Synthesis of chemically edited derivatives of the endogenous regulator of inflammation 9-PAHSA

Article abstract of DOI:10.1038/s41429-019-0180-1

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a growing class of natural products found in organisms ranging from plants to humans. The roles these endogenous derivatives of fatty acids play in biology and their novel pathways for controlling inflammation have increased our understanding of basic human physiology. FAHFAs incorporate diverse fatty acids into their structures, however, given their recent discovery non-natural derivatives have not been a focus and as a result structure-activity relationships remain unknown. The importance of the long chain hydrocarbons extending from the ester linkage as they relate to anti-inflammatory activity is unknown. Herein the systematic removal of carbons from either the hydroxy fatty acid or fatty acid regions of the most studied FAHFA, palmitic acid ester of 9-hydroxystearic acid (9-PAHSA), was achieved and these synthetic, abridged analogs were tested for their ability to attenuate IL-6 production. Reduction of the carbon chain lengths of the 9-hydroxystearic acid portion or palmitic acid hydrocarbon chain resulted in lower molecular weight analogs that maintained anti-inflammatory activity or in one case enhanced activity.

Full text of DOI:10.1038/s41429-019-0180-1

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0180-1
SPECIAL FEATURE: BRIEF COMMUNICATION
Synthesis of chemically edited derivatives of the endogenous
regulator of inflammation 9-PAHSA
Huijing Wang1 Tina Chang2 Srihari Konduri1 Jianbo Huang1 Alan Saghatelian2 Dionicio Siegel1
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Received: 21 February 2019 / Revised: 14 March 2019 / Accepted: 18 March 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a growing class of natural products found in organisms ranging from
plants to humans. The roles these endogenous derivatives of fatty acids play in biology and their novel pathways for
controlling inflammation have increased our understanding of basic human physiology. FAHFAs incorporate diverse fatty
acids into their structures, however, given their recent discovery non-natural derivatives have not been a focus and as a result
structure-activity relationships remain unknown. The importance of the long chain hydrocarbons extending from the ester
linkage as they relate to anti-inflammatory activity is unknown. Herein the systematic removal of carbons from either the
hydroxy fatty acid or fatty acid regions of the most studied FAHFA, palmitic acid ester of 9-hydroxystearic acid (9-PAHSA),
was achieved and these synthetic, abridged analogs were tested for their ability to attenuate IL-6 production. Reduction of
the carbon chain lengths of the 9-hydroxystearic acid portion or palmitic acid hydrocarbon chain resulted in lower molecular
weight analogs that maintained anti-inflammatory activity or in one case enhanced activity.
Introduction
diabetic characteristics, such as insulin sensitivity and glu-
cose homeostasis when compared with their wild-type
counterparts [1]. Further studies of AG4OX mice revealed
that a class of lipid-based metabolites was differentially
upregulated by more than 16-fold in their AT. Upon
structural reconstruction based on the detected masses,
fragmentations of these metabolites were derived from a
novel class of natural lipids called FAHFAs, which had
contributed to the favorable metabolic phenotypes in
AG4OX mice. Structurally unique, a FAHFA (Fig. 1)
is composed of a fatty acid chain and a hydroxyl fatty
acid chain linked by an ester bond. Targeted liquid
chromatography-mass spectrometry (LC-MS) found that at
least 16 different FAHFA families exist, which consisted of
four fatty acids and four hydroxy-fatty acids in different
combinations [2]. For each family, multiple regioisomers
can be found that differ in the site of the ester functional
group at junctions of chains. For instance, palmitic acid
esters of hydroxy stearic acids (PAHSAs) was one of the
most abundant FAHFA families reported in which eight
distinct PAHSA regioisomers (−5, −7, −8, −9, −10, −11,
−12, and −13) were identified to be the most upregulated
family members in adipose tissue of AG4OX mice [2].
Previous evidence has highlighted the therapeutic
potential of FAHFAs in mediating symptoms of insulin
resistance and inflammation. Compared with vehicle treated
FAHFAs (Fatty Acid esters of Hydroxy Fatty Acids) are
endogenous bioactive lipids with anti-diabetic and anti-
inflammatory activities. FAHFAs were discovered and
structurally elucidated through lipidomic analyses of adi-
pose tissues (AT) obtained from transgenic mice over-
expressing the glucose transporter 4 in adipose tissue
(AG4OX). Though obese, AG4OX mice exhibited anti-
This study is dedicated to Professor Samuel Danishefsky for his
tireless efforts to advance the field of organic chemistry and his
tremendous scientific contributions to the exploration of structurally
complex, and biologically important, natural products.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0180-1) contains supplementary
material, which is available to authorized users.
* Dionicio Siegel
drsiegel@ucsd.edu
1
Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California San Diego, 9500 Gilman Drive, La Jolla,
CA 92093-0934, USA
2
Clayton Foundation Laboratories for Peptide Biology, Salk
Institute for Biological Studies, 10010 North Torrey Pines Road,
La Jolla, CA 92037-1002, USA

H. Wang et al.
O
The similarity in pharmacodynamic activity could arise
through pseudosymmetry of FAHFAs where the fatty acid
and the long alkyl chain extending after the ester linkage of
the hydroxy-fatty acid would engender similar non-polar
interactions. Herein we report the syntheses of 9-PAHSA
analogs with varying lengths of the hydrocarbon chains
through an efficient synthetic route and the analogs poten-
tial in modulating the secretion of a proinflammatory
cytokine, IL-6, in LPS-stimulated RAW 264.7 cells.
O
9
CH3
HO2C
HO2C
HO2C
CH3
CH3
CH3
9-PAHSA
5-PAHSA
O
O
5
CH3
O
CH3
O
9
9-OAHSA
Results and discussion
Fig. 1 Representative FAHFA structures; 9-PAHSA, 5-PAHSA, and
9-OAHSA
Chemistry
mice, acute oral administration of 5-PAHSA or 9-PAHSA
improved glucose tolerance and enhanced insulin sensitivity
in insulin-resistant mice on high-fat diet (HFD). Specifi-
cally, consecutive gavage of 9-PAHSA in HFD-fed mice
demonstrated significant reduction in levels of proin-
flammatory cytokines, such as TNF and IL-1β in their AT
macrophages. Treatment of LPS-stimulated bone-marrow-
derived dendritic cells (BMDCs) with 9-PAHSA also
inhibited BMDC maturation and abrogated expressions of
cellular inflammation markers such as, CD80, CD86, CD40,
and MHCII at a dose-dependent manner. In addition, a
mouse colitis model was used to investigate the anti-
inflammatory activities of 9-PAHSA and determine whether
the effects were broadly applicable or disease specific. Mice
with chemically induced colitis that had been treated with
9-PAHSA showed significant improvements in clinical
outcomes, suggesting that the inflammation was remediated
by the FAHFA treatment [3]. As demonstrated by Kuda
et al. treatment of docosahexaenoic acid ester of 13-hydroxy
linoleic acid (13-DHAHLA) could also inhibit the secretion
of proinflammatory cytokines in LPS-induced mouse
macrophage cell line RAW 264.7, and in fact, at a higher
potency than 9-PAHSA [4]. Together, FAHFAs are
general anti-inflammatory agents with varying degrees of
activities that are modulated by their structural composi-
tions. In this study, our goal is to delineate the structure-
activity relationships in FAHFAs, specifically PAHSAs,
and the anti-inflammatory abilities attributed to their lengths
of carbon chains.
Synthesis of 9-PAHSA analogs through methylene editing
The synthesis of a set of 9-PAHSA analogs with differing
carbon chain length in either the 9-hydroxystearic acid
region after the ester linkage or in the palmitic acid frag-
ment required a systematically applicable synthetic strategy.
Previous syntheses of 9-PAHSA have been achieved and
succeeded in providing the material needed for biological
evaluation. In 2014 9-PAHSA was prepared in 11 steps
starting from 1,9-nonanediol and the synthesis was pivotal
in the early identification and biological characterization of
FAHFAs, however, with this step count the initial strategy
would not be applicable to the syntheses of a set of analogs
(Fig. 2a) [2]. In 2017, an improved synthetic strategy was
used to investigate the stereochemistry of endogenous
9-PAHSA (Fig. 2b) [5]. The use of nonracemic epi-
chlorohydrin and two epoxide opening reactions with
Grignard reagents proved successful in generating materials
which were used to characterize endogenous 9-PAHSA.
However, a further reduction of steps is required as
removing even one step could have dramatic implications
on the number of compounds needed to make a focused set
of 9-PAHSA analogs. These syntheses and related synth-
eses of FAHFAs [4, 6–8] provided insight into route
selection. To this goal a revised synthesis of 9-PAHSA and
derivatives was achieved over three steps initiated by
Grignard addition into methyl 9-oxononanoate (Fig. 2c).
To determine the importance of fatty acid and hydroxy
fatty acid hydrocarbon within 9-PAHSA as it relates to anti-
inflammatory activity methylenes were iteratively removed
from the tail portions of palmitic acid and 9-hydroxy stearic
acid (Fig. 3). Within 9-hydroxy stearic acid the carbons
spanning the region between the carboxylic acid and ester
linkage were left unperturbed. The syntheses of every
analog began with the addition of Grignard reagents
selectively into the aldehyde of methyl 9-oxononanoate
[9, 10], affording different hydroxy-fatty acid methyl esters
represented by 13 in good yields (Scheme 1). Esterification
The lack of an understanding of the structural compo-
nents needed for activity led to the syntheses and testing of
synthetic 9-PAHSA analogs. We aim to investigate whether
modifications in hydrocarbon chains can influence the
analogs’ abilities to mitigate inflammation following LPS
stimulation in vitro. Preliminary data generated indicated
that the R and S 9-PAHSA were equipotent in biological
assays (glucose uptake and GPR40 activation) but
were hydrolyzed at different rates in liver lysates [5].

Synthesis of chemically edited derivatives of the endogenous regulator of inflammation 9-PAHSA
a
O
Fig. 2 9-PAHSA syntheses
starting in 2014 to access
material for biological
evaluation
Yore et al (2014)
O
CH3
11 steps
HO
OH
7
HO2C
CH3
5.5 % yield
nonane-1,9-diol
7
9-PAHSA
b
Nelson et al (2017)
O
5 steps
O
O
CH3
Cl
66 % yield
HO2C
(S)-2-(chloromethyl)oxirane
CH3
7
R-9-PAHSA
c
this work
MeO2C
O
CH3
3 steps
70-77 % yield
O
CHO
m
HO2C
methyl 9-oxononanoate
CH3
n
7
been implicated in chronic inflammatory conditions such
as rheumatoid arthritis and cancer [11]. The rationale
behind selecting IL-6 as an inflammatory marker in
our experiments is that previous knowledge about FAH-
FAs, specifically 9-PAHSA and DHAHLA, and their
abilities to regulate IL-6 secretion in vitro has been
established [3, 4]. Based on previous studies, we sought to
understand the magnitude of the anti-inflammatory abil-
ities of synthetic 9-PAHSA analogs and whether
the activities were correlative to the number of hydro-
carbons present in the structure. To quantitate the anti-
inflammatory capabilities of these analogs, RAW 264.7
cells were stimulated with DMSO or LPS (100 ng/mL),
an endotoxin derived from Gram-negative bacteria.
Simultaneously, LPS-stimulated cells were treated with
synthetic analogs at either 25 or 50 µM for 20 h before the
culture media were collected and subjected to IL-6
quantification using a mouse IL-6 enzyme-linked immu-
nosorbent assay, or ELISA (Fig. 5) (for 50 µM data see
supporting information). To monitor potential cytotoxic
effects from the treatment, adherent cells were tested
for viability after media removal using an MTT assay
(see supporting information).
Reduction of the chain length of the 9-hydroxystearic
acid region of 9-PAHSA resulted analogs 1–4 that
approximately maintained the anti-inflammatory activity of
9-PAHSA. Similar activity could be obtained by editing of
the palmitic acid chain of 9-PAHSA with the effect max-
imized by replacement of palmitic acid with hexanoic acid,
compound 10, that shows statistical improvement in activity
compared to 9-PAHSA (5) (Student’s t-test, #p < 0.05,
compared to 9-PAHSA). This provides insight into the
structure-activity relationships of 9-PAHSA and additional
assessment of anti-inflammatory activities of the analogs in
the future will help statistically resolve the effects of the
bio-similar analogs relative to 9-PAHSA (5) given the
background of the IL-6 measurements.
truncate
O
O
CH3
HO2C
7
CH3
truncate
Fig. 3 Methylene deletion from 9-PAHSA to assess the importance of
hydrocarbon
of secondary alcohol 13 with the required acid chloride and
pyridine provided a FAHFA methyl ester 14 which when
subjected to selective hydrolysis of more accessible methyl
ester yielded the 9-PAHSA analogs 1–12 in relatively short
order. Deletion of hydrocarbon from palmitoyl chloride
(C16) down to acetyl chloride (C2) made abridged fatty
acid ester derivatives of 9-PAHSA providing 6–12 using the
methyl ester of 9-hydroxy stearic acid. More effort was
required to access derivatives with excised carbon from 9-
hydroxy stearic acid tail. Synthesis of 9-hydroxy stearic
acid (C18), 9-hydroxy heptadecanoic acid (C17), 9-hydroxy
pentadecanoic acid (C15), 9-hydroxy tridecanoic acid
(C13), and 9-hydroxy undecanoic acid (C11) were all
achieved to prepare abridged derivatives 1–4 and 9-PAHSA
(5) (Fig. 4). Similarly, all the steps proceeded in good
yield, providing the material for testing the IL-6 lowering
capabilities of the analogs. Using this strategy 11 analogs
and 9-PAHSA were synthesized through a total of 17
intermediates.
Biology
Interleukin-6 (IL-6) is a proinflammatory cytokine with
important roles in both innate and adaptive immune
responses and functions as the major mediator during the
acute phase of inflammation. Elevated levels of IL-6 have

H. Wang et al.
OH
b
a
MeO2C
CH3 9-oxononanoate
CHO
MeO2C
CH3
MeO2C
CH3
n
methyl oleate
13
n=0, 2, 4, 6, 7
O
O
CH3
O
CH3
m
m
d
c
O
MeO2C
CH3
HO2C
n
CH3
n
14
1-12
m = 0, 2, 4, 6, 8, 10, 12, 14
a
Scheme 1 Optimized syntheses of truncated 9-PAHSA analogs and 9-PAHSA starting from methyl 9-oxononanate. Conditions: (a) O3, NMO,
DCM, –78 °C, 93% yield; (b) alkyl-magnesium bromide, THF, –78 °C, 85–90% yield; (c) acyl chloride, pyr., CH2Cl2, 0 °C to 23 °C, 90–95%
yield; (d) LiOH·H2O, THF:H2O (1:1, v/v), 0 °C to 23 °C, 88–93% yield.
O
O
O
O
O
O
CH3
CH3
CH3
CH3
O
CH3
HO2C
HO2C
HO2C
HO2C
CH3
HO2C
CH3
7
7
1
7
O
O
O
CH3
CH3
O
HO2C
HO2C
CH3
7
7
8
2
O
CH3
CH3
O
CH3
7
9
7
3
O
O
O
O
CH3
O
HO2C
HO2C
HO2C
CH3
CH3
7
7
10
4
O
O
O
CH3
O
CH3
HO2C
HO2C
CH3
9-PAHSA (5)
CH3
7
7
11
12
O
CH3
O
CH3
CH3
CH3
7
7
6
Fig. 4 9-PAHSA and abridged analogs prepared from methyl 9-oxononanate possessing different hydrocarbon in the palmitic acid or 9-hydroxy
stearic acid region of 9-PAHSA
Conclusion
hydroxy stearic acid chain) to bioactivity. The synthetic
strategy used provided access to 11 truncate 9-PAHSA
analogs and the parent compound in sufficient quantities
to test for the compounds’ abilities to reduce inflammation.
Treatment of RAW 264.7 cells with LPS induced
an inflammatory response. Subsequent treatment with
The lack of structure-activity relationships for FAHFAs
prompted the syntheses and testing the 9-PAHSA variants
with methylene editing to determine the specific contribu-
tion of two region of hydrocarbon (fatty acid ester chain and

Synthesis of chemically edited derivatives of the endogenous regulator of inflammation 9-PAHSA
bubbled through the solution for additional 10 min. The
reaction mixture was allowed to warm to room temperature
and aged for 2 h. The reaction was concentrated and the
residue was dissolved in ethyl acetate (200 mL) and washed
with water (80 mL) and brine (50 mL), dried over sodium
sulfate, filtered, and concentrated to give a yellow oil. The oil
was purified by chromatography on silica gel (hexane:EtOAc
2:1) to provide methyl 9-oxononanoate (2.9 g, 93% yield) as a
colorless oil. The spectral data were same with the one in
published literature [9, 10].
Fig. 5 Testing methylene edited FAHFAs for anti-inflammatory activ-
ities. Relative IL-6 levels were quantitated after LPS-stimulated RAW
264.7 cells were co-treated with synthetic 9-PAHSA analogs for 20 h.
As a positive control, cells were treated with 25 µM of Dexamethasone,
a glucocorticoid used in the clinic as an anti-inflammatory medication.
Methylene edited FAHFAs were tested with most lipids showing a
reduction in IL-6 levels compared to LPS treated cells (Student’s t-test
compared to LPS control, *p-value < 0.05) furthermore compound 10
shows statistical improvement in activity compared to 9-PAHSA (5)
(Student’s t-test, #p < 0.05, compared to 9-PAHSA)
General procedure for preparation of derivatives of
the methyl ester of 9-hydroxy stearic acid (13)
To a stirred solution of methyl 9-oxononanoate [9] (500 mg,
2.7 mmol, 1.0 equiv) in dry THF (50 mL) was added the
selected Grignard reagent (4.0 mmol, 1.5 equiv) under a
nitrogen atmosphere at –78 °C. The reaction mixture was
stirred for 1 h at –78 °C and then saturated aqueous NH4Cl
solution (10 mL) was added in a single portion. The resulting
mixture was warmed to 23 °C and repeatedly extracted with
EtOAc (20 mL × 3). The combined organic extracts were
washed with brine (15 mL), dried over sodium sulfate, fil-
tered, and concentrated. The crude compound was purified via
careful silica gel column chromatography using hexane:
EtOAc to give the esters 13a-e. All the Grignard reagents we
used are shown below and commercially available, ethyl-
magnesium bromide solution (C2H5BrMg, 1.0 M in THF),
butylmagnesium chloride solution (C4H9ClMg, 2.0 M in
THF), hexylmagnesium bromide solution (C6H13BrMg,
0.8 M in THF), octylmagnesium bromide solution
(C8H17BrMg, 1.0 M in diethyl ether) and nonylmagnesium
bromide solution (C9H19BrMg, 1.0 M in diethyl ether).
Methyl 9-hydroxyundecanoate (13a): Rf = 0.2 (silica gel,
60:10 hexanes:EtOAc, KMnO4); 522 mg, 90% yield, col-
orless oil; 1H NMR (600 MHz, CDCl3) δ 3.65 (s, 3 H), 3.50
(m, 1H), 2.29 (t, J = 7.6 Hz, 2 H), 1.65–1.55 (m, 2 H),
1.54–1.35 (m, 4 H), 1.30 (m, 8 H), 0.92 (t, J = 7.5 Hz, 3 H);
13C NMR (151 MHz, CDCl3) δ 174.5, 73.4, 51.6, 37.0,
34.2, 30.3, 29.6, 29.3, 29.2, 25.7, 25.0, 10.0; IR (film,
cm−1): 2928, 2855, 1738, 1460, 1436, 1245, 1197, 1171;
HRMS (ESI) calc. for C12H24O3Na [M + Na]+: 239.1618,
obs. 239.1619.
9-PAHSA analogs and assessment of the resulting IL-6
levels provided a measurement of the anti-inflammatory
activity of each of the compounds. The removal of hydro-
carbon was successful in producing analogs that maintained
anti-inflammatory activity while also reducing the mole-
cular weight. One compound, 10, displayed statistically
improved activity relative to 9-PAHSA demonstrating the
potential importance of this approach. The finding that the
reduction of the chain length of 9-hydroxystearic acid of
9-PAHSA and editing of the palmitic acid chain results in
active compounds provides avenues for optimization of
9-PAHSA and other FAHFAs. In addition, continued
assessment of this series of compounds in related assays
evaluating anti-inflammatory activity will provide refine-
ment on the importance of the hydrocarbons of 9-PAHSA
as it relates to anti-inflammatory effects.
Experimental
Synthesis of methyl 9-oxononanoate
4-Methylmorpholine N-oxide monohydrate (5.9 g, 50.6 mmol,
3.0 equiv) was dehydrated by heating at 90 °C under high
vacuum overnight. Following the ozonolysis conditions of
Drussault [12], a 250 mL round-bottom flask equipped with
stir bar was charged with methyl oleate (5.0 g, 16.9 mmol, 1.0
equiv), the anhydrous 4-methylmorpholine N-oxide mono-
hydrate prepared above, and anhydrous DCM (100 mL).
Stirring was initiated, affording a clear solution. The reaction
mixture was cooled to –78 °C for 15 min before bubbling in a
mixture of ozone/oxygen. Conversion was complete within
10 min. The ozone generator was turned off and oxygen was
Methyl 9-hydroxytridecanoate (13b): Rf = 0.2 (silica gel,
60:10 hexanes:EtOAc, KMnO4); 557 mg, 85% yield, light
yellow oil; 1H NMR (600 MHz, CDCl3) δ 3.64 (s, 3H), 3.55
(m, 1H), 2.27 (t, J = 7.5 Hz, 2 H), 1.59 (m, 2H), 1.47–1.34
(m, 4 H), 1.34–1.21 (m, 12 H), 0.88 (t, J = 6.9 Hz, 3 H); 13
C
NMR (151 MHz, CDCl3) δ 174.4, 72.0, 51.6, 37.5, 37.3,
34.2, 29.6, 29.3, 29.2, 28.0, 25.7, 25.0, 22.9, 14.2; IR
(film, cm−1): 2928, 2856, 1740, 1436, 1249, 1197, 1171;
HRMS (ESI) calc. for C14H28O3Na [M + Na]+: 267.1931,
obs. 267.1933.

H. Wang et al.
Methyl 9-hydroxypentadecanoate (13c): Rf = 0.3 (silica
gel, 60:10 hexanes:EtOAc, KMnO4); 643 mg, 88% yield,
light yellow oil; 1H NMR (600 MHz, CDCl3) δ 3.66 (s, 3H),
3.58 (m, 1H), 2.30 (td, J = 7.6, 2.1 Hz, 2 H), 1.61 (m, 2H),
1.48–1.35 (m, 4 H), 1.29 (t, J = 9.0 Hz, 16 H), 0.88 (td, J =
6.9, 2.6 Hz, 3 H); 13C NMR (151 MHz, CDCl3) δ 174.5,
72.1, 51.6, 37.6, 37.5, 34.2, 32.0, 29.6, 29.5, 29.4, 29.2,
25.8, 25.7, 25.1, 22.8, 14.3. IR (film, cm−1): 2926, 2854,
1740, 1458, 1436, 1197, 1171; HRMS (ESI) calc. for
C16H32O3Na [M + Na]+: 295.2244, obs. 295.2243.
27.1, 25.4, 25.3, 25.0, 22.8, 14.2, 9.7; IR (film, cm−1):
2923, 2853, 1733, 1463, 1246, 1172; HRMS (ESI) calc. for
C28H55O4 [M + H]+: 455.4095, obs. 455.4095.
13-Methoxy-13-oxotridecan-5-yl palmitate (14b): Rf =
0.7 (silica gel, 80:20 hexanes:EtOAc, KMnO4); 160 mg,
1
92% yield, colorless oil; H NMR (600 MHz, CDCl3) δ
4.87 (m, 1H), 3.66 (s, 3 H), 2.29 (m, 4H), 1.67–1.58 (m, 4
H), 1.55–1.47 (m, 4 H), 1.29 (s, 36 H), 0.88 (m, 6H); 13C
NMR (151 MHz, CDCl3) δ 174.3, 173.7, 74.0, 51.5, 34.8,
34.2, 34.1, 34.0, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3,
29.2, 29.1, 27.6, 25.3, 25.0, 22.8, 22.7, 14.2, 14.1; IR (film,
cm−1): 2923, 2853, 1733, 1465, 1172; HRMS (ESI) calc.
for C30H59O4 [M + H]+: 483.4408, obs. 483.4405.
Methyl 9-hydroxyheptadecanoate (13d): Rf = 0.2 (silica
gel, 70:10 hexanes:EtOAc, KMnO4); 693 mg, 86% yield;
1
white solid; H NMR (600 MHz, CDCl3) δ 3.64 (s, 3 H),
3.55 (m, 1H), 2.28 (t, J = 7.6 Hz, 2 H), 1.60 (m, 2H),
1.44–1.33 (m, 4 H), 1.26 (m, 20H), 0.86 (t, J = 7.0 Hz, 3
H); 13C NMR (151 MHz, CDCl3) δ 174.5, 72.0, 51.6, 37.6,
37.5, 34.2, 32.0, 29.8, 29.7, 29.6, 29.4, 29.3, 29.2, 25.8,
25.7, 25.0, 22.8, 14.2; IR (film, cm−1): 2926, 2853,
1740,1436, 1197, 1171; HRMS (ESI) calc. for C18H36O3Na
[M + Na]+: 323.2557, obs. 323.2558.
15-Methoxy-15-oxopentadecan-7-yl palmitate (14c):
Rf = 0.6 (silica gel, 80:20 hexanes:EtOAc, KMnO4); 174
mg, 95% yield, colorless oil; 1H NMR (600 MHz, CDCl3) δ
4.89–4.83 (m, 1 H), 3.66 (s, 3 H), 2.28 (m, 4H), 1.60 (m,
4H), 1.49 (m, 4H), 1.28 (m, 40H), 0.87 (m, 6H); 13C NMR
(151 MHz, CDCl3) δ 174.4, 173.9, 74.1, 51.6, 34.9, 34.3,
34.2, 32.1, 32.0, 29.9, 29.8, 29.7, 29.5, 29.3, 29.2, 25.5,
25.4, 25.3, 25.0, 22.8, 14.3; IR (film, cm−1): 2922, 2853,
1733, 1465, 1170; HRMS (ESI) calc. for C32H62O4Na
[M + Na]+: 533.4540, obs. 533.4536.
Methyl 9-hydroxyoctadecanoate (13e): Rf = 0.2 (silica gel,
70:10 hexanes:EtOAc, KMnO4); 751 mg, 89% yield; white
1
solid; H NMR (600 MHz, CDCl3) δ 3.65 (s, 3 H), 3.56
(m, 1H), 2.29 (t, J = 7.6 Hz, 2 H), 1.60 (m, 2H), 1.40 (m, 4H),
1.28 (m, 22H). 0.86 (t, J = 7.0 Hz, 3 H); 13C NMR (151
MHz, CDCl3) δ 174.5, 72.1, 51.6, 37.6, 37.5, 34.2, 32.1, 29.8,
29.7, 29.6, 29.5, 29.3, 29.2, 25.8, 25.7, 25.0, 22.8, 14.3; IR
(film, cm−1): 2917, 2848, 1740, 1436, 1173; HRMS (ESI)
calc. for C19H38O3Na [M + Na]+: 337.2713, obs. 337.2713.
Methyl 9-(palmitoyloxy)heptadecanoate (14d): Rf = 0.6
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 180 mg, 93%
yield, colorless oil; H NMR (600 MHz, CDCl3) δ 4.84
(m, 1H), 3.64 (s, 3 H), 2.27 (m, 4H), 1.59 (m, 4H),
1.53–1.43 (m, 4 H), 1.28 (s, 44 H), 0.86 (m, 6H); 13C NMR
(151 MHz, CDCl3) δ 174.4, 173.8, 74.1, 51.6, 34.8, 34.3,
32.1, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 25.4,
25.3, 25.0, 22.8, 14.3, 14.2; IR (film, cm−1): 2922, 2853,
1733, 1465, 1170; HRMS (ESI) calc. for C34H66O4Na
[M + Na]+: 561.4853, obs. 561.4849.
1
General procedure for the preparation the methyl
ester of 9-PAHSA and analogous derivatives 14
To a stirred solution of 9-hydroxyl methyl ester 13a-e (0.36
mmol, 1.0 equiv) in dry dichloromethane (15 mL) was
added neat, dry pyridine (1.8 mmol, 5.0 equiv) at 0 °C and
the solution was stirred for 15 min. Neat acyl chloride (0.43
mmol, 1.2 equiv) was added to the reaction mixture. The
reaction was allowed to warm to 23 °C and stirred 12 h. The
unreacted acyl chloride was quenched with saturated aqu-
eous ammonium chloride (10 mL). The mixture was
extracted with dichloromethane (10 mL × 3). The combined
organic extracts were dried over sodium sulfate, filtered,
and concentrated. The crude product was purified via silica
gel column chromatography using hexane:EtOAc to give
the methyl esters of 9-PAHSA derivatives 14a–l.
Methyl 9-(palmitoyloxy)octadecenoate (14e): Rf = 0.6
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 188 mg, 95%
yield, colorless oil; H NMR (600 MHz, CDCl3) δ 4.85
(m, 1H), 3.66 (s, 3 H), 2.28 (m, 4H), 1.60 (m, 4H), 1.49
(m, 4H), 1.30–1.18 (m, 46 H), 0.87 (m, 6H); 13C NMR
(151 MHz, CDCl3) δ 174.4, 173.9, 74.1, 51.6, 34.9, 34.3,
34.2, 32.1, 31.9, 29.8, 29.7, 29.5, 29.4, 29.3, 29.2, 25.4,
25.3, 25.0, 22.8, 22.7, 14.3; IR (film, cm−1): 2922, 2853,
1733, 1465, 1171; HRMS (ESI) calc. for C35H68O4Na
[M + Na]+: 575.5010, obs. 575.5006.
1
Methyl 9-(tetradecanoyloxy)octadecenoate (14 f): Rf =
0.7 (silica gel, 80:20 hexanes:EtOAc, KMnO4); 172 mg,
1
91% yield, colorless oil; H NMR (600 MHz, CDCl3) δ
11-Methoxy-11-oxoundecan-3-yl palmitate (14a): Rf =
0.7 (silica gel, 80:20 hexanes:EtOAc, KMnO4); 155 mg,
95% yield, colorless oil;1H NMR (600 MHz, CDCl3) δ 4.78
(m, 1H), 3.63 (s, 3 H), 2.26 (m, 4H), 1.58 (m, 4H),
1.54–1.44 (m, 4 H), 1.29 (m, 32 H), 0.85 (m, 6H); 13C NMR
(151 MHz, CDCl3) δ 174.3, 173.8, 75.2, 51.5, 34.8, 34.1,
33.7, 32.0, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1,
4.89–4.82 (m, 1 H), 3.66 (s, 3 H), 2.28 (m, 4H), 1.66–1.57
(m, 4 H), 1.49 (m, 4H), 1.30–1.20 (m, 42 H), 0.87 (m, 6H);
13C NMR (151 MHz, CDCl3) δ 174.4, 173.9, 74.2, 51.7,
34.9, 34.3, 34.2, 32.1, 32.0, 29.8, 29.7, 29.5, 29.3, 29.2,
25.4, 25.3, 25.1, 22.8, 14.3; IR (film, cm−1): 2922, 2853,
1733, 1464, 1170; HRMS (ESI) calc. for C33H64O4Na
[M + Na]+: 547.4697, obs. 547.4692.

Synthesis of chemically edited derivatives of the endogenous regulator of inflammation 9-PAHSA
Methyl 9-(dodecanoyloxy)octadecenoate (14 g): Rf = 0.7
1 H), 3.66 (s, 3 H), 2.29 (m, 2H), 2.03 (s, 3 H), 1.62
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 168 mg, 94%
yield, colorless oil; H NMR (600 MHz, CDCl3) δ 4.85
(m, 2H), 1.49 (m, 4H), 1.27 (m, 22H), 0.87 (t, J = 7.0 Hz, 3
H); 13C NMR (151 MHz, CDCl3) δ 174.5, 171.1, 74.6, 51.6,
34.3, 34.2, 32.0, 29.7, 29.5, 29.3, 29.2, 25.5, 25.4, 25.0,
22.8, 21.5, 14.3; IR (film, cm−1): 2925, 2855, 1737, 1436,
1240; HRMS (ESI) calc. for C21H41O4 [M + H]+:
357.2999, obs. 357.2996.
1
(m, 1H), 3.66 (s, 3 H), 2.28 (m, 4H), 1.60 (m, 4H), 1.49
(m, 4H), 1.25 (m, 38H), 0.87 (m, 6H); 13C NMR (151 MHz,
CDCl3) δ 174.4, 173.9, 74.1, 51.6, 34.9, 34.3, 34.2, 32.1,
32.0, 29.8, 29.7, 29.5, 29.3, 29.2, 25.5, 25.4, 25.3, 25.1,
22.8, 14.3; IR (film, cm−1): 2922, 2853, 1733, 1465, 1169;
HRMS (ESI) calc. for C31H60O4Na [M + Na]+: 519.4384,
obs. 519.4379.
General procedure for preparation 9-PAHSA and
truncated 9-PAHSA analogs
Methyl 9-(decanoyloxy)octadecenoate (14 h): Rf = 0.7
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 156 mg, 93%
yield, colorless oil; H NMR (600 MHz, CDCl3) δ 4.85
(m, 1H), 3.66 (s, 3 H), 2.28 (m, 4H), 1.61 (m, 4H), 1.49
(m, 4H), 1.29 (s, 34 H), 0.87 (m, 6H); 13C NMR (151 MHz,
CDCl3) δ 174.5, 173.9, 74.1, 51.6, 34.9, 34.3, 34.2, 32.0,
29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 25.5, 25.4, 25.3, 25.1,
22.8, 14.2. IR (film, cm−1): 2924, 2855, 1734, 1465, 1170;
HRMS (ESI) calc. for C29H57O4 [M + H]+: 469.4251, obs.
469.4244.
The methyl esters 14a–l (0.22 mmol) were dissolved in THF:
H2O (5 mL: 5 mL) and cooled to 0 °C for 15 min before
adding solid LiOH (1.1 mmol, 5 equiv) in one portion. The
reaction was allowed to warm to 23 °C and stirred 12 h.
Excess hydroxide was quenched with 1 N HCl. The product
was extracted with ethyl acetate (5 mL × 3) and combined
organic extracts were washed with brine (5 mL), dried over
sodium sulfate, filtered, and concentrated. The crude products
were purified by silica gel column chromatography, eluting
with hexanes:EtOAc to yield compounds 1–12.
1
Methyl 9-(octanoyloxy)octadecenoate (14i):
Rf = 0.7
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 150 mg, 95%
yield, colorless oil; H NMR (600 MHz, CDCl3) δ 4.86
9-(Palmitoyloxy)undecanoic acid (1): Rf = 0.2 (silica gel,
80:20 hexanes:EtOAc, KMnO4); 87 mg, 90% yield, white
solid; H NMR (600 MHz, CDCl3) δ 4.84–4.77 (m, 1 H),
2.34 (t, J = 7.5 Hz, 2 H), 2.28 (t, J = 7.5 Hz, 2 H), 1.62 (m,
4H), 1.58–1.47 (m, 4 H), 1.31–1.23 (m, 32 H), 0.87 (m,
6H); 13C NMR (151 MHz, CDCl3) δ 180.3, 174.0, 75.3,
34.8, 34.2, 33.7, 32.1, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3,
29.1, 27.1, 25.4, 25.3, 24.7, 22.8, 14.3, 9.7; IR (film, cm−1):
2926, 2854, 1930, 1711, 1465; HRMS (ESI) calc. for
C27H51O4 [M–H]–: 439.3793, obs. 439.3790.
1
1
(m, 1H), 3.66 (s, 3 H), 2.28 (m, 4H), 1.60 (m, 4H), 1.48
(m, 4H), 1.32–1.21 (m, 30 H), 0.87 (m, 6H); 13C NMR
(151 MHz, CDCl3) δ 174.5, 173.9, 74.2, 51.7, 34.9, 34.3,
34.2, 32.0, 31.9, 29.7, 29.5, 29.3, 29.2, 29.1, 25.5, 25.4,
25.3, 25.1, 22.8, 14.3, 14.2; IR (film, cm−1): 2924, 2854,
1732, 1463, 1168; HRMS (ESI) calc. for C27H53O4 [M +
H]+: 441.3938, obs. 441.3936.
Methyl 9-(hexanoyloxy)octadecenoate (14j):
Rf = 0.7
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 133 mg, 90%
yield, colorless oil; 1H NMR (600 MHz, CDCl3) δ
4.91–4.80 (m, 1 H), 3.65 (s, 3 H), 2.28 (m, 4H), 1.60
(m, 4H), 1.53–1.45 (m, 4 H), 1.31–1.22 (m, 26 H), 0.87
(m, 6H); 13C NMR (151 MHz, CDCl3) δ 174.4, 173.9, 74.1,
51.6, 34.8, 34.3, 34.2, 32.0, 31.5, 29.7, 29.5, 29.3, 29.2,
25.5, 25.4, 25.0, 22.8, 22.5, 14.3, 14.1; IR (film, cm−1):
2925, 2855, 1732, 1463, 1171; HRMS (ESI) calc. for
C25H49O4 [M + H]+: 413.3625, obs. 413.3625.
Methyl 9-(butyryloxy)octadecenoate (14k): Rf = 0.7
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 127 mg, 92%
yield, colorless oil; 1H NMR (600 MHz, CDCl3) δ 4.86 (m,
1H), 3.66 (s, 3 H), 2.27 (m, 4H), 1.63 (m, 4H), 1.49 (m,
4H), 1.28 (m, 22H), 0.94 (t, J = 7.4 Hz, 3 H), 0.87 (t, J =
7.0 Hz, 3 H); 13C NMR (151 MHz, CDCl3) δ 174.5, 173.7,
74.2, 51.6, 36.8, 34.3, 34.2, 32.0, 29.7, 29.5, 29.3, 29.2,
25.5, 25.4, 25.0, 22.8, 14.3, 13.9; IR (film, cm−1): 2926,
2855, 1733, 1459, 1182; HRMS (ESI) calc. for C23H45O4
[M + H]+: 385.3312, obs. 385.3308.
9-(Palmitoyloxy)tridecanoic acid (2): Rf = 0.2 (silica gel,
80:20 hexanes:EtOAc, KMnO4); 91 mg, 89% yield, white
solid; H NMR (600 MHz, CDCl3) δ 4.86 (m, 1H), 2.33 (t,
1
J = 7.5 Hz, 2 H), 2.27 (t, J = 7.4 Hz, 2 H), 1.60 (m, 4H),
1.52–1.47 (m, 4 H), 1.27 (m, 36H), 0.89–0.85 (m, 6 H); 13
C
NMR (151 MHz, CDCl3) δ 180.1, 174.0, 74.1, 34.9, 34.3,
34.2, 34.0, 32.1, 29.8, 29.7, 29.5, 29.3, 29.1, 27.6, 25.4,
25.3, 24.8, 22.9, 22.8, 14.3, 14.2; IR (film, cm−1): 2925,
2855, 1931, 1709, 1465; HRMS (ESI) calc. for C29H55O4
[M–H]–: 467.4106, obs. 467.4102.
9-(Palmitoyloxy)pentadecanoic acid (3): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 100 mg, 92% yield,
1
colorless oil; H NMR (600 MHz, CDCl3) δ 4.86 (m, 1H),
2.34 (t, J = 7.5 Hz, 2 H), 2.27 (t, J = 7.5 Hz, 2 H), 1.61
(m, 4H), 1.53–1.47 (m, 4 H), 1.31–1.22 (m, 40 H), 0.87
(m, 6H); 13C NMR (151 MHz, CDCl3) δ 179.6, 173.9, 74.2,
34.9, 34.3, 33.9, 32.1, 29.9, 29.8, 29.7, 29.5, 29.4, 29.3,
29.1, 25.5, 25.4, 25.3, 24.8, 22.8, 14.3; IR (film, cm−1):
2925, 2854, 1930, 1710, 1465; HRMS (ESI) calc. for
C31H59O4 [M–H]–: 495.4419, obs. 495.4414.
Methyl 9-acetoxyoctadecanoate (14 l): Rf = 0.7 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 124 mg, 95% yield,
9-(Palmitoyloxy)heptadecanoic acid (4): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 106 mg, 92% yield,
1
colorless oil; H NMR (600 MHz, CDCl3) δ 4.89–4.79 (m,

H. Wang et al.
colorless oil; 1H NMR (600 MHz, Chloroform-d) δ 4.86 (m,
1H), 2.34 (d, J = 7.8 Hz, 2 H), 2.27 (t, J = 7.5 Hz, 2 H),
1.62 (m, 4H), 1.50 (m, 4H), 1.29 (m, 44H), 0.88 (m, 6H).
13C NMR (151 MHz, CDCl3) δ 179.5, 174.0, 74.2, 34.9,
34.3, 32.1, 32.0, 29.9, 29.8, 29.7, 29.5, 29.4, 29.3, 29.1,
25.5, 25.4, 25.3, 24.8, 22.8, 14.3. IR (film, cm−1): 2922,
2853, 1732, 1709, 1465, 1377, 1174; HRMS (ESI) calc. for
C33H63O4 [M–H]–: 523.4732, obs. 523.4727.
(m, 6H); 13C NMR (151 MHz, CDCl3) δ 179.5, 174.0, 74.2,
34.9, 34.3, 34.0, 32.0, 31.9, 29.7, 29.5, 29.3, 29.1, 25.5,
25.4, 25.3, 24.8, 22.8, 14.3, 14.2; IR (film, cm−1): 2924,
2855, 1732, 1709, 1464, 1169; HRMS (ESI) calc. for
C26H49O4 [M–H]–: 425.3636, obs. 425.3632.
9-(Hexanoyloxy)octadecanoic acid (10): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 81 mg, 93% yield,
1
colorless oil; H NMR (600 MHz, CDCl3) δ 4.86 (m, 1H),
9-(Palmitoyloxy)octadecanoic acid (5): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 108 mg, 92% yield,
colorless oil; 1H NMR (600 MHz, CDCl3) δ 4.92–4.80
(m, 1 H), 2.31 (dt, J = 38.9, 7.7 Hz, 4 H), 1.61 (m, 4H), 1.50
(m, 4H), 1.27 (m, 46H), 0.87 (m, 6H); 13C NMR (151 MHz,
CDCl3) δ 179.7, 174.0, 74.2, 35.0, 34.4, 34.2, 32.0, 30.0,
29.9, 29.8, 29.6, 29.5, 29.4, 29.2, 25.5, 25.4, 24.9, 22.9,
22.8, 14.4, 14.3; IR (film, cm−1): 2917, 2848, 1733, 1714,
1470, 1156; HRMS (ESI) calc. for C34H65O4 [M–H]–:
537.4888, obs. 537.4887.
2.34 (t, J = 7.5 Hz, 2 H), 2.28 (t, J = 7.5 Hz, 2 H), 1.62
(m, 4H), 1.54–1.46 (m, 4 H), 1.32–1.22 (m, 26 H), 0.88
(m, 6H); 13C NMR (151 MHz, CDCl3) δ 180.2, 174.0, 74.2,
34.8, 34.3, 34.2, 32.0, 31.5, 29.7, 29.4, 29.3, 29.1, 25.4,
25.0, 24.7, 22.8, 22.5, 14.2, 14.1; IR (film, cm−1): 2925,
2855, 1732, 1709, 1464, 1174; HRMS (ESI) calc. for
C24H45O4 [M–H]–: 397.3323, obs. 397.3321.
9-(Butyryloxy)octadecanoic acid (11): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 73 mg, 90% yield,
1
colorless oil; H NMR (600 MHz, CDCl3) δ 4.89–4.84 (m,
9-(Tetradecanoyloxy)octadecanoic acid (6): Rf = 0.2
(silica gel, 80:20 hexanes:EtOAc, KMnO4); 100 mg, 89%
yield, colorless oil; 1H NMR (600 MHz, CDCl3) δ
4.89–4.83 (m, 1 H), 2.34 (td, J = 7.5, 3.1 Hz, 2 H), 2.27 (td,
J = 7.6, 3.1 Hz, 2 H), 1.61 (m, 4H), 1.49 (m, 4H), 1.28 (m,
42H), 0.87 (m, 6H). 13C NMR (151 MHz, CDCl3) δ 179.9,
174.0, 74.2, 34.9, 34.3, 34.1, 32.1, 29.8, 29.7, 29.5, 29.3,
29.1, 25.5, 25.4, 25.3, 24.8, 22.8, 14.3; IR (film, cm–1):
2923, 2854, 1733, 1710, 1465, 1177; HRMS (ESI) calc. for
C32H61O4 [M–H]–: 509.4575, obs. 509.4572.
1 H), 2.34 (t, J = 7.5 Hz, 2 H), 2.26 (t, J = 7.4 Hz, 2 H),
1.63 (m, 4H), 1.54–1.46 (m, 4 H), 1.27 (m, 22H), 0.94
(t, J = 7.4 Hz, 3 H), 0.87 (t, J = 7.0 Hz, 3 H); 13C NMR
(151 MHz, CDCl3) δ 179.8, 173.8, 74.2, 36.8, 34.3, 34.1,
32.0, 29.7, 29.5, 29.4, 29.3, 29.1, 25.5, 25.4, 24.8, 22.8,
18.8, 14.3, 13.9; IR (film, cm−1): 2926, 2855, 1733, 1709,
1465, 1184; HRMS (ESI) calc. for C22H41O4 [M–H]–:
369.3010, obs. 369.3008.
9-Acetoxyoctadecanoic acid (12): Rf = 0.2 (silica gel,
80:20 hexanes:EtOAc, KMnO4); 66 mg, 88% yield, color-
less oil; 1H NMR (600 MHz, CDCl3) δ 4.88–4.82 (m, 1 H),
2.34 (t, J = 7.5 Hz, 2 H), 2.04 (s, 3 H), 1.62 (m, 2H), 1.50
(m, 4H), 1.27 (m, 22H), 0.87 (t, J = 7.0 Hz, 3 H). 13C NMR
(151 MHz, CDCl3) δ 180.0, 171.2, 74.6, 34.3, 34.2, 34.1,
32.0, 29.7, 29.4, 29.3, 29.1, 25.5, 25.4, 24.8, 22.8, 21.5,
14.3; IR (film, cm−1): 2926, 2855, 1737, 1710, 1464, 1242;
HRMS (ESI) calc. for C20H37O4 [M–H]–: 341.2697, obs.
341.2698.
9-(Dodecanoyloxy)octadecanoic acid (7): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 95 mg, 90% yield,
1
colorless oil; H NMR (600 MHz, CDCl3) δ 4.86 (m, 1H),
2.33 (td, J = 7.6, 1.5 Hz, 2 H), 2.27 (td, J = 7.5, 1.5 Hz,
2 H), 1.61 (m, 4H), 1.50 (m, 4H), 1.26 (m, 38 H), 0.87
(m, 6H). 13C NMR (151 MHz, CDCl3) δ 180.2, 174.0, 74.2,
34.9, 34.3, 34.2, 32.1, 32.0, 29.8, 29.7, 29.5, 29.4, 29.3,
29.1, 25.5, 25.4, 25.3, 24.8, 22.8, 14.3; IR (film, cm−1):
2923, 2854, 1433, 1709, 1465, 1174; HRMS (ESI) calc. for
C30H57O4 [M–H]–: 481.4266, obs. 481.4260.
Biology
9-(Decanoyloxy)octadecanoic acid (8): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 88 mg, 88% yield,
colorless oil; H NMR (600 MHz, CDCl3) δ 4.86 (m, 1H),
Culturing
1
2.34 (t, J = 7.5 Hz, 2 H), 2.27 (t, J = 7.5 Hz, 2 H), 1.62
(m, 4H), 1.49 (m, 4H), 1.29 (m, 34H), 0.87 (m, 6H); 13C
NMR (151 MHz, CDCl3) δ 178.7, 173.9, 74.2, 34.9, 34.3,
33.9, 32.1, 32.0, 29.7, 29.6, 29.5, 29.4, 29.3, 29.1, 25.5,
25.4, 25.3, 24.8, 22.8, 14.3; IR (film, cm−1): 2925, 2855,
1733, 1710, 1465, 1106; HRMS (ESI) calc. for C28H53O4
[M–H]–: 453.3949, obs. 453.3948.
RAW 264.7 cells were cultured in RPMI 1640 (Gibco),
supplemented with L-glutamine (2 mM), 10% FBS at 37 C
and 5% CO2. All experiments were performed on or prior to
passage 15.
Quantification of IL-6 upon LPS stimulation assay
9-(Octanoyloxy)octadecanoic acid (9): Rf = 0.2 (silica
gel, 80:20 hexanes:EtOAc, KMnO4); 85 mg, 91% yield,
RAW 264.7 cells were seeded onto 48-well plates (2.5 × 104
cells per well) a day prior to treatment. Adherent cells were
then treated with DMSO or with LPS (100 ng/mL) and
individual compounds at 25 µM or 50 µM. Supernantants
were collected 20 h post-treatment and subjected to IL-6
1
colorless oil; H NMR (600 MHz, CDCl3) δ 4.86 (m, 1H),
2.34 (t, J = 7.5 Hz, 2 H), 2.28 (t, J = 7.5 Hz, 2 H), 1.61
(m, 4H), 1.54–1.46 (m, 4 H), 1.32–1.21 (m, 30 H), 0.87

Synthesis of chemically edited derivatives of the endogenous regulator of inflammation 9-PAHSA
quantification using mouse IL-6 ELISA MAXTM Deluxe
Kits (BioLegend). Adherent cells were subjected to MTT
assay to measure cell viabilities.
2. Yore MM, Syed I, Moraes-Vieira PM, Zhang T, Herman MA,
Homan EA, et al. Discovery of a class of endogenous mammalian
lipids with anti-diabetic and anti-inflammatory effects. Cell.
2014;159:318–32.
3. Lee J, Moraes-Vieira PM, Castoldi A, Aryal P, Yee EU, Vickers
C, et al. Branched fatty acid esters of hydroxy fatty acids (FAH-
FAs) protect against colitis by regulating gut innate and adaptive
immune responses. J Biol Chem. 2016;291:22207–17.
4. Kuda O, Brezinova M, Rombaldova M, Slavikova B, Posta M,
Beier P, et al. Docosahexaenoic acid-derived fatty acid esters of
hydroxy fatty acids (FAHFAs) with anti-inflammatory properties.
Diabetes. 2016;65:2580–90.
5. Nelson AT, Kolar MJ, Chu Q, Syed I, Kahn BB, Saghatelian A,
et al. Stereochemistry of endogenous palmitic acid ester of 9-
hydroxystearic acid and relevance of absolute configuration to
regulation. J Am Chem Soc. 2017;139:4943–7.
Post-treatment analysis of cell proliferation using MTT
Assay
3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bro-
mide (MTT) was dissolved in sterile PBS (5 mg/mL) and
filtered through a 0.22 μm Sterile Millex Filter to prepare a
500 µg/mL solution in RPMI. Prepared solution was added
to adherent cells and incubated at 37 °C for 4 h. Sterile
DMSO was then supplied upon removal of MTT solution.
Relative cell viabilities were quantified using a plate reader
at an absorbance of 570 nm.
6. Balas L, Feillet-Coudray C, Durand T. Branched fatty acyl esters
of hydroxyl fatty acids (FAHFAs), appealing beneficial endo-
genous fat against obesity and type-2 diabetes. Chem A Eur J.
2018;24:9463–76.
Acknowledgements This work was supported by the National Insti-
tutes of Health Grants R56 DK110150 (to D. S. and A. S.). The
authors thank Brendan Duggan for assistance with NMR spectroscopy.
This work was supported by the University of California, San Diego.
7. Balas L, Bertrand-Michel J, Viars F, Faugere J, Lefort C, Caspar-
Bauguil S, et al. Regiocontrolled syntheses of FAHFAs and
LC-MS/MS differentiation of regioisomers. Org Biomol Chem.
2016;14:9012–20.
8. Weibel DB, Shevy LE, Schroeder FC, Meinwald J. Synthesis
of mayolene-16 and mayolene-18: larval defensive lipids from the
European cabbage butterfly. J Org Chem. 2002;67:5896–900.
9. Louis K, Vivier L, Clacens J-M, Brandhorst M, Dubois J-L, Vigier
KDO, et al. Sustainable route to methyl-9-hydroxononanoate
(polymer precursor) by oxidative cleavage of fatty acid methyl
ester from rapeseed oil. Green Chem. 2014;16:96–101.
10. Tamura S, Kaneko M, Shiomi A, Yang GM, Yamaura T, Mur-
akami N. Unprecedented NES non-antagonistic inhibitor for
nuclear export of Rev from Sida cordifolia. Bioorg Med Chem
Lett. 2010;20:1837–9.
11. Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation,
immunity, and disease. Cold Spring Harb Perspect Biol. 2014;6:
a016295.
12. Schwartz C, Raible J, Mott K, Dussault PH. Fragmentation of
carbonyl oxides by N-oxides: an improved approach to alkene
ozonolysis. Org Lett. 2006;8:3199–201.
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Conflict of interest The authors declare that they have no conflict of
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References
1. Shepherd PR, Gnudi L, Tozzo E, Yang H, Leach F, Kahn BB.
Adipose cell hyperplasia and enhanced glucose disposal in
transgenic mice overexpressing GLUT4 selectively in adipose
tissue. J Biol Chem. 1993;268:22243–6.