, in which the sn-1 position is also protected by the
Introduction
The major lysophospholipid lysophosphatidic acid (LPA) is an
important phospholipid biological mediator. It has been shown that
some of the fundamental functions of LPA in the nervous, vascular,
immune, and reproductive systems involve binding to LPA receptors
of the G protein–coupled receptor superfamily1. In addition, LPA
receptors are involved in neuro-inflammation, nerve injury,
schizophrenia, atherosclerosis, wound healing, cancer, airway
diseases, and obesity2.
formation of a cyclic ether9. Li and colleagues synthesized LPA
analogue 4 as a photo-affinity probe using an enzymatic and
chemical synthetic approach. The primary alcohol at the sn-1
position is protected by a methoxymethyl group to facilitate use in
biological assays10. All of these primary alcohols are protected to
prevent acyl migration; thus, these strategies are useful for SAR
study1112
.
To understand the biological function of LPA, LPA itself should be
used instead of an analogue. However, there are currently no
methods available for synthesizing useful quantities of 2-acyl LPA,
In the two species of LPA, 1-lyso/2-acyl LPA and 2-lyso/1-acyl LPA,
a long-chain fatty acid is condensed at the sn-2 and sn-1 positions,
respectively. The related phospholipid, phosphatidic acid (PA),
possesses an unsaturated fatty acid at the sn-2 position and a
saturated acid at the sn-1 position. Because LPA is both a
biosynthetic precursor and metabolite of PA, the condensed fatty
acids of 2-acyl and 1-acyl LPA are unsaturated and saturated fatty
acids, respectively3, 4, 5. However, studies of LPA conducted to date
have been limited by acyl migration, in which an acyl group readily
migrates from the sn-2 to sn-1 position6. Acyl migration impedes the
synthesis of 2-acyl LPA, thus hampering efforts to determine the
biological role of this important molecule.
To prevent the migration reaction, organic chemists have
designed and synthesized analogues of 2-acyl LPA (Fig. 1). The first
trial has been done by Xu and co-workers, who synthesized difluoro-
methyl compound 1, in which the primary alcohol at the sn-1 position
is substituted with a difluoro-methyl group7. They successfully
showed that their synthetic analogues have PPARγ activity. Qian and
a.Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical
University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan.
E-mail: yamamoto@ac.shoyaku.ac.jp.
Fig. 1 The structures of lysophosphatidic acids (LPAs) and LPA
analogues. 2-acyl LPA and 1-acyl LPA possesses an acyl group
at sn-2 and sn-1 position. 2-DL and 1-DL contain DHA as acyl
group. Many kinds of 2-acyl LPA analogues have been designed
†Electronic Supplementary Information (ESI) available: Scheme S1, 1H NMR chart of
2-DL with (C4H9)4N+ ion (Fig. S1), 1H and 13C NMR chart of new compounds including
crude and purified 2-DL (Na+) and 2-DL(K+) and 1H chart of 2-DL (NH4+) after 18
months (Figure S2): 10.1039/x0xx00000x
and synthesized (1-4).
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although enzymatic methods permit the synthesis of small amounts
of 2-acyl LPA.
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DOI: 10.1039/C7OB01771E
Aoki and co-workers compared LPA levels in subjects with normal
coronary arteries or stable angina pectoris using mass spectrometry.
They revealed that serum levels of 2-acyl LPA containing
docosahexaenoic acid (DHA) at the sn-2 position (2-acyl DHA LPA) are
higher in patients with acute coronary syndrome13. However,
obtaining a more comprehensive understanding of the physiologic
role of 2-acyl LPA will require the development of a synthesis method
capable of providing sufficient amounts of the compound. Here, we
report a method that enables control of acyl migration for the
chemical synthesis of 2-acyl DHA LPA as a representative of 2-acyl
LPA.
Scheme 2 Synthesis of 1-DL for the confirmation of occurring acyl
migration (Scheme 1).
NMR analysis of the product did not show the expected spectrum for
2-DL16. Only trace peaks for the sn-2 proton were observed at 5 ppm,
which is the peak position corresponding to precursor 7. We
hypothesized that the primary product was 1-DL, which resulted
from acyl migration during the final deprotection step. To confirm
the occurrence of acyl migration, we synthesized 1-acyl DHA LPA (1-
DL), as shown in Scheme 2.
Condensation of diol 8 and DHA chloride was performed using 2,4,6-
trimethylpyridine at −78°C in DCM to give 9 as a single isomer at 58%
yield (Scheme 2)15. Deprotection of 9 was carried out under the same
conditions shown in Scheme 1 to furnish DHA LPA at 79% yield. The
ratio of major to minor products was 85:15. The major product was
confirmed as 1-DL by ESI-MS and NMR analyses. The spectra for 1-DL
were identical to those of the major product in the DHA LPA
synthesis shown in Scheme 1. These data indicate that acyl migration
occurred under the conventional deprotection conditions used. The
minor products in the DHA LPA synthesis pathways shown in
Schemes 1 and 2 were identical. Thus, the minor product was
simultaneously determined to be 2-DL.
Results
Synthesis of protected 2-DL
A sufficient quantity of a biomolecule is necessary to elucidate its
physiologic function. Synthesis of 2-acyl LPA analogues has been
reported, but synthesis of true 2-acyl LPA has not. We concluded that
acyl migration is the most important issue that must be addressed in
developing a suitable method for the synthesis of 2-acyl LPA. We
therefore planned the synthesis of 2-acyl DHA LPA (2-DL), which
contains the polyunsaturated fatty acid, DHA.
Compound 5, possessing an R-hydroxy group, was obtained in
three steps using a previously reported procedure (Scheme 1)
Condensation of 5 and DHA was carried out using EDC•HCl and
DMAP in DCM to give protected 2-DHA LPA 6 at 85% yield. TBDPS
was removed from 6 by treatment with TBAF in the presence of AcOH
in THF to furnish alcohol 7 at 80% yield.
14, 15
.
Deprotection and acyl migration
We deprotected dimethyl phosphate
7
using N,O-
Suppression of acyl migration
Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and TMSBr for the
substitution of TMS, followed by treatment with MeOH and H2O to
produce the deprotected compound. ESI-MS analysis confirmed the
In a study involving lyso-bis-phosphatidic acid 10, Chevallier and co-
workers found that acyl migration was suppressed under basic
conditions in the presence of pyridine (Fig. 2)17. Okudaira et al.
examined the pH dependence of acyl migration using 1-oleoyl-
lysophosphatidic acid (11) and related lysophospholipids18. In
contrast to the report of Chevallier’s group, they showed that acyl
1
compound was LPA condensed with DHA (DHA-LPA). However, H-
migration was suppressed at pH
4 and −20°C in formic
acid/ammonium formate buffer. Intriguingly, previous reports had
demonstrated suppression under basic conditions, whereas the
latter demonstrated suppression under acidic conditions. This
apparent contradiction suggested to us that pH is not important for
suppressing acyl migration. We envisaged that suppression of acyl
migration is due to the formation of a salt between phosphate and
ammonium or pyridinium ions.
We therefore attempted deprotection of 7 in the presence of
ammonium ion. Deprotection was carried out in 100 mM ammonium
formate buffer (pH 4.0) in MeOH, conditions similar to those
reported by Okudaira et al. for preservation of 2-acyl LPA.18 (Scheme
Scheme1 Synthesis of 2-DL using a general deprotection
condition. The given yield for DHA-LPA is mixture of major and
minor isomer.
1
3). H-NMR analysis showed that the major product was 2-DL. This
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a)
result could indicate that acyl migration was suppressed at least until
the NMR analysis,
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DOI: 10.1039/C7OB01771E
1st fraction
2-DL (NH4+ salt)
sn-1
sn-3
sn-2
Scheme 3 Deprotection of 7 and 12 in presence of ammonium ion,
NH4+ or nBu4N+, for the synthesis of 2-DL.
3.5
4.0
5.0
4.5
(ppm)
b)
after the compound passed through the reverse-phase column at
room temperature (rt) using MeOH-H2O as the solvent. We then
attempted synthesis of 2-DL using an alternative scheme in which the
deprotection reaction order was exchanged (Scheme 3). Removal of
phosphonate methyl groups was carried out using BSTFA and TMSBr,
followed by treatment with MeOH and H2O to give compound 12 at
89% yield16. The remaining TBDPS was removed using TBAF/AcOH to
2nd fraction
1-DL (Free)
4.5
1
give DHA LPA at 94% yield. H-NMR analysis in CD3OD showed that
the peak at 5 ppm was retained, and there was a corresponding peak
for tetrabutylammonium ion (Fig. S1). The major DHA LPA was 2-DL
(2-DL/1-DL = 79:21), indicating that acyl migration was suppressed
by changing the deprotection reaction order. In addition, 1H-NMR
and high-resolution ESI-MS data revealed that one phosphate ion
forms a salt with one ammonium ion.
5.0
4.0
3.5
(ppm)
Fig. 3 1H NMR of in situ experiment (Scheme 4). a) The chart of 1st
fraction is identical to that of 2-DL :NH4+ in an experiment obtained
in Scheme 3. b) The chart of 2nd fraction, is identical to that of 1-DL
in an experiment obtained by Scheme 2.
Comparison of salt and free form in situ
We designed another experiment to confirm that acyl migration is
suppressed by formation of a salt containing ammonium ion. We
expected that moderate suppression of acyl migration would result
from adding only 0.5 equivalents of ammonium formate to the
reaction. Indeed, deprotection proceeded readily. Interestingly, the
product was separated into two fractions by reverse-phase open-
column chromatography (Scheme 4). Unexpectedly, the reaction
yielded ammonium salt and free forms of DHA LPA at 50 and 28%
yields, respectively, for a total yield of 78%. 1H-NMR analysis
revealed that the major species in the first fraction was 2-DL
ammonium salt (2-DL/1DL = 80:20), whereas the major species in the
second fraction was the free form of 1-DL (2-DL/1DL = 25:75) (Fig. 3).
Synthesis of pure 1-DL
Certainly, acyl migration from 1-DL to 2-DL also occurs (Scheme 2).
Thus, we applied this condition to synthesize 1-DL. The deprotection
Scheme 4 The deprotection reaction in the presence of 0.5
equivalent of ammonium ion. The salt form and free form of
DHA-LPA were separated by column chromatography.
Fig. 2 The structures of liso-bis-phosphatidic acid (10) and 1-
oleoyl-lysophosphatidic acid (11).
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applied to the reaction (entries 8 and 9), the reaction produced yields
DOI: 10.1039/C7OB01771E
of 86 and 84% and 2-DL/1-DL ratios of 83:17 and 88:12, respectively.
In addition to these yields, under crude conditions, the ratios were
94:6 in both cases, better than that obtained using ammonium
formate (entries 1-6).
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Table 1 Determination of limitation for the suppression of acyl
migration of 2-DLa
We also examined the effect of divalent cations. Unfortunately,
sticky products were obtained in reactions applying calcium formate
or magnesium formate, which are insoluble in both organic solvents
and water; thus, we could not determine the structure of the
product.
We compared with and without ammonium formate buffer for
storage condition of 2-DL. In 100 mM ammmoium formate (pH4.0)
20 mM of 2-DL, no acyl migration occur at -80 °C at least 18 months.
However 2-DL ammonium salt alone occurs more than 40% of the
compound migrated to 1-DL in 5 months.
Yielda,b
(%)
76
Ratioc
Entry
Salt
pH Temp.
2-DL : 1-DL
1
2
3
4
5
6
7
8
9
HCO2NH4 4.0
HCO2NH4 4.0
rt
79 : 21_______
78 : 22________
83 : 17 (89 : 11)d
81 : 19________
83 : 17________
83 : 17 (90 : 10) d
19 : 81 (22 : 78) d
83 : 17 (94 : 6) d
0 °C
78
HCO2NH4 4.0 −78 °C
70
HCO2NH4 7.8
HCO2NH4 7.8
rt
73
Discussion
0 °C
78
LPAs are important chemical mediator biomolecules. Studies of LPAs
usually employ the 1-acyl form instead of the 2-acyl form. This is the
first report of organic synthesis of 2-acyl LPA. Our results revealed
that salt formation involving LPA suppresses acyl migration.
However, the mechanism remains to be elucidated. One possibility is
that the phosphate group directly enhances migration.It is also
possible that formation of the salt induces a conformational change.
When we applied 0.5 equivalents of ammonium salt, both salt and
free forms of DHA-LPA were isolated using ODS-silica gel column
chromatography. The salt form of DHA-LPA suppressed acyl
migration, but the free-LPA did not. If the ion pairs are exchangeable,
a certain level of suppression would reflect the presence of both salt
and free-LPA. This indicates that the ion pairs are not exchangeable
in situ; in other words, formation of the salt between phosphate and
ammonium ions is stable. Thus, it can be inferred that the salt form
LPA exists in a stable conformation, which inhibits the acyl migration
reaction.
There is concern as to whether synthetic 2-acyl LPA is meaningful in
biological assaysbecause acyl migration readily occurs with 2-acyl
LPA. Our experiments (Table 1) were performed over the pH 4.0-7.8,
which covers neutral conditions. This suggests that 2-acyl LPA can
exist at physiologic pH.
Several species of cations are present in serum at significant
concentrations. Reported concentration of Na+, K+, Ca2+and Mg2+ in
serum (cells) are 142 mM (10 mM), 4.5 mM (141 mM), 2.25 mM (0.5
mM) and 1.15 mM (30 mM)19, 20, 21, respectively. The physiologic
concentrations of Na+ and K+ thus exceed those of our reaction
conditions (100 mM). Thus, these monovalent cations could play a
role in suppressing acyl migration.
HCO2NH4 7.8 −78 °C
60
−
−
rt
rt
rt
65
HCO2Na
HCO2K
6.8
7.1
86
88 : 12 (94 : 6) d
84
a95% MeOH aq. was used for entries 1-6 and 8,9and 90% MeOH aq.
was used for entry 7 as a solvent. bIsolated yield of DHA-LPA as
mixture of 2-DL and 1-DL. cRatio was determined by 1H NMR
analysis.d Ratio of 2-DL and 1-DL under crude condition.
reaction in the presence of ammonium ion yielded pure 1-DL at 93%
yield (Scheme S1).
Determination of limitation for suppression of the acyl migration of
2-DL
We also examined the importance of pH, temperature, and cation
dependence for the suppression of acyl migration (Table 1). At pH
4.0, the reaction produced LPA at both temperatures (rt [0°C] and
−78°C) at moderate yield, with a 2-DL/1-DL ratio of about 8:2 (entries
1-3). These conditions did not produce a significant difference in yield
or ratio. At pH 7.8, the reaction also proceeded at all temperatures
examined (entries 4-6). No significant improvements in yield (~80%)
or ratio (2-DL/1-DL
= 8:2) were observed with change in
temperature. Overall, no significant differences were observed
between pH 4.0 and 7.8 (entries 1-6). Interestingly, the crude 2-DL/1-
DL ratio was 9:1 (entries 3 and 6, parentheses). This could indicate
that slight acyl migration occurred during purification using open-
column chromatography. In the reaction without salt, acyl migration
occurred even under crude conditions (entry 7, parentheses).
We then conducted experiments to determine the effect of
counter cations on the suppression of acyl migration. Thus, we
planned to use physiologically relevant metal ions instead of
ammonium ion. When sodium formate and potassium formate were
Hama and co-workers showed that activation of LPA receptors by
LPAs is regulated by the albumin concentration, and they suggested
that albumin plays a shuttling LPA22 to LPA receptors. Their
hypothesis is persuasive, given that crystal structure analysis
revealed six fatty acid binding sites in albumin complexed with
myristic acid23. Crystal structures of albumin in complex with a
glycerophospholipid, lysophosphatidylethanolamine. Intriguingly,
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lysophosphatidylethanolamine forms
ARTICLE
a
salt bridge between 127.76 (3 carbons), 127.78 (2 carbons), 127.83, 127.99, 128.04,
View Article Online
DOI: 10.1039/C7OB01771E
phosphate and an arginine residue. The ammonium concentration in 128.06, 128.20, 128.22, 128.24, 128.51, 129.32, 129.85 (2 carbons),
blood is low (~40 μM) 24, but the concentration of serum albumin, 131.93, 132.84, 132.88, 135.45 (2 carbons), 135.52 (2 carbons),
1
which contains multiple lipophilic binding sites, is 0.6 mM25. Thus, 2- 172.11; HRMS (ESI+) calcd. for 12C43 H6123Na116O731P128Si1 771.38218,
acyl LPA could be stabilized by formation of a salt bridge with basic found 771.38662.
albumin residues as well as by the formation of salts with various
cations such as Na+ and K+ and so on.
(R)-1-((Dimethoxyphosphoryl)oxy)-3-hydroxypropan-2-
yl(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate (7).
Synthesized 2-DL, contains approximately 10% (crude: 6%) of 1- To a solution of compound 6 (1.0 g, 1.34 mmol) and acetic acid (573
DL, but pure 1-DL would be a useful control to compare the biological μL, 10.0 mmol) in THF (33.9 mL) was added TBAF (1.0 M solution in
data.
THF, 2.0 mL, 2.0 mmol). The mixture was stirred for 5 h. After
evaporation, the residue was chromatographed on silica gel (10 g,
firstly hexane/ AcOEt, 2 : 1, secondly hexane/ AcOEt, 1 : 2) to give 7
(54.8 mg, 80%). 1H NMR (CDCl3): δ0.97 (3H, t, J = 7.5 Hz, -CH=CH-CH2-
CH3), 2.00–2.15 (2H, m, -CH=CH-CH2-CH3), 2.32–2.49 (4H, m, -CH=CH-
CH2-CH2-CO2-), 2.72–2.94 (10H, m, -CH=CH-CH2-CH=CH-), 3.70–3.87
(8H, s, -P-O-CH3, -O-CH2-CH(OCO)-CH2-O-P-), 4.25 (2H, dd, J = 8.9, 4.7
Conclusions
We succeeded first synthesis of 2-DL and 1-DL by suppression
of acyl migration. It revealed that formation of salt is important
for the suppression of acyl migration. Since our synthetic target
was involving DHA, which is one the most unstable poly Hz, -O-CH2-CH(OCO)-CH2-O-P-), 5.00–5.09 (1H, m, -O-CH2-CH(OCO)-
CH2-O-P- ), 5.26–5.50 (12H, m, -CH=CH-). 13C NMR (CDCl3): δ = 14.29,
20.56, 22.68, 25.55, 25.60, 25.64 (3 carbons), 34.06, 54.54, 54.62,
60.09, 65.46 (d, J = 5.4 Hz), 72.54 (d, J = 6.3 Hz), 127.02, 127.73,
127.87, 127.98, 128.07 (2 carbons), 128.27 (2 carbons), 128.32,
unsaturated fatty acid, this method may be applicable to the
synthesis of LPAs containing various kinds of unsaturated fatty
acids.
128.55, 129.44, 132, 172.50; HRMS (ESI+) calcd. for
Experimental section
12
C
1H4323Na116O731P1 533.26441, found 533.26141.
27
(R)-1-((tert-butyldiphenylsilyl)oxy)-3-(phosphonooxy)propan-2-
(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate
yl
General Information:
(12). Compound 6 (0.116 g, 0.155 mmol) was dissolved in CH2Cl2 (2.2
mL) and treated with BSTFA (152 μL, 0.57 mmol) and TMSBr (67 μL,
0.51 mmol) at 0 °C. The mixture was stirred for 2 h at rt. After
evaporation, the residue was dissolved in H2O/MeOH, 1 : 9 (4.3 mL)
and stirred for 30 min. After evaporation, the residue was
chromatographed on reversed phase silica gel (1.0 g, MeOH/H2O, 7 :
1) to give 12 (99.0 mg, 89%). 1H NMR (CDCl3): δ0.96 (3H, t, J = 7.5 Hz,
-CH=CH-CH2-CH3), 1.01 (9H, s, -Si-C(CH3)3), 1.95–2.16 (2H, m, -
CH=CH-CH2-CH3), 2.18–2.48 (4H, m, -CH=CH-CH2-CH2-CO2-), 2.67–
2.94 (10H, m, -CH=CH-CH2-CH=CH-), 3.76 (2H, d, J = 4.2 Hz, -O-CH2-
CH(OCO)-CH2-O-P-), 4.04–4.34 (2H, m, -O-CH2-CH(OCO)-CH2-O- P-),
5.09–5.21 (1H, m, -O-CH2-CH(OCO)-CH2-O-P-), 5.23–5.46 (12H, m, -
CH=CH-), 7.28–7.45 (6H, m, m-Ar-H and p-Ar-H), 7.54–7.74 (4H, m, o-
Ar-H). 13C NMR (CDCl3): δ = 14.48, 19.37, 20.75, 22.70, 25.73 (2
carbons), 25.81 (3 carbons), 26.90 (3 carbons), 34.25, 62.10, 65.52,
72.85, 127.21, 127.97 (5 carbons), 128.07, 128.28 (2 carbons), 128.34,
128.41, 128.45 (2 carbons), 128.75, 129.46, 130.01 (2 carbons),
132.21, 133.07, 133.14, 135.67 (2 carbons), 135.74 (2 carbons),
DHA were prepared by hydrolysis of DHA ethyl ester. All other
reagents were purchased from commercial sources. 1H NMR spectra
were recorded at 300 MHz using TMS as an internal standard.
13CNMR spectra were recorded at 75 MHz with CDCl3 (77.0 ppm) and
CD3OD with TMS as reference. High resolution mass spectra were
obtained with a JEOL AccuTOF LC-plus JMS-T100LP spectrometer. All
air and moisture sensitive reactions were carried out under nitrogen
atmosphere.
(R)-1-((tert-Butyldiphenylsilyl)oxy)-3-
((dimethoxyphosphoryl)oxy)propan-2-yl(4Z,7Z,10Z,13Z,16Z,19Z)-
docosa-4,7,10,13,16,19-hexaenoate (6).To a solution of compound
5 (1.00 g, 2.28 mmol), DHA (0.82 g, 2.51 mmol) and DMAP (0.168 g,
1.37 mmol) in CH2Cl2 (37.9 mL) was added EDC・HCl (0.656 g, 3.42
mmol). The mixture was stirred for 2 h. After quenching with water,
the mixture was extracted with CH2Cl2. The organic layer was washed
with water and brine, dried over MgSO4, and concentrated. The
residue was flash-chromatographed on silica gel (100 g, firstly
hexane/ AcOEt, 5 : 1, secondly hexane/ AcOEt, 3 : 1) to give 6 (1.46
1
173.43; HRMS (ESI–) calcd. for 12C41 H5616O731P128Si1 719.35329, found
1
g, 85%). H NMR (CDCl3): δ0.97 (3H, t, J = 7.5 Hz, -CH=CH-CH2-CH3),
719.35110.
1.05 (9H, s, -Si-C(CH3)3), 1.99–2.14 (2H, m, -CH=CH-CH2-CH3), 2.25–
2.43 (4H, m, -CH=CH-CH2-CH2-CO2-), 2.74–2.92 (10H, m, -CH=CH-CH2-
CH=CH-), 3.71 (3H, s, -P-O-CH3), 3.75 (3H, s, -P-O-CH3), 3.78 (2H, d, J
= 5.5 Hz, -O-CH2-CH(OCO)-CH2-O-P-), 4.18–4.37 (2H, m, -O-CH2-
CH(OCO)-CH2-O-P-), 5.09–5.19 (1H, m, -O-CH2-CH(OCO)-CH2-O-P-),
5.25–5.46 (12H, m, -CH=CH-), 7.34–7.48 (6H, m, m-Ar-H and p-Ar-H ),
7.59–7.71 (4H, m, o-Ar-H); 13C NMR (CDCl3): δ = 14.27, 19.18, 20.54,
22.58, 25.53, 25.56, 25.62 (3 carbons), 26.69 (3 carbons), 34.02,
54.25, 54.33, 61.72, 65.63 (d, J = 5.4 Hz), 72.18 (d, J = 7.3 Hz), 127,
(R)-1-Hydroxy-3-(phosphonooxy)propan-2-
yl(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate (2-
DL(Tetrabutylammonium salt)). To a solution of compound 12 (40
mg, 0.056 mmol) and acetic acid (9.6 μL, 0.17 mmol) in THF (1.4 mL)
was added TBAF (1.0 M solution in THF, 167μL, 0.167 mmol). The
mixture was stirred for 1 h. After evaporation, the residue was
reversed phase silica gel (0.8 g, MeOH/ H2O,
chromatographed on silica gel (0.9 g, firstly only AcOEt, secondly
MeOH/ AcOEt, 1 : 1) to give DHA-LPA (Tetrabutylammonium salt)(
3 : 1) and
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2-DL : 1-DL = 79 : 21, 38 mg, 94%). 1H NMR (CD3OD): δ0.97 (3H, t, J = 25.70, 25.75 (3 carbon), 34.06, 54.71 (d, J = 2.4 Hz), 54.79 (d, J = 2.4
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7.5 Hz, -CH=CH-CH2-CH3), 1.02 (12 H, t, J = 7.3 Hz, N+-CH2-CH2-CH2- Hz), 64.60, 68.67 (d, J = 5.9 Hz), 68.84 (d, J = 5.7 Hz), 127.13, 127.84,
DOI: 10.1039/C7OB01771E
CH3), 1.34–1.49 (8H, m, N+-CH2-CH2-CH2-CH3), 1.58–1.74 (8H, m, N+- 127.98, 128.10, 128.19, 128.20, 128.37, 128.39, 128.44, 128.68,
CH2-CH2-CH2-CH3), 2.02–2.15 (2H, m, -CH=CH-CH2-CH3), 2.31–2.45 129.59, 132.14, 173.14; HRMS (ESI+) calcd. for C27H43Na1O7P1
(4H, m, -CH=CH-CH2-CH2-CO2-), 2.77–2.93 (10H, m, -CH=CH-CH2- 533.26441, found 533.26329.
CH=CH-), 3.19–3.28 (8H, m, N+-CH2-CH2-CH2-CH3), 3.66–3.80 (2H, m,
(R)-2-Hydroxy-3-(phosphonooxy)propyl(4Z,7Z,10Z,13Z,16Z,19Z)-
-O-CH2-CH(OCO)-CH2- O-P-), 3.94–4.04 (2H, m, -O-CH2-CH(OCO)-CH2- docosa-4,7,10,13,16,19-hexaenoate (1-DL (free)). Compound 8 (50
O-P-), 4.93–5.02 (1H, m, -O-CH2- CH(OCO)-CH2-O-P-), 5.25–5.45 (12H, mg, 0.099 mmol) was dissolved in CH2Cl2 (1.4 mL) and treated with
m, -CH=CH-); 13C NMR (CDCl3): δ =172.8, 132.2, 129.3, 128.7, 128.4 BSTFA (96.2 μL, 0.36 mmol) and TMSBr (42.7 μL, 0.324 mmol) at 0 °C.
(2 carbons), 128.3 (2 carbons), 128.26, 128.2, 128.0, 127.2, 74.10, The mixture was stirred for 2 h at rt. After evaporation, the residue
74.05, 69.8, 65.9, 61.4, 61.3, 59.4, 58.8 (2 carbons), 34.4, 25.8 (4 was dissolved in H2O/MeOH, 1 : 9 (2.7 mL) and stirred for 30 min.
carbons), 25.72, 25.69, 24.2 (4 carbons), 22.9, 20.7, 19.8 (4 carbons), After evaporation, the residue was chromatographed on reversed
14.4, 13.9 (4 carbons); HRMS (ESI–) calcd. for C25H38O7P1 481.23551, phase silica gel (0.9 g, firstly MeOH/H2O, 2 : 1, secondly MeOH/H2O,
found 481.23659; HRMS (ESI+) calcd. for C16H36N1 242.28477, found 3 : 1) to give 1-DL (1-DL : 2-DL = 85 : 15, 38 mg, 79%). 1H NMR
242.28935.
(R)-1-Hydroxy-3-(phosphonooxy)propan-2-
(CD3OD): δ0.97 (3H, t, J = 7.5 Hz, -CH=CH-CH2-CH3), 2.01–2.15 (2H, m,
-CH=CH-CH2-CH3), 2.32–2.49 (4H, m, -CH=CH-CH2-CH2-CO2-), 2.73–
yl(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate (2- 2.96 (10H, m, -CH=CH-CH2-CH=CH-), 3.91–4.04 (3H, m, -OCO-CH2-
DL (ammonium salt)). Compound 7 (0.10 g, 0.196 mmol) was CH(OH)-CH2-O-P-), 4.11 (1H, 1/2 ABqt, J = 18.1, 5.3 Hz, -OCO-CH2-
dissolved in CH2Cl2 (2.7 mL) and treated with BSTFA (193 μL, 0.725 CH(OH)-CH2-O-P-), 4.19 (1H, 1/2 ABqt, J = 18.1, 4.2 Hz, -OCO-CH2-
mmol) and TMSBr (85.3 μL, 0.646 mmol) at 0 °C. The mixture was CH(OH)-CH2-O-P-), 5.24–5.46 (12H, m, -CH=CH-); HRMS (ESI–) calcd.
stirred for 2 h at rt. After evaporation, the residue was dissolved in for C25H38O7P1 481.23551, found 481.23826.
100 mM HCO2NH4 H2O/MeOH (pH 7.8) (4.9 mL) and stirred for 30 min.
After evaporation, the residue was chromatographed on reversed docosa-4,7,10,13,16,19-hexaenoate
(R)-2-Hydroxy-3-(phosphonooxy)propyl(4Z,7Z,10Z,13Z,16Z,19Z)-
(1-DL(ammonium salt)).
phase silica gel (1.6 g, MeOH/ H2O, 2 : 1) to give 2-DL (ammonium Compound 8 (100 mg, 0.196 mmol) was dissolved in CH2Cl2 (2.7 mL)
salt) (2-DL : 1-DL = 88 : 12, 89 mg, 91%). 1H NMR (CD3OD): δ0.97 (3H, and treated with BSTFA (192 μL, 0.72 mmol) and TMSBr (85 μL, 0.64
t, J = 7.5 Hz, -CH=CH-CH2-CH3), 2.01–2.16 (2H, m, -CH=CH-CH2-CH3), mmol) at 0 °C. The mixture was stirred for 2 h at rt. After evaporation,
2.29–2.47 (4H, m, -CH=CH-CH2-CH2-CO2-), 2.75–2.96 (10H, m, - the residue was dissolved in acidic 100 mM HCO2NH4 H2O/MeOH (pH
CH=CH-CH2-CH=CH-), 3.65–3.80 (2H, m, -O-CH2-CH(OCO)-CH2-O-P-), 7.8) (4.9 mL) and stirred for 30 min. After evaporation, the residue
3.98 (2H, dd, J = 6.2, 5.3 Hz, -O-CH2-CH(OCO)-CH2-O-P-), 4.94–5.03 was chromatographed on reversed phase silica gel (1.6 g, MeOH/
1
(1H, m, -O-CH2-CH(OCO)-CH2-O-P-), 5.24–5.47 (12H, m, -CH=CH-); 13
C
H2O, 2 : 1) to give 1-DL(ammonium salt) (90.7 mg, 93%). H NMR
NMR (CDCl3): δ = 14.47, 20.74, 22.70, 25.72, 25.79 (4 carbons), 34.19, (CD3OD): δ0.97 (3H, t, J = 7.5 Hz, -CH=CH-CH2-CH3), 2.01–2.15 (2H, m,
60.52, 63.62, 73.13, 127.19, 127.97, 128.03, 128.16, 128.22, 128.25, -CH=CH-CH2-CH3), 2.30–2.46 (4H, m, -CH=CH-CH2-CH2-CO2-), 2.74–
128.48 (2 carbons), 128.59, 128.75, 129.59, 132.18, 173.34; HRMS 2.95 (10H, m, -CH=CH-CH2-CH=CH-), 3.83–3.92 (2H, m -OCO-CH2-
(ESI–) calcd. for C25H38O7P1 481.23551, found 481.23163.
(R)-3-((Dimethoxyphosphoryl)oxy)-2-
hydroxypropyl(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-
CH(OH)-CH2-O-P-), 3.92–4.02 (1H, m, -OCO-CH2-CH(OH)-CH2-O-P-),
4.11 (1H, 1/2 ABqt, J = 24.13, 6.0 Hz, -OCO-CH2-CH(OH)-CH2-O-P-),
4.17 (1H, 1/2 ABqt, J = 24.13, 4.3 Hz, -OCO-CH2-CH(OH)-CH2-O-P-),
hexaenoate (9). To a mixture of DHA (0.40 g, 1.2 mmol) in CH2Cl2 (4.6 5.24–5.47 (12H, m, -CH=CH-); 13C NMR (CDCl3): δ = 14.46, 20.73,
mL) and DMF (50 μL) was slowly added oxalyl chloride (2.0 M in 22.68, 25.71, 25.79 (4 carbon), 34.05, 65.10, 66.95, 69.04, 127.19,
CH2Cl2, 0.73 mL, 1.46 mmol). The reaction mixture was stirred for 1 128.02 (2 carbon), 128.18, 128.22, 128.25, 128.47 (2 carbon), 128.55,
h. After evaporation, this compound was used as such in next step of 128.74, 129.51, 132.17, 173.73; HRMS (ESI–) calcd. for C25H38O7P1
the synthesis. A solution of diol 8 (0.24 g, 1.22 mmol) in CH2Cl2 (6.1 481.23551, found 481.23454.
mL) was cooled to -78 °C. To the mixture was carefully added DHA-Cl
(R)-1-Hydroxy-3-(phosphonooxy)propan-2-
dissolved in CH2Cl2 (6.0 mL) at -78 °C. The mixture was stirred for 2 h yl(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate (2-
at -78 °C. After evaporation, the mixture was poured into 2M HCl aq. DL (sodium salt)). Compound 7 (10.0 mg, 0.0196 mmol) was
and extracted with AcOEt. The organic layer was washed with brine, dissolved in CH2Cl2 (0.3 mL) and treated with BSTFA (19.3 μL, 0.0727
dried over MgSO4, and evaporated. The residue was mmol) and TMSBr (8.5 μL, 0.0644 mmol) at 0 °C. The mixture was
chromatographed on silica gel (10 g, firstly hexane/ AcOEt, 2 : 1, stirred for 2 h at rt. After evaporation, the residue was dissolved in
secondly hexane/ AcOEt, 1 : 2, finally only AcOEt) to give 9 (0.36 g, 100 mM HCO2Na H2O/MeOH (pH 6.8) (0.5 mL) and stirred for 30 min.
58%). 1H NMR (CDCl3): δ0.97 (3H, t, J = 7.5 Hz, CH3-), 2.01–2.14 (2H, After evaporation, the residue was chromatographed on reversed
m, -CH=CH-CH2-CH3), 2.32–2.48 (4H, m, -CH=CH-CH2-CH2-CO2-), phase silica gel (0.9 g, MeOH/ H2O, 2 : 1) to give 2-DL (sodium salt)
2.74–2.92 (10H, m, -CH=CH-CH2-CH=CH-), 3.18 (1H, d, J = 4.8 Hz, - 2-DL : 1-DL = 83 : 17 (8.5 mg, 86%). 1H NMR was identical to the 2-
OCO-CH2-CH(OH)-CH2-O-P-), 3.79 (3H, s, -P-O-CH3), 3.82 (3H, s, -P-O- DL (ammonium salt); MS (ESI–) m/z: 481.2 [M-H]–. HRMS (ESI–) calcd.
CH3), 4.03–4.29 (5H, m, -OCO-CH2-CH(OH)-CH2-O-P-), 5.26–5.48 (12H, for C25H38O7P1 481.23551, found 481.23226. MS (ESI+) m/z: 527.2
m, -CH=CH-); 13C NMR (75 MHz, CDCl3): δ = 14.39, 20.67, 22.78, 25.66,
6 | J. Name., 2012, 00, 1-3
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[M−H+2Na]+; HRMS (ESI+) calcd. for C25H38Na2O7P1 527.21505, found
527.21181.
7
8
9
Y. Xu and G. D. Prestwich, J. Org. Chem., 2002, 67, 7158–
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Prestwich, Org. Lett., 2003, 5, 4685–4688.
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Shimizu, J. Aoki, H. Arai and M. Shibasaki, Angew. Chemie -
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G. G. Durgam, T. Virag, M. D. Walker, R. Tsukahara, S.
Yasuda, K. Liliom, L. A. Van Meeteren, W. H. Moolenaar, N.
Wilke, W. Siess, G. Tigyi and D. D. Miller, J. Med. Chem.,
2005, 48, 4919–4930.
G. Jiang, Y. Xu, Y. Fujiwara, T. Tsukahara, R. Tsukahara, J.
Gajewiak, G. Tigyi and G. D. Prestwich, ChemMedChem,
2007, 2, 679–690.
M. Kurano, A. Suzuki, A. Inoue, Y. Tokuhara, K. Kano, H.
Matsumoto, K. Igarashi, R. Ohkawa, K. Nakamura, T. Dohi,
K. Miyauchi, H. Daida, K. Tsukamoto, H. Ikeda, J. Aoki and
Y. Yatomi, Arterioscler. Thromb. Vasc. Biol., 2015, 35, 463–
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(R)-1-Hydroxy-3-(phosphonooxy)propan-2-
yl(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate (2-
DL (potassium salt)). Compound 7 (10.2 mg, 0.0200 mmol) was
dissolved in CH2Cl2 (0.3 mL) and treated with BSTFA (19.3 μL, 0.0727
mmol) and TMSBr (8.5 μL, 0.0644 mmol) at 0 °C. The mixture was
stirred for 2 h at rt. After evaporation, the residue was dissolved in
100 mM HCO2K H2O/MeOH (pH 7.1, 0.5 mL) and stirred for 30 min.
After evaporation, the residue was chromatographed on reversed
phase silica gel (0.9 g, MeOH/ H2O, 2 : 1) to give2-DL (potassium salt)
mixture 2-DL : 1-DL = 7.68 : 1 (8.7 mg, 84%). 2-DL (potassium salt) 1H
10
11
NMR was identical to the 2-DL (ammonium salt); MS (ESI–) m/z :
12
13
12
481.2 [M-H]–. HRMS (ESI–) calcd. for
C
1H3816O731P1 481.23551,
25
found 481.23456; MS (ESI+) m/z : 559.2 [M−H+2K]+. HRMS (ESI+)
calcd. for C25H38K2O7P1 559.16293, found 559.16209; MS (ESI+) m/z :
527.2 [M−H+2Na]+. HRMS (ESI+) calcd. for C25H38Na2O7P1 527.21505,
found 527.21731.
In situ experiment for suppression of migration. Compound 9 (50
mg, 0.098 mmol) was dissolved in CH2Cl2 (1.4 mL) and treated with
BSTFA (96.2 μL, 0.362 mmol) and TMSBr (43 μL, 0.324 mmol) at 0 °C.
The mixture was stirred for 2 h at rt. After evaporation, the residue
was dissolved in acidic 100 mM HCO2NH4 H2O/MeOH (pH 7.8) (0.49
mL) and stirred for 30 min. After evaporation, the residue was
chromatographed on reversed phase silica gel (0.9 g, firstly MeOH/
H2O, 2 : 1, secondly MeOH/ H2O, 3 : 1) to give first fraction and second
fraction. First fraction gave 2-DL : 1-DL = 81 : 19 (24.6 mg, 0.0492
mmol) and second fraction gave 2-DL : 1-DL = 24 : 76 (13.3 mg, 0.0276
mmol). The first fraction MS (ESI–) m/z : 481.2 [M-H]–. HRMS (ESI–)
14
15
16
17
18
1
calcd. for 12C25 H3816O731P1 481.23551, found 481.23066. The second
fraction MS (ESI–) m/z: 481.2 [M-H]–. HRMS (ESI–) calcd. for
19
12
C
1H3816O731P1 481.23551, found 481.23629.
25
Acknowledgements
This work was financially supported by the Platform for Drug
Discovery, Informatics, and Structural Life Science from the
Japan Agency for Medical Research and Development (AMED)
to K.Y., and T.I., and a Grant-in-Aid for Scientific Research (No.
25860090) from MEXT to T.I..
20
21
22
23
24
25
Notes and references
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