Saturated Oxo Fatty Acids (SOFAs): A Previously Unrecognized Class of Endogenous Bioactive Lipids Exhibiting a Cell Growth Inhibitory Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c02058

The discovery of novel bioactive lipids that promote human health is of great importance. Combining suspect and targeted lipidomic liquid chromatography-high-resolution mass spectrometry (LC-HRMS) approaches, a previously unrecognized class of oxidized fatty acids, the saturated oxo fatty acids (SOFAs), which carry the oxo functionality at various positions of the long chain, was identified in human plasma. A library of SOFAs was constructed, applying a simple green photochemical hydroacylation reaction as the key synthetic step. The synthesized SOFAs were studied for their ability to inhibit in vitro the cell growth of three human cancer cell lines. Four oxostearic acids (OSAs) were identified to inhibit the cell growth of human lung carcinoma A549 cells. 6OSA and 7OSA exhibited the highest cell growth inhibitory potency, suppressing the expression of both STAT3 and c-myc, which are critical regulators of cell growth and proliferation. Thus, naturally occurring SOFAs may play a role in the protection of human health.

Full text of DOI:10.1021/acs.jmedchem.0c02058

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Article
Saturated Oxo Fatty Acids (SOFAs): A Previously Unrecognized Class
of Endogenous Bioactive Lipids Exhibiting a Cell Growth Inhibitory
Activity
Charikleia S. Batsika, Christiana Mantzourani,∥ Dimitrios Gkikas,∥ Maroula G. Kokotou,∥
Olga G. Mountanea, Christoforos G. Kokotos, Panagiotis K. Politis, and George Kokotos*
Cite This: J. Med. Chem. 2021, 64, 5654−5666
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ABSTRACT: The discovery of novel bioactive lipids that promote
human health is of great importance. Combining “suspect” and
targeted lipidomic liquid chromatography-high-resolution mass
spectrometry (LC-HRMS) approaches, a previously unrecognized
class of oxidized fatty acids, the saturated oxo fatty acids (SOFAs),
which carry the oxo functionality at various positions of the long
chain, was identified in human plasma. A library of SOFAs was
constructed, applying a simple green photochemical hydroacylation
reaction as the key synthetic step. The synthesized SOFAs were
studied for their ability to inhibit in vitro the cell growth of three
human cancer cell lines. Four oxostearic acids (OSAs) were identified
to inhibit the cell growth of human lung carcinoma A549 cells. 6OSA and 7OSA exhibited the highest cell growth inhibitory potency,
suppressing the expression of both STAT3 and c-myc, which are critical regulators of cell growth and proliferation. Thus, naturally
occurring SOFAs may play a role in the protection of human health.
(R)-hydroxydecanoic acid (2, 3HDA, Figure 1a) has been
demonstrated to trigger immunity in Arabidopsis plants.9 9-
Hydroxystearic acid (9HSA) was identified in 1991 as a
peroxidation product in Lewis lung carcinoma cells,10 and later
studies demonstrated its ability to upregulate p21WAF1 in
HT29 cancer cells11 and inhibit cell growth in human colon
cancer, targeting histone deacetylase 1.12
Most recently, in milk, we have identified new families of
previously unrecognized saturated hydroxy fatty acids
(SHFAs), namely, hydroxystearic acids (HSAs) and hydrox-
ypalmitic acids (HPAs), which exhibit cell growth inhibitory
activity and the ability to inhibit the cytokine-induced β-cell
apoptosis.13 Among the various positional isomers of HPAs
and HSAs, 7-(R)-hydroxystearic acid (3, 7HSA, Figure 1a)
exhibited the highest potency both in cell growth inhibition
and in suppressing β-cell death.13 Due to their potential role in
the promotion and protection of human health, an analytical
liquid chromatography-high-resolution mass spectrometry
INTRODUCTION
■
Fatty acids exhibit biological roles and actions on their own in
their free form, in addition to their roles as key components of
the various lipid classes (triacylglycerols, phospholipids, etc.).1
A great attention has been paid to the major classes of
saturated, monounsaturated, and polyunsaturated fatty acids
and their roles as energy sources, membrane components,
signaling molecules, and regulators of gene expression. Since
fatty acids are widespread in nature and are essential
ingredients of foods and natural products, research on their
role as bioactive nutrients and their impact on human health is
an ever-expanding field. Oxidized saturated fatty acids, carrying
functional groups like a hydroxyl or an oxo (keto)
functionality, have attracted limited attention so far. Recently,
a novel class of endogenous lipids exhibiting antidiabetic and
anti-inflammatory properties has been identified,2−5 indicating
that previously unrecognized classes of lipids might be present
in the lipidome exhibiting unexplored biological properties so
far. These compounds are named fatty acid esters of hydroxy
fatty acids (FAHFAs, 1, Figure 1a) and are composed of a
hydroxy fatty acid (HFA) acylated by a fatty acyl chain, and
their properties have been recently reviewed.6,7 HFAs
constitute another class of potential bioactive lipids, and
scientific efforts are in progress to identify their activities.
Medium-chain 2-hydroxy or 3-hydroxy fatty acids have been
reported to activate receptors like GPR84 more effectively than
their corresponding nonhydroxylated compounds,8 while 3-
Received: December 10, 2020
Published: April 21, 2021
https://doi.org/10.1021/acs.jmedchem.0c02058
© 2021 American Chemical Society
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Figure 1. Oxidized saturated fatty acids and their bioactivities: (a) examples of known hydroxy fatty acids, (b) examples of known oxo fatty acids,
and (c) the general structure of oxo fatty acids designed in this work.
(LC-HRMS) method was developed for the quantitation of
these new bioactive lipids in cow and goat milk.14
vitro cell growth inhibitory activity of SOFAs. Herein, we
present the direct one-step synthesis of SOFAs from
commercially available aldehydes and ω-alkenoic acids through
a photochemical hydroacylation reaction under mild con-
ditions on water and a structure−activity relationship study
demonstrating that among the SOFAs studied, 6-oxostearic
acid (6OSA) presents the most potent cell growth inhibitory
results. Furthermore, we report that this effect of SOFAs
involves the STAT3 signaling pathway, as well as the c-myc
signaling pathway, with major players in a pro-proliferative
cascade.
An unexplored up-to-date class of naturally occurring fatty
acids is that of saturated oxo (keto) fatty acids (SOFAs).
Although 3-oxo fatty acids are generally considered as
intermediates in β-oxidation (fatty acid metabolism),15 little
is known about the origin of a range of minor SOFAs found in
natural sources, differing in the chain length and positions of
the oxo functionality. 3-Oxostearic (3-oxooctadecanoic) acid
(4, 3OSA, Figure 1b) has been studied by ultra-performance
liquid chromatography quadrupole time-of-flight mass spec-
trometry (UPLC/QTOF-MS) in serum, together with various
other metabolites that might function as potential biomarkers
in gestational diabetes mellitus.16,17 An example of a natural
source of SOFAs is milk. The occurrence of ketostearic acids in
milk has been reported since the sixties.18,19 16-Ketostearic
acid (5, 16OSA, Figure 1b) is a representative SOFA identified
in the rumen.19 More recently, 10-ketostearic acid (10OSA)
and 8-ketopalmitic acid (8OPA) have been determined in milk
by gas chromatography-mass spectrometry (GC-MS).20
Furthermore, 6-ketostearic acid (or lactarinic acid) (6,
6OSA, Figure 1b) has been isolated and characterized from
the fruiting bodies of the fungi Lactarius theiogalus.21 However,
nothing is known about the bioactivities of SOFAs.
Inspired by our most recent research that led to the
discovery of bioactive SHFAs,13 where we followed a research
workflow involving: (a) “suspect” LC-HRMS analysis in a food
matrix, (b) organic synthesis, (c) targeted LC-HRMS analysis
using reference compounds in a natural matrix, and (d) a study
of in vitro bioactivities; the aim of the present study was to
apply the same successful strategy to investigate SOFAs
(Figure 1c) and to uncover their potential bioactivities. Thus,
first, we adopted a lipidomic approach to identify previously
unrecognized SOFAs existing in natural sources. Then, we
developed a general efficient method for the synthesis of
SOFAs because easy access to such compounds could allow an
extended study of their bioactivities. Finally, we studied the in
RESULTS
■
Suspect Liquid Chromatography-High-Resolution
Mass Spectrometry Analysis. In recent years, lipidomics
has been recognized as one of the fastest-growing research
fields in life sciences, contributing to the study of disease
mechanisms and the identification of novel biomarkers,
therapeutic targets, and bioactive lipids.22 Detection, identi-
fication, and subsequent quantification of lipids have become
essentials in biomedical research. Without any doubt, the
success story of lipidomics is closely related to the develop-
ment of new analytical methods, as well as the availability of
new mass spectrometry equipment.23 In particular, HRMS
offers high sensitivity and high mass accuracy24 required for
mediator-lipidomics, which focuses on the detection and
quantification of low abundance bioactive lipid species.
Following a suspect analysis approach involving an LC-
HRMS technique, we successfully identified new families of
bioactive SHFAs in milk.13 In the present work, our aim was to
explore if families of SOFAs are present in human plasma and
cow milk.
A suspect LC-MS approach with reversed-phase chromatog-
raphy and HRMS operating in the full scan negative mode was
utilized. Initially, we screened human plasma and cow milk
samples by LC-HRMS and monitored for peaks corresponding
to the exact masses (deprotonated molecule) of two SOFAs,
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namely, oxopalmitic (OPA, m/z 269.2122) and oxostearic
(OSA, m/z 297.2435) acids. The accurate mass for each
formula was searched using a 0.01 mass width. Figure 2a,b
Figure 3. EICs for mass corresponding to OPA (m/z 269.2122). (a,
b) Human plasma samples and (c, d) cow milk samples.
approach for their synthesis. Starting from malonic esters, a few
SOFAs were synthesized in a multistep procedure and in
mediocre yields.25,26 Other methods used polyunsaturated
fatty or cyclic ketones as starting materials, leading to the
synthesis of specific SOFAs.27,28 The shortest approach
described to date involved a thermal radical initiation protocol
that employed aldehydes and ω-ester protected olefins,
utilizing benzoyl peroxide as the initiator; however, this
method provided products in low yields.29 A method reported
in 2014 starts from dibromoalkanes requiring multiple steps.30
Within the last decade, photochemistry has attracted special
attention as an alternative green synthetic approach,
uncovering new reactivities.31,32 Recently, a mild, metal-free
photochemical protocol that is easy to operate, employing
household lamps as the irradiation source and phenylglyoxylic
acid as the initiator, was reported for the hydroacylation of
olefins, without the need of directing groups, additives, or
metal catalysts.33 We immediately recognized the potential of
this method, which could lead to a fast and reliable
methodology to synthesize a library of various members of
the SOFA family. The key-step consisted of a photochemical
hydroacylation reaction between commercially available ω-
alkenoic acids (1a−f) and aldehydes (2a−h) to provide the
target compounds 3a−k under mild conditions on water in
Figure 2. EICs for mass corresponding to OSA (m/z 297.2435). (a,
b) Human plasma samples and (c, d) cow milk samples.
presents the extracted ion chromatograms (EICs), correspond-
ing to the exact mass of OSA, for two representative human
plasma samples, while Figure 2c,d shows the EICs for two
representative cow milk samples. Several different peaks were
observed either in plasma or in milk samples, indicating the
presence of different various isobaric regioisomers (i.e., with
the oxo group at various positions of the octadecyl chain).
Similarly, Figure 3a−d presents the EICs, corresponding to the
exact mass of OPA, for two human plasma samples and two
cow milk samples, respectively. Again, different peaks were
clearly observed, suggesting the existence of various
regioisomers.
The suspect analysis, indicating the presence of multiple
regioisomers of OSA and OPA in both plasma and milk,
boosted us to generate the prerequisite LC/HRMS reference
synthetic compounds.
Synthesis of Saturated Oxo Fatty Acids. A limited
number of methods for the synthesis of SOFAs exist in the
literature, which cannot provide a reliable, efficient, general
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48−68% yield (Scheme 1). The synthesis of 7OSA was scaled
up (gram scale) to give the product in 50% yield (see
14-oxopalmitic acid (14OPA)] were used in the present study.
The analytes, their exact masses, and their retention times are
summarized in Table 1.
As shown in Figure 4a, the 17 reference compounds were
distinctly separable using this chromatographic technique.
Seven different plasma samples were examined in triplicates,
and a representative chromatogram of a human plasma sample
is illustrated in Figure 4b. 6OSA was found to be the most
abundant in plasma and its contents were ranged from 4.9 to
19.5 ng/mL. 4OSA, 9OSA, and 10OSA were also detected at
levels not exceeding 6.5 ng/mL. Details of the analytical
method will be published elsewhere.
Scheme 1. General, One-Step Photochemical Synthesis of
SOFAs
a
Cell Growth Inhibitory Effects of Saturated Oxo Fatty
Acids on Human Cancer Cell Lines. As mentioned in the
introduction, we have most recently studied the cell growth
inhibitory activity of various HSAs and HPAs, and we have
found that 7RHSA exhibited the highest potency on epithelial
lung carcinoma (A549) cell line with an IC50 value of 38 μM.13
In the present work, we studied the effect of a number of
synthesized OPAs and OSAs on the proliferation of three
human cancer cell lines: epithelial lung carcinoma (A549),
colorectal adenocarcinoma (Caco-2), and astrocytoma
(SF268). The MTT assay was employed to assess viable cell
numbers.34 The growth inhibitory effects of seven OSAs and
five OPAs (50 μM), together with 7HSA and 9HSA for
comparison, on A549 are summarized in Figure 5a. 5-Oxo-, 6-
oxo-, 7-oxo, and 8-oxostearic acids exhibited inhibition higher
than 50%, similar to that of 7HSA and clearly higher than that
of 9HSA. The effect of these compounds on A549 at
concentrations ranging from 10 to 75 μM is shown in Figure
5b. The determination of their IC50 values, which were
calculated from the curves depicted in Figure 5c, revealed that
5OSA, 6OSA, 7OSA, and 8OSA exhibited approximately
similar inhibitory potency on A549 cells (IC50 values of 49, 26,
28, and 37 μM, respectively). Palmitic acid (PA) and stearic
acid (SA) are included for comparison purposes in Figure 5a.
PA had no effect, while a very weak effect was observed for SA,
underlying the importance of an oxo functionality in the long
chain for cell growth inhibitory activity. 6OSA, 7OSA, and
8OSA exhibited lower growth inhibitory activity on Caco-2
cells (Figure 5d) and even lower activity on SF268 cells
(Figure 5e), indicating a cell-specific effect. In addition, the
effect of 6OSA and 7RHSA (for comparison purposes) was
studied on human umbilical vein endothelial cells (HUVEC)
as an example of normal, noncancerous cells. Neither 6OSA
nor 7RHSA presented significant cell growth inhibition on
HUVEC up to 75 μM concentration (see Figure S1,
Supporting Information).
a
(a) Phenylglyoxylic acid (20 mol %), H2O, hv (CFL lamps), 48 h.
Supporting Information). For the synthesis of 10OPA (6),
terminal alkenol 4 was used as the starting material instead of
the corresponding ω-alkenoic acid, leading to hydroxy-ketone
5, which after Jones oxidation afforded the desired product 6
(Scheme 2). Modified processes were also developed using ω-
alkenols as the starting materials (Schemes 3 and 4). Benzoyl
and TBDMS-protected ω-alkenols (7, 11a−c) were synthe-
sized and subjected to the photochemical process. Removal of
the benzoyl or TBDMS group, followed by Jones oxidation, led
to the desired 4OSA, 10OSA, 16OSA, and 14OPA. The
required intermediates 11b,c were prepared by the oxidation of
monoprotected alcohols 16a,b, followed by appropriate
olefination (Scheme 5).
Determination of Various Oxostearic and Oxopal-
mitic Acids in Plasma. A rapid LC-HRMS analytical method
was developed for the simultaneous determination of OSAs
and OPAs in human plasma. This method allowed the
determination of 17 different SOFAs within a 10 min sample
run. Ten regioisomers of OSA [3-oxostearic acid (3OSA), 4-
oxostearic acid (4OSA), 5-oxostearic acid (5OSA), 6-
oxostearic acid (6OSA), 7-oxostearic acid (7OSA), 8-
oxostearic acid (8OSA), 9-oxostearic acid (9OSA), 10-
oxostearic acid (10OSA), 12-oxostearic acid (12OSA), and
16-oxostearic acid (16OSA)] and seven regioisomers of OPA
[5-oxopalmitic acid (5OPA), 6-oxopalmitic acid (6OPA), 7-
oxopalmitic acid (7OPA), 8-oxopalmitic acid (8OPA), 9-
oxopalmitic acid (9OPA), 10-oxopalmitic acid (10OPA), and
To shed light on the mode of action of OSAs, mRNA
expression analysis was employed to unravel the signaling
a
Scheme 2. Photochemical Synthesis of 10OPA
a
(a) Phenylglyoxylic acid (20 mol %), H2O, hv (CFL lamps), 48 h and (b) Jones reagent, acetone.
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a
Scheme 3. Photochemical Synthesis of 4OSA
a
(a) Phenylglyoxylic acid (20 mol %), H2O, hv (CFL lamps), 48 h; (b) NaOH, 1,4-dioxane-H2O; and (c) Jones reagent, acetone.
a
Scheme 4. Photochemical Synthesis of 10OSA, 16OSA, and 14OPA
a
(a) Phenylglyoxylic acid (20 mol %), H2O, hv (CFL lamps), 48 h; (b) TBAF, dry tetrahydrofuran (THF); and (c) Jones reagent, acetone.
a
Scheme 5. Synthesis of Compounds 11b,c
a
(a) TBDMS-Cl, imidazole, 4-(dimethylamino)pyridine (DMAP), dry dimethylformamide (DMF); (b) PCC, dry CH2Cl2; and (c) MePPh3Br, n-
BuLi (1.6 M in hexane), dry THF.
(PIK3CA), p53, mTOR, PTEN, AKT1, c-myc, and STAT3,
(Figure 6a−k), was examined by real-time RT-qPCR.
Fascinatingly, STAT3 and c-myc were significantly reduced
in the presence of 6OSA and 7OSA (Figure 6j,k). Additionally,
the down-regulation of STAT3 expression was also observed in
protein levels of both total and phosphorylated STAT3 (p-
STAT3) (Figure 6l−n). Both c-myc and STAT3 are major
regulators of tumor development and its resistance in various
treatments.35,36
Table 1. Chromatographic Details for Saturated Oxo Fatty
Acids
a
compound
abbreviation
exact mass [M − H]−
tr
5-oxopalmitic acid
6-oxopalmitic acid
7-oxopalmitic acid
8-oxopalmitic acid
9-oxopalmitic acid
10-oxopalmitic acid
14-oxopalmitic acid
3-oxostearic acid
4-oxostearic acid
5-oxostearic acid
6-oxostearic acid
7-oxostearic acid
8-oxostearic acid
9-oxostearic acid
10-oxostearic acid
12-oxostearic acid
16-oxostearic acid
5OPA
6OPA
7OPA
8OPA
9OPA
10OPA
14OPA
3OSA
4OSA
5OSA
6OSA
7OSA
8OSA
9OSA
10OSA
12OSA
16OSA
269.2122
269.2122
269.2122
269.2122
269.2122
269.2122
269.2122
297.2435
297.2435
297.2435
297.2435
297.2435
297.2435
297.2435
297.2435
297.2435
297.2435
5.13
4.97
4.85
4.76
4.69
4.64
4.51
6.22
5.99
5.80
5.64
5.53
5.44
5.37
5.31
5.23
5.15
DISCUSSION
■
Although 3-OFAs are considered as intermediates in fatty acid
metabolism,15 only a few examples are known in the
literature,18−21 demonstrating the presence of other SOFAs,
carrying the oxo functionality at other positions of the long
alkyl chain, in natural sources. Neither isolation nor study of
their biological properties has been performed so far. To assess
the biological properties of a family of lipids like SOFAs and to
perform structure−activity relationship studies, first, a general
and efficient synthetic route is highly desirable. We
demonstrate here a fast, green, reliable, photochemical process,
which allows the synthesis of a library of SOFAs in one-step.
This process uses commercially available ω-alkenoic acids and
aldehydes as starting materials, water as the solvent, common
household lamps as the irradiation source, and phenylglyoxylic
acid as the photoinitiator, and leads directly to SOFAs in
a
Retention time.
pathway(s) that are affected by 6OSA and 7OSA. A variety of
different crucial cell cycle regulators and signaling molecules,
namely, cyclin D1, cyclin E1, EGFR, ERBB2 (Her2), PI3K
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Figure 4. Extracted ion chromatograms (EICs) of saturated OPAs and OSAs. (a) Standard solution (500 ng/mL). (b) Representative plasma
sample. OSA (blue), OPA (magenta).
efficient to high yields. Alternative procedures have also been
developed, employing alternative starting materials, for
example, ω-alkenols; however, the same photochemical key-
step reaction was used. Thus, a variety of OSAs and OPAs
were synthesized.
respectively. This potency is similar to that of 7RHSA (IC50 38
μM).13 The structure−activity relationship study led to the
conclusion that the position of the oxo functionality at the long
saturated chain is a critical factor for the antiproliferative
activity. Derivatives carrying the oxo functionality at the fifth to
eighth carbon atom were the most active, indicating that the
relative spatial arrangement of the oxo and the carboxy
functionalities is an essential structural prerequisite. Compar-
ing SOSAs with SHFAs,13 we observe that a similar distance
between the carboxy group and either the oxo or the hydroxyl
group is necessary for the bioactivity.
In the level of gene regulation, both 6OSA and 7OSA were
found to suppress the expression of critical regulators of cell
growth and proliferation, namely, STAT3 and c-myc. Both
these genes are highly active in a plethora of cancers and
promote tumor growth.35,36 Specifically, STAT3 is a master
regulator of tumor invasiveness through immunosuppression
and angiogenesis.37,38 Additionally, the oncogene c-myc is one
of the most crucial players in the development of tumor.
Specifically, it is implicated in tumor initiation, promotes cell
cycle progression, and controls cell metabolism.39,40 Thus,
both of them are promising pharmacological targets for cancer
therapy35,36 and a variety of small-molecule inhibitors have
been developed and studied as potential therapeutics.41,42 The
present study uncovered that previously unrecognized oxidized
forms of the long-chain fatty acid, stearic acid, in particular
6OSA and 7OSA, are able to suppress cell growth inhibition
and may become new leads for cancer treatment.
Only one member of SOFAs, 3OSA, has been reported so
far, including this oxidized fatty acid in studies in human
plasma, together with other potential biomarkers.16,17 Applying
a suspect lipidomic approach and taking advantage of the
sensitivity and accuracy of an HRMS technique, we showcase
for the first time that various OSAs and OPAs are present in
human plasma. We developed an LC-HRMS analytical
method, which permitted the rapid and successful simulta-
neous determination of ten OSAs and seven OPAs in human
plasma. 6OSA has been proven to be the most abundant in
human plasma at concentrations varying from 4.9 to 19.5 ng/
mL. In addition, three other OSAs (4OSA, 9OSA, and
10OSA) were detected at lower concentrations (up to 6.5 ng/
mL).
Most recently, we reported the presence of SHFAs, a
previously unrecognized class of lipids, in milk,13,14 and
demonstrated that SHFAs inhibit in vitro the cell growth of
cancer cells, with 7HSA exhibiting the most potent effect. We,
therefore, sought that SOFAs might possess a similar biological
activity. Studying the in vitro effect of our synthetic SOFAs on
the proliferation of three different human cancer cell lines
(A549, Caco-2, and SF268), we found that four OSAs (5OSA,
6OSA, 7OSA, 8OSA) inhibit the cell growth of human
epithelial lung carcinoma A549 cells. This effect was proved to
be cell-specific. 6OSA and 7OSA were found to be the most
potent derivatives exhibiting IC50 values of 26 and 28 μM,
General pathway(s) for the generation of various SOFAs
through the metabolism of dietary fatty acids by humans is not
known. However, such oxidized fatty acid forms may result
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Figure 5. Cell growth-inhibitory potency of OPAs and OSAs. (a) Screening of OPAs and OSAs in A549 cancer cell line at 50 μM. (b) Human
cancer cell line A549. (c) IC50 values (μM) of 5OSA, 6OSA, 7OSA, and 8OSA in A549 cells. IC50 values were derived from the dose−response
relationship for a minimum of three experiments (95% CI log IC50 −4.71 to −4.46, −4.64 to −4.46, −4.69 to −4.16, respectively). (d) Human
cancer cell line Caco-2. (e) Human cancer cell line SF268. Cells were treated with increasing concentrations (10, 25, 35, 50, 75 μM) of the test
compounds for 72 h and cell viability was determined by the MTT assay for a minimum of three experiments (avg standard error of the mean
(SEM)). No effect was observed for any compound at 10 μM and this concentration is not shown in the graph for SF268 and Caco-2 cell lines.
from microbes in the gastrointestinal tract. It has been reported
that OFAs may be produced as intermediates during gut
microbial fatty acid metabolism.43 A gut lactic acid bacterium,
Lactobacillus plantarum, has been shown to produce OFAs and
HFAs, for example, 10OSA, from unsaturated fatty acids, such
as linoleic acid, through multienzymatic reactions in saturation
metabolism.43 Apart from the possibility that SOFAs are gut
microbial metabolites of dietary unsaturated acids, the
existence of particular oxidative enzymes able to lead to
regioselective oxidation at a particular position of a saturated
long chain or a nonregioselective chemical C−H activation
mechanism cannot be excluded. In any case, as we demonstrate
in this work, a class of SOFAs is identified for the first time in
human plasma and some of them possess cell growth inhibitory
properties and might play a role in human health.
CONCLUSIONS
■
In summary, adopting both suspect and targeted analytical LC-
HRMS methods, we identified in human plasma a previously
unrecognized class of oxidized fatty acids, the saturated oxo
fatty acids (SOFAs), which carry the oxo functionality at
various positions of the long aliphatic chain. Efficient synthetic
methods, employing a simple photochemical hydroacylation
reaction as the key-step, allowed the easy construction of a
library of SOFAs. The photochemical reaction has been scaled
to prepare SOFAs on a gram scale. Four OSAs were found to
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Figure 6. Effect of 6OSA and 7OSA on cell cycle regulators. (a−k) Relative expression levels of Cyclin D1, Cyclin E1, EGFR, ERBB2 (Her2),
AKT1, PIK3CA (PI3K), PTEN, mTOR, p53, c-myc, and STAT3 mRNA in DMSO, 6OSA-treated (50 μM), and 7OSA-treated (50 μM) A549
cells, measured with quantitative real-time RT-PCR. (l) Western blots for phospho-STAT3, STAT3, and β-actin protein in DMSO-, 6OSA (50
μM)-, and 7OSA (50 μM)-treated A549 cells. (m, n) Relative expression of phospho-STAT3 and STAT3 proteins in DMSO, 6OSA-treated (50
μM), and 7OSA-treated (50 μM) A549 cells. For all cases, *p < 0.05, **p < 0.01, and ***p < 0.001.
apparatus and were uncorrected. 1H and 13C NMR spectra were
inhibit in vitro the cell growth of human epithelial lung
recorded on a Varian Mercury (200 and 50 MHz, respectively) or a
carcinoma A549 cells. Among SOFAs tested, 6OSA and 7OSA
Bruker AVANCE NEO (400 and 100 MHz, respectively) in CDCl3.
exhibited the highest cell growth inhibitory potency. Studying
Chemical shifts δ are given in ppm and coupling constants (J) in Hz.
the effect of these OSAs on different crucial cell cycle
Peak multiplicities are described as follows: s, singlet, t, triplet, and m,
regulators and signaling molecules, we demonstrated that both
multiplet. High-resolution mass spectra were obtained on a Bruker
Maxis Impact QTOF spectrometer or an AB Sciex 4600 Triple TOF
6OSA and 7OSA suppressed the expression of STAT3 and c-
myc. Thus, oxidized fatty acids, such SOFAs and the previously
identified SHFAs,13 may play roles in the protection and
promotion of human health.
mass spectrometer. The purity of all compounds subjected to
biological tests was determined by analytical HPLC and was found
to be ≥95%.
Compounds 744 and 11a45 were synthesized as previously
described and their analytical data were in accordance with the
literature.
EXPERIMENTAL SECTION
■
General. All commercially available products and solvents were
purchased from Sigma-Aldrich, Fluka, Merck, and Alfa Aesar. Solvents
were used as received or dried over molecular sieves (4 Å). All water
or air-sensitive reactions were performed under an argon atmosphere
with dry solvents and anhydrous conditions. All reactions were
monitored by thin-layer chromatography (TLC) performed on
aluminum-backed silica plates (0.2 mm, 60 F254). Purification by
flash chromatography was performed on Merck silica gel 60 (230−
400 mesh). Melting points were determined using a Buchi 530
General Procedure for the Photochemical Reaction of
Aldehydes with Alkenes. To a glass vial with a screw cap
containing the alkene (0.50 mmol) and the aldehyde (1.50 mmol) in
water (1 mL), phenylglyoxylic acid (15 mg, 0.10 mmol) was added.
The vial was sealed with a screw cap and left stirring under household
bulb irradiation (2 × 85 W household lamps) for 48 h. The reaction
mixture was extracted with CH2Cl2 (3 × 3 mL). The combined
organic layers were dried over Na2SO4, and the solvent was removed
in vacuo. The desired oxo fatty acids 3a−3k were isolated by
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Article
trituration with hexane (3−5 mL) (cooled with external ice bath).
Compound 5 and compound 9 as well as ketones 12a−12c were
purified by flash chromatography on silica gel eluting with petroleum
ether (bp 40−60 °C)/ethyl acetate 5/5, 95/5, and 95/5, respectively.
5-Oxooctadecanoic Acid (3a). White solid; mp 79−80 °C; lit.30
80 °C; yield 53%; 1H NMR (400 MHz, CDCl3) δ 2.49 (2H, t, J = 7.2
Hz, CH2COOH), 2.38 (4H, t, J = 7.3 Hz, 2 × COCH2), 1.94−1.85
(2H, m, CH2), 1.60−1.51 (2H, m, CH2), 1.31−1.23 (20H, m, 10 ×
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3) δ
210.6, 179.2, 43.1, 41.4, 33.1, 32.1, 29.8, 29.7, 29.6, 29.5, 29.4, 24.0,
8-Oxohexadecanoic Acid (3j). White solid; mp 77−78 °C; yield
48%; 1H NMR (400 MHz, CDCl3): δ 2.43−2.31 (6H, m, 3 ×
COCH2), 1.68−1.50 (6H, m, 3 × CH2), 1.39−1.21 (14H, m, 7 ×
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
211.7, 179.7, 43.0, 42.8, 34.0, 32.0, 29.5, 29.4, 29.3, 29.0, 24.6, 24.0,
−
23.7, 22.8, 14.2; HRMS (ESI−): m/z calcd for C16H29O3 , 269.2122;
[M − H]− found, 269.2114.
9-Oxohexadecanoic Acid (3k). White solid; mp 70−71 °C; yield
53%; 1H NMR (400 MHz, CDCl3): δ 2.41−2.30 (6H, m, 3 ×
COCH2), 1.68−1.51 (6H, m, 3 × CH2), 1.37−1.21 (14H, m, 7 ×
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
211.8, 179.7, 43.0, 42.9, 34.1, 31.8, 29.4, 29.2, 29.0, 24.8, 24.1, 23.9,
−
22.8, 18.7, 14.2; HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435;
[M − H]− found, 297.2431.
−
22.8, 14.2; HRMS (ESI−): m/z calcd for C16H29O3 , 269.2122; [M −
6-Oxooctadecanoic Acid (3b). White solid; mp 84−85 °C; lit.30
84 °C; yield 63%; 1H NMR (400 MHz, CDCl3): δ 2.45−2.32 (6H, m,
3 × COCH2), 1.67−1.52 (6H, m, 3 × CH2), 1.30−1.22 (18H, m, 9 ×
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
211.1, 179.1, 43.1, 42.3, 33.9, 32.1, 29.8, 29.6, 29.5, 29.4, 24.4, 24.0,
H]− found, 269.2118.
16-Hydroxyhexadecan-7-one (5). White solid; mp 76−78 °C;
yield 51%; 1H NMR (200 MHz, CDCl3): δ 6.67 (1H, br s, OH), 3.60
(2H, t, J = 6.4 Hz, CH2OH), 2.42−2.24 (4H, m, 2 × COCH2), 1.66−
1.13 (22H, m, 11 × CH2), 0.84 (3H, t, J = 5.7 Hz, CH3); 13C NMR
(50 MHz, CDCl3): δ 212.2, 62.9, 42.9, 34.2, 32.6, 31.7, 31.5, 29.4,
29.3, 29.0, 28.8, 25.8, 24.8, 23.9, 22.5, 14.1; HRMS (ESI+): m/z calcd
−
23.3, 22.8 14.3; HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435;
[M − H]− found, 297.2430.
7-Oxooctadecanoic Acid (3c). White solid; mp 78−79 °C; lit.30 78
°C; yield 52%; 1H NMR (400 MHz, CDCl3): δ 2.43−2.32 (6H, m, 3
× COCH2), 1.68−1.50 (6H, m, 3 × CH2), 1.38−1.20 (18H, m, 9 ×
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
211.5, 179.3, 43.0, 42.5, 33.9, 32.1, 29.8, 29.6, 29.5, 29.4, 28.8, 24.6,
+
for C16H32NaO2 , 279.2295; [M + Na]+ found, 279.2289.
4-Oxooctadecyl Benzoate (9). Colorless oil; yield 46%; H NMR
1
(200 MHz, CDCl3): δ 8.06−7.90 (2H, m, COArH), 7.60−7.31 (3H,
m, ArH), 4.30 (2H, t, J = 6.4 Hz, CH2COO), 2.61−2.25 (4H, m, 2 ×
COCH2), 2.12−1.95 (2H, m, CH2), 1.65−1.06 (24H, m, 12 × CH2),
0.85 (3H, t, J = 6.5 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 210.1,
166.5, 133.0, 130.2, 129.6, 128.4, 64.3, 43.0, 39.0, 32.0, 29.8, 29.7,
29.5, 29.4, 29.3, 29.1, 23.9, 23.0, 22.8, 14.2; HRMS (ESI+): m/z calcd
−
24.1, 23.5, 22.8, 14.2; HRMS (ESI−): m/z calcd for C18H33O3 ,
297.2435; [M − H]− found, 297.2429.
8-Oxooctadecanoic Acid (3d). White solid; mp 80−81 °C; lit.30
79 °C; yield 54%; 1H NMR (400 MHz, CDCl3): δ 2.43−2.30 (6H, m,
3 × COCH2), 1.68−1.50 (6H, m, 3 × CH2), 1.40−1.18 (18H, m, 9 ×
CH2), 0.87 (3H, t, J = 6.6 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
211.7, 179.6, 43.0, 42.8, 34.0, 32.0, 29.7, 29.6, 29.4, 29.0, 24.6, 24.1,
+
for C25H40NaO3 , 411.2870; [M + Na]+ found, 411.2865.
18-((tert-Butyldimethylsilyl)oxy)octadecan-9-one (12a). Color-
1
less oil; yield 52%; H NMR (200 MHz, CDCl3): δ 3.58 (2H, t, J
= 6.5 Hz, CH2OTBDMS), 2.37 (4H, t, J = 7.4 Hz, 2 × COCH2),
1.61−1.18 (26H, m, 13 × CH2), 0.91−0.80 (12H, m, 4 × CH3), 0.03
(6H, s, 2 × CH3); 13C NMR (50 MHz, CDCl3): δ 211.8, 63.4, 42.9,
33.0, 32.0, 29.5, 29.4, 29.3, 26.1, 25.9, 24.0, 22.8, 18.5, 14.2, −5.2;
HRMS (ESI+): m/z calcd for C24H50NaO2Si+, 421.3472; [M + Na]+
found, 421.3467.
−
23.7, 22.8, 14.2; HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435;
[M − H]− found, 297.2427.
9-Oxooctadecanoic Acid (3e). White solid; mp 76−77 °C; lit.30
74 °C; yield 67%; 1H NMR (400 MHz, CDCl3): δ 2.42−2.30 (6H, m,
3 × COCH2), 1.67−1.50 (6H, m, 3 × CH2), 1.35−1.23 (18H, m, 9 ×
CH2), 0.87 (3H, t, J = 6.6 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
211. 7, 179.7, 42.9, 42.7, 34.0, 31.9, 29.4, 29.3, 29.0, 28.9, 24.6, 23.9,
18-((tert-Butyldimethylsilyl)oxy)octadecan-3-one (12b). Color-
1
less oil; yield 47%; H NMR (200 MHz, CDCl3): δ 3.59 (2H, t, J
−
23.8, 22.7, 14.1; HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435;
= 6.6 Hz, CH2OTBDMS), 2.48−2.32 (4H, m, 2 × COCH2), 1.59−
1.21 (26H, m, 13 × CH2), 1.04 (3H, t, J = 7.3 Hz, CH3) 0.88 (9H, s,
3 × CH3), 0.04 (6H, s, 2 × CH3); 13C NMR (50 MHz, CDCl3): δ
212.2, 63.5, 42.6, 36.0, 33.0, 29.8, 29.6, 29.4, 26.1, 25.9, 24.1, 18.5,
8.0, −5.1; HRMS (ESI+): m/z calcd for C24H50NaO2Si+, 421.3472;
[M + Na]+ found, 421.3465.
[M − H]− found, 297.2434.
12-Oxooctadecanoic Acid (3f). White solid; mp 80−82 °C; lit.46
78.7−81.4 °C; yield 68%; 1H NMR (400 MHz, CDCl3): δ 2.41−2.31
(6H, m, 3 × COCH2), 1.68−1.50 (6H, m, 3 × CH2), 1.36−1.22
(18H, m, 9 × CH2), 0.88 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100
MHz, CDCl3): δ 212.1, 180.0, 43.0, 42.9, 34.2, 31.8, 29.5, 29.4, 29.3,
29.2, 29.1, 24.8, 24.0, 22.6, 14.2; HRMS (ESI−): m/z calcd for
16-((tert-Butyldimethylsilyl)oxy)hexadecan-3-one (12c). Color-
1
less oil; yield 40%; H NMR (200 MHz, CDCl3): δ 3.59 (2H, t, J =
−
C18H33O3 , 297.2435; [M − H]− found, 297.2435.
6.5 Hz, CH2OTBDMS), 2.48−2.31 (4H, m, 2 × COCH2), 1.63−1.19
(22H, m, 11 × CH2), 1.04 (3H, t, J = 7.3 Hz, CH3), 0.88 (9H, s, 3 ×
CH3), 0.04 (6H, s, 2 × CH3); 13C NMR (50 MHz, CDCl3): δ 212.2,
63.5, 42.6, 36.0, 33.0, 29.8, 29.6, 29.4, 26.1, 25.9, 24.1, 18.5, 8.0, −5.1;
HRMS (ESI+): m/z calcd for C22H46NaO2Si+, 393.3159; [M + Na]+
found, 393.3149.
General Procedure for the Deprotection of tert-Butyldime-
thylsilyl (TBDMS) Group. The appropriate protected alcohol (1.00
mmol) was added to a flame-dried flask in dry THF (3 mL). A
solution of tetra-n-butylammonium fluoride (1.00 mmol, 1 M in
THF) was added dropwise. The reaction mixture was left stirring for 1
h at r.t. and then the solvent was removed under reduced pressure.
The crude reaction mixture was purified by flash chromatography on
silica gel eluting with petroleum ether (bp 40−60 °C)/ethyl acetate
5/5 to give the desired hydroxy-ketone.
5-Oxohexadecanoic Acid (3g). White solid; mp 83−84 °C; lit.47
86.5−87.5 °C; yield 56%; 1H NMR (400 MHz, CDCl3): δ 2.48 (2H,
t, J = 7.2 Hz, CH2COOH), 2.40−2.34 (4H, m, 2 × COCH2), 1.92−
1.84 (2H, m, CH2), 1.58−1.50 (2H, m, CH2), 1.30−1.21 (16H, m, 8
× CH2), 0.86 (3H, t, J = 6.7 Hz,CH3); 13C NMR (100 MHz, CDCl3):
δ 210.7, 179.5, 43.0, 41.4, 33.2, 32.0, 29.7, 29.6, 29.5, 29.4, 29.3, 24.0,
−
22. 8, 18.7, 14.2; HRMS (ESI−): m/z calcd for C16H29O3 , 269.2122;
[M − H]− found, 269.2118.
6-Oxohexadecanoic Acid (3h). White solid; mp 79−80 °C; lit.48
1
81.5−82 °C; yield 53%; H NMR (400 MHz, CDCl3): δ 2.45−2.31
(6H, m, 3 × COCH2), 1.66−1.50 (6H, m, 3 × CH2), 1.31−1.22
(14H, m, 7 × CH2), 0.87 (3H, t, J = 6.6 Hz, CH3); 13C NMR (100
MHz, CDCl3): δ 211.1, 179.2, 43.1, 42.3, 33.9, 32.0, 29.7, 29.6, 29.4,
−
24.3, 24.0, 23.3, 22.8, 14.2; HRMS (ESI−): m/z calcd for C16H29O3 ,
269.2122; [M − H]− found, 269.2114.
18-Hydroxyoctadecan-9-one (13a). White solid; mp 70−71 °C;
7-Oxohexadecanoic Acid (3i). White solid; mp 76−77 °C; lit.28
1
yield 81%; H NMR (200 MHz, CDCl3): δ 3.59 (2H, t, J = 6.5 Hz,
1
75−75.5 °C; yield 52%; H NMR (400 MHz, CDCl3): δ 2.47−2.28
CH2OH), 2.35 (4H, t, J = 7.4 Hz, 2 × COCH2), 1.86 (1H, br s, OH),
1.61−1.43 (6H, m, 3 × CH2), 1.33−1.16 (20H, m, 10 × CH2), 0.84
(3H, t, J = 7.0 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 212.0, 63.0,
42.9, 32.8, 31.9, 29.5, 29.4, 29.3, 29.2, 25.8, 24.0, 23.9, 22.7, 14.2;
(6H, m, 3 × COCH2), 1.70−1.52 (6H, m, 3 × CH2), 1.38−1.22
(14H, m, 7 × CH2), 0.88 (3H, t, J = 6.4 Hz, CH3); 13C NMR (100
MHz, CDCl3): δ 211.5, 177.8, 43.1, 42.6, 33.6, 32.0, 29.6, 29.5, 29.4,
28.8, 24.6, 24.1, 23.5, 22.8, 14.3; HRMS (ESI−): m/z calcd for
+
HRMS (ESI+): m/z calcd for C18H36NaO2 , 307.2608; [M + Na]+
−
C16H29O3 , 269.2122; [M − H]− found, 269.2117.
found, 307.2601.
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18-Hydroxyoctadecan-3-one (13b). White solid; mp 73−74 °C;
which was purified by flash chromatography on silica gel eluting with
petroleum ether (bp 40−60 °C)/ethyl acetate 8/2 to afford the
desired mono-TBDMS-protected diol.
1
yield 79%; H NMR (200 MHz, CDCl3): δ 3.60 (2H, t, J = 6.6 Hz,
CH2OH), 2.46−2.32 (4H, m, 2 × COCH2), 1.94 (1H, br s, OH),
1.60−1.43 (6H, m, 3 × CH2), 1.29−1.20 (20H, m, 10 × CH2), 1.01
(3H, t, J = 7.4 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 212.3, 63.0,
42.5, 35.9, 32.9, 29.7, 29.5, 29.4, 25.8, 24.0, 7.9; HRMS (ESI+): m/z
14-((tert-Butyldimethylsilyl)oxy)tetradecan-1-ol (16a).51 Color-
less oil; yield 31%; 1H NMR (200 MHz, CDCl3) δ 3.64−3.52 (4H, m,
2 × OCH2), 2.15 (1H, br s, OH), 1.50−1.41 (4H, m, 2 × CH2),
1.37−1.16 (20H, m, 10 x CH2), 0.87 (9H, s, 3 × CH3), 0.02 (6H, s, 2
× CH3); 13C NMR (50 MHz, CDCl3) δ 63.5, 63.0, 33.0, 32.9, 29.8,
29.7, 29.6, 26.1, 25.9, 18.5, −5.2.
+
calcd for C18H36NaO2 , 307.2608; [M + Na]+ found, 307.2600.
16-Hydroxyhexadecan-3-one (13c). White solid; mp 68−69 °C;
1
yield 89%; H NMR (200 MHz, CDCl3): δ 3.61 (2H, t, J = 6.5 Hz,
12-((tert-Butyldimethylsilyl)oxy)dodecan-1-ol (16b).52 Colorless
oil; yield 31%; 1H NMR (200 MHz, CDCl3) δ 3.56 (4H, t, J = 6.5 Hz,
2 × OCH2), 2.39 (1H, br s, OH), 1.58−1.39 (4H, m, 2 × CH2),
1.35−1.16 (16H, m, 8 × CH2), 0.85 (9H, s, 3 × CH3), 0.01 (6H, s, 2
× CH3); 13C NMR (50 MHz, CDCl3) δ 63.4, 62.8, 32.9, 32.8, 29.7,
29.6, 29.5, 26.1, 25.9, 18.4, −5.2.
CH2OH), 2.46−2.30 (4H, m, 2 × COCH2), 1.68 (1H, br s, OH),
1.60−1.47 (4H, m, 2 × CH2), 1.33−1.18 (18H, m, 9 × CH2), 1.02
(3H, t, J = 7.3 Hz, CH3); 13C NMR (50 MHz, CDCl3): δ 212.3, 63.1,
42.6, 36.0, 32.9, 29.7, 29.5, 29.4, 25.8, 24.1, 8.0; HRMS (ESI+): m/z
+
calcd for C16H32NaO2 , 279.2295; [M + Na]+ found, 279.2288.
General Procedure for the Oxidation of Hydroxy Ketones.
To a round-bottomed flask containing the alcohol (1.00 mmol)
diluted in acetone (10 mL) at 0 °C, the Jones reagent (2 M, 1.5 mL,
3.00 mmol) was added, and the reaction mixture was left stirring
vigorously for 1 h at r.t. Then, the reaction mixture was quenched
with a saturated solution of NaHSO3 (10 mL) and the reaction
mixture was extracted with Et2O (3 × 20 mL), washed with brine (1
× 50 mL), and dried over Na2SO4. The combined organic layers were
then evaporated to give a crude mixture, which was purified by
trituration with hexane (3−5 mL) (cooled with external ice bath) to
afford the desired oxo fatty acids.
General Procedure for the Oxidation of Mono-TBDMS-
Protected Diols. TBDMS-protected diol 16a,b (1.00 mmol) was
dissolved in dry CH2Cl2 (5 mL) and the solution was added to a
stirred solution of PCC (431 mg, 2.00 mmol) in dry CH2Cl2 (10
mL). The reaction mixture was stirred for 1 h at r.t. Then, the solution
was filtered through a funnel packed with Celite and silica and the
residue was washed with CH2Cl2 (30 mL). The filtrate was
concentrated and evaporated to give a crude mixture, which was
purified by flash chromatography on silica gel eluting with petroleum
ether (bp 40−60 °C)/ethyl acetate 8/2 to give the desired aldehyde.
14-((tert-Butyldimethylsilyl)oxy)tetradecanal (17a).53 Colorless
oil; yield 78%; 1H NMR (200 MHz, CDCl3) δ 9.73 (1H, t, J = 1.9 Hz,
CHO), 3.57 (2H, t, J = 6.5 Hz, CH2OTBDMS), 2.46−2.31 (2H, m,
CH2CHO), 1.69−1.40 (4H, m, 2 × CH2), 1.36−1.18 (18H, m, 9 ×
CH2), 0.87 (9H, s, 3 × CH3), 0.02 (6H, s, 2 × CH3); 13C NMR (50
MHz, CDCl3) δ 203.0, 63.3, 43.9, 32.9, 29.6, 29.4, 29.3, 29.2, 26.0,
25.8, 22.1, 18.5, −5.2.
10-Oxohexadecanoic Acid (6). White solid; mp 76−77 °C; lit.49
74.5−75.5 °C; yield 72%; 1H NMR (400 MHz, CDCl3): δ 2.42−2.31
(6H, m, 3 × COCH2), 1.68−1.50 (6H, m, 3 × CH2), 1.37−1.22
(14H, m, 7 × CH2), 0.88 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100
MHz, CDCl3): δ 211.9, 179.0, 43.0, 42.9, 34.0, 31.8, 29.3, 29.2, 29.1,
−
24.8, 24.0, 22.7, 14.2; HRMS (ESI−): m/z calcd for C16H29O3 ,
269.2122; [M − H]− found, 269.2119.
4-Oxooctadecanoic Acid (10). White solid; mp 95−96 °C, lit.50
95.5−96.2 °C; yield 71%; 1H NMR (400 MHz, CDCl3) δ 2.74−2.68
(2H, m, COCH2), 2.66−2.60 (2H, m, CH2COOH), 2.43 (2H, t, J =
7.5 Hz, COCH2), 1.63−1.53 (2H, m, CH2), 1.33−1.22 (22H, m, 11
× CH2), 0.88 (3H, t, J = 6.6 Hz, CH3); 13C NMR (100 MHz, CDCl3)
δ 209.2, 178.1, 36.9, 32.1, 29.8, 29.6, 29.5, 29.3, 27.8, 24.0, 22.8, 14.3;
12-((tert-Butyldimethylsilyl)oxy)dodecanal (17b).54 Colorless oil;
1
yield 77%; H NMR (200 MHz, CDCl3) δ 9.72 (1H, t, J = 1.9 Hz,
CHO), 3.56 (2H, t, J = 6.6 Hz, CH2OTBDMS), 2.44−2.33 (2H, m,
CH2CHO), 1.68−1.38 (4H, m, 2 × CH2), 1.35−1.16 (14H, m, 7 ×
CH2), 0.86 (9H, s, 3 × CH3), 0.01 (6H, s, 2 × CH3); 13C NMR (50
MHz, CDCl3) δ 203.0, 63.4, 44.0, 33.0, 29.8, 29.6, 29.5, 29.3, 26.1,
25.9, 25.8, 18.5, −5.2.
−
HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435; [M − H]−
found, 297.2432.
General Procedure for the Olefination of TBDMS-Protected
Aldehydes. To a flame-dried round-bottomed flask containing
methyltriphenylphosphonium bromide (536 mg, 1.50 mmol) in dry
THF (6 mL), n-BuLi (1.6 M in hexane, 1.3 mL, 2.00 mmol) was
added slowly at 0 °C, under an argon atmosphere. The resulting deep
orange solution was left to stir at this temperature for 1 h. A solution
of TBDMS-protected aldehyde 17a,b (1.00 mmol) in dry THF (5
mL) was then added slowly over 10 min, and the reaction mixture was
left stirring for 16 h at r.t. The reaction mixture was quenched by the
addition of NH4Cl (5 mL) and the aqueous layer was extracted with
EtOAc (3 × 20 mL). The combined organic layers were dried over
Na2SO4 and evaporated to give a crude mixture, which was purified by
flash chromatography on silica gel eluting with petroleum ether (bp
40−60 °C)/ethyl acetate 95/5 to afford the desired product.
tert-Butyldimethyl(pentadec-14-en-1-yloxy)silane (11b). Color-
less oil; yield 58%; 1H NMR (200 MHz, CDCl3) δ 5.93−5.70 (1H, m,
CH), 5.05−4.88 (2H, m, CH2), 3.60 (2H, t, J = 6.6 Hz,
CH2OTBDMS), 2.12−1.94 (2H, m, CHCH2), 1.56−1.19 (22H,
m, 11 × CH2), 0.89 (9H, s, 3 × CH3), 0.05 (6H, s, 2 × CH3); 13C
NMR (100 MHz, CDCl3) δ 139.4, 114.2, 63.5, 34.0, 33.1, 29.8, 29.7,
29.6, 29.3, 29.1, 26.2, 26.0, −5.1; HRMS (ESI+): m/z calcd for
C21H44NaOSi+, 363.3054; [M + Na]+ found, 363.3049.
10-Oxooctadecanoic Acid (14a). White solid; mp 79−80 °C; lit.30
80 °C; yield 83%; 1H NMR (400 MHz, CDCl3): δ 2.41−2.30 (6H, m,
3 × COCH2), 1.66−50 (6H, m, 3 × CH2), 1.36−1.19 (18H, m, 9 ×
CH2), 0.87 (3H, t, J = 6.7 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
212.0, 179.9, 43.0, 42.9, 34.1, 32.0, 29.5, 29.4, 29.3, 29.2, 29.1, 24.8,
−
24.0, 22.8, 14.2; HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435;
[M − H]− found, 297.2442.
16-Oxooctadecanoic Acid (14b). White solid; mp 83−84 °C; lit.30
84 °C; yield 81%; 1H NMR (400 MHz, CDCl3): δ 2.45−2.31 (6H, m,
3 × COCH2), 1.67−1.51 (4H, m, 2 × CH2), 1.36−1.22 (20H, m, 10
× CH2), 1.04 (3H, t, J = 7.3 Hz, CH3); 13C NMR (100 MHz,
CDCl3): δ 212.4, 180.0, 42.6, 36.0, 34.2, 29.7, 29.6, 29.5, 29.3, 29.1,
−
24.8, 24.1, 8.0; HRMS (ESI−): m/z calcd for C18H33O3 , 297.2435;
[M − H]− found, 297.2439.
14-Oxohexadecanoic Acid (14c). White solid; mp 82−83 °C;
1
yield 88%; H NMR (400 MHz, CDCl3): δ 2.45−2.30 (6H, m, 3 ×
COCH2), 1.66−1.50 (4H, m, 2 × CH2), 1.32−1.21 (16H, m, 8 ×
CH2), 1.03 (3H, t, J = 7.3 Hz, CH3); 13C NMR (100 MHz, CDCl3): δ
212.3, 180.1, 42.6, 36.0, 34.2, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1,
−
8.0; HRMS (ESI−): m/z calcd for C16H29O3 , 269.2122; [M − H]−
found, 269.2128.
General Procedure for the Mono-TBDMS-Protection of
Diols. To a flame-dried round-bottomed flask containing diol 15a,b
(1.00 mmol) in dry DMF (0.5 mL) under an argon atmosphere,
TBDMS-Cl (75 mg, 0.50 mmol), imidazole (68 mg, 1.00 mmol), and
DMAP (6 mg, 0.05 mmol) were added and the reaction mixture was
left stirring for 16 h at r.t. Then, the reaction mixture was quenched
with a saturated solution of NaHCO3 (10 mL), and the aqueous layer
was extracted with EtOAc (3 × 20 mL). The combined organic layers
were dried over Na2SO4 and evaporated to give a crude mixture,
tert-Butyldimethyl(tridec-12-en-1-yloxy)silane (11c). Colorless
1
oil; yield 56%; H NMR (200 MHz, CDCl3) δ 5.88−5.70 (1H, m,
CH), 5.05−4.85 (2H, m, CH2), 3.60 (2H, t, J = 6.4 Hz,
CH2OTBDMS), 2.08−1.98 (2H, m, CHCH2), 1.55−1.22 (18H,
m, 9 × CH2), 0.90 (9H, s, 3 × CH3), 0.05 (6H, s, 2 × CH3); 13C
NMR (50 MHz, CDCl3) δ 139.3, 114.2, 63.5, 34.0, 33.1, 29.8, 29.7,
29.6, 29.3, 29.1, 26.1, 26.0, −5.1; HRMS (ESI+): calcd for
C19H40NaOSi+, 335.2741; [M + Na]+ found, 335.2733.
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Article
Suspect Screening Analysis and Determination of SOFAs.
Liquid chromatography with tandem mass spectrometry (LC-MS/
MS) measurements were performed with an AB Sciex Triple TOF
4600 combined with a micro-LC Eksigent, an autosampler set at 5 °C,
and a thermostated column compartment. Electrospray ionization
(ESI) in a negative mode was used for all of the MS experiments. The
data acquisition method consisted of a TOF-MS full scan m/z 50−
850 and several information-dependent acquisition (IDA)-TOF-MS/
MS product ion scans using 40 V collision energy (CE) with 15 V
collision energy spread (CES) used for each candidate ion in each
data acquisition cycle (1091). A Halo C18 2.7 μm, 90 Å, 0.5 × 50
mm2 column from Eksigent was used, and the mobile phase consisted
of a gradient (A: acetonitrile/0.01% formic acid/isopropanol 80/20
v/v; B: H2O/0.01% formic acid). The data acquisition was carried out
with MultiQuant 3.0.2 and PeakView 2.1 from AB SCIEX. EICs were
obtained with the use of MultiQuant 3.0.2, which created the base
peak chromatograms for the masses that achieve a 0.01 mass accuracy
width. The relative tolerance of the retention time was set within a
margin of 2.5%. Seven samples of human plasma and seven samples
of cow milk were analyzed by LC/HRMS. Seven brand products of
fresh (pasteurized) whole cow milk (fat content 3.5−3.7%) were
collected from the local market in Athens, Greece.
to nitrocellulose membranes (Amersham) using the semidry transfer
system (Bio-Rad). The membranes were blocked with BSA
(Applichem, A1391), 5% dissolved in Tris-buffered saline (1×)
containing 0.1% Tween-20 for 1 h at r.t. The membranes were
incubated with primary antibodies at 4 °C overnight, followed by
secondary antibodies for 1.30 h at r.t. The primary antibodies in the
Western blot were rabbit antiphospho-STAT3 (Cell Signaling, 9145)
(1:1000 dilution), rabbit anti-STAT3 (Santa Cruz, sc-482) (1:1000
dilution), and mouse anti-β-actin (Sigma, A5441) (1:20 000 dilution).
The secondary antibodies were rabbit anti-mouse IgG (Sigma,
A9044) (1:20 000 dilution) and goat anti-rabbit IgG (Sigma,
A6154) (1:10 000 dilution) IgGs.
Statistics. Comparisons between the two data groups (with n ≥ 3
independent experiments) were analyzed using a Student’s t-test.
Values of <0.05 were considered significant and are indicated in the
graphs as *p < 0.05, **p < 0.01, or ***p < 0.001. All data represent
the mean SEM (n ≥ 3).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02058.
Cell Culture. A549, Caco-2, and SF268 cells were cultured in a 37
°C humidified incubator with 5% CO2 cells in Dulbecco’s modified
Eagle’s medium (DMEM) (4.5 g/L glucose, Biosera) medium
supplemented with 10% (v/v) heat-inactivated FBS (Biosera) and
pen−strep (100 U/mL and 100 μg/mL, respectively; Invitrogen).
MTT Assay. All three cell lines used were cultured as described.
After 24 h, the medium was changed to one containing the desired
compounds at selected concentrations, including control samples with
the equivalent vehicle amount. All of the compounds tested were
dissolved in DMSO to prepare a stock solution of 10 mM. The final
solutions were prepared by appropriate dilution of the stock solution
in the media, maintaining a final 0.5% DMSO content. A triplicate for
each condition was always executed in the same plate. Following 72 h,
MTT was added at a final concentration of 0.25 mg/mL and
incubated for 3 h. The medium was removed and DMSO was added
to dissolve the formazan crystals. The formazan absorbance was
measured at 570 nm using an ELISA Plate reader (ELX800,
BIOTEC), while background absorbance was measured at 690 nm.
Cell Growth Inhibition Analysis. Data analysis was performed
using Microsoft Excel (Office Professional Plus 2016). The 690 nm
background absorbance was subtracted from the 570 nm values, and
the average of the three repeats per condition was calculated. The
data-set was normalized to DMSO control cells as a 100% survival
value. The curves were generated using GraphPad Prism version 6.01,
and the IC50 values were calculated from the resultant plots. One-way
and two-way analysis of variance (ANOVA) statistical analysis with
multiple comparisons were performed on the data-set comparing the
various concentrations of the compounds investigated to DMSO
control.
RNA Extraction and Real-Time RT-qPCR Analysis. Total RNA
was isolated by TRI reagent solution (AM9738, Ambion/RNA, Life
Technologies) according to the manufacturer’s instructions, followed
by treatment with RQ1 DNase (Promega, Madison, WI). The RNA
concentration and purity were measured by Nanodrop 2000c
(Thermo), and 1.5 μg was used for cDNA synthesis using the
SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad)
together with random hexamer primers. Quantitative Real-time RT-
qPCR analysis was performed in a LightCycler 96 Instrument
(Roche). Measured values were normalized using β-actin mRNA
levels as internal references. Primers used for real-time RT-qPCR are
shown in Table S4 (Supporting Information).
Two-way and one-way ANOVA statistical analysis; cell
growth inhibitory potency of 6OSA and 7RHSA on
human umbilical vein endothelial cells (Huvec); primers
used for real-time RT-qPCR; large scale synthesis of 7-
1
oxooctadecanoic acid (3c); H NMR and 13C NMR
spectra of the compounds synthesized; and HPLC traces
of SOFAs studied in this work (PDF)
Molecular formula strings and inhibition data (CSV)
AUTHOR INFORMATION
■
Corresponding Author
George Kokotos − Center of Excellence for Drug Design and
Discovery, Department of Chemistry, National and
Kapodistrian University of Athens, Panepistimiopolis, Athens
15771, Greece; orcid.org/0000-0003-3753-7082;
Phone: +30 210 7274462; Email: gkokotos@chem.uoa.gr;
Fax: +30 210 7274761
Authors
Charikleia S. Batsika − Center of Excellence for Drug Design
and Discovery, Department of Chemistry, National and
Kapodistrian University of Athens, Panepistimiopolis, Athens
15771, Greece
Christiana Mantzourani − Center of Excellence for Drug
Design and Discovery, Department of Chemistry, National
and Kapodistrian University of Athens, Panepistimiopolis,
Athens 15771, Greece
Dimitrios Gkikas − Center of Basic Research, Biomedical
Research Foundation of the Academy of Athens, Athens
11527, Greece; Department of Biology, University of Patras,
Patras 26504, Greece
Maroula G. Kokotou − Center of Excellence for Drug Design
and Discovery, Department of Chemistry, National and
Kapodistrian University of Athens, Panepistimiopolis, Athens
15771, Greece
Immunoblotting. The total protein was isolated from treated and
untreated A549 cell with lysis buffer RIPA. The homogenates were
centrifuged at 17 000g for 15 min at 4 °C. The supernatants were
collected, and the protein concentration was measured with the
Bradford protein assay (Bio-Rad protein assay). Then, 35 μg of
protein samples were loaded each time into sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred
Olga G. Mountanea − Center of Excellence for Drug Design
and Discovery, Department of Chemistry, National and
Kapodistrian University of Athens, Panepistimiopolis, Athens
15771, Greece
Christoforos G. Kokotos − Center of Excellence for Drug
Design and Discovery, Department of Chemistry, National
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Article
novel class of lipids with antidiabetic and anti-inflammatory effects.
Diabetes 2016, 65, 1808−1815.
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M.; Beier, P.; Janovska, P.; Veleba, J.; Kopecky, J., Jr.; Kudova, E.;
Pelikanova, T.; Kopecky, J. Docosahexaenoic acid−derived fatty acid
esters of hydroxy fatty acids (FAHFAs) with anti-inflammatory
properties. Diabetes 2016, 65, 2580−2590.
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Sontheimer, A.; Aryal, P.; Wellenstein, K.; Kolar, M. J.; Nelson, A. T.;
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Peroni, O. D.; Saghatelian, A.; Kahn, B. B. Palmitic acid
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beneficial effects on glucose homeostasis. Cell Metab. 2018, 27, 419−
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and Kapodistrian University of Athens, Panepistimiopolis,
Athens 15771, Greece
Panagiotis K. Politis − Center of Basic Research, Biomedical
Research Foundation of the Academy of Athens, Athens
11527, Greece
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c02058
Author Contributions
∥C.M., D.G., and M.G.K. contributed equally.
Notes
The authors declare no competing financial interest.
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T.; Kuda, O. Understanding FAHFAs: From structure to metabolic
regulation. Prog. Lipid Res. 2020, 79, No. 101053.
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fatty acid esters of hydroxy fatty acids (FAHFAs) in biological
samples, plants and foods. Biomolecules 2020, 10, No. 1092.
(8) Köse, M.; Pillaiyar, T.; Namasivayam, V.; De Filippo, E.;
ACKNOWLEDGMENTS
■
The research presented was carried out within the framework
of a Stavros Niarchos Foundation grant to the National and
Kapodistrian University of Athens (G.K.). This work was
partially supported by a Fondation Sante grant and the
Hellenic Foundation for Research and Innovation (H.F.R.I.)
under the “First Call for H.F.R.I. Research Projects to support
Faculty members and Researchers and the procurement of
high-cost research equipment grant” (Grant number: 655)
(C.G.K.) and 1782 (P.K.P.). The research work was supported
by the Hellenic Foundation for Research and Innovation
(H.F.R.I.) under the H.F.R.I. PhD Fellowship grant (Fellow-
ship Number: 1338) (O.G.M.).
Sylvester, K.; Ulven, T.; Kugelgen, I.; Muller, C. E. An agonist
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ABBREVIATIONS
■
AKT, a serine/threonine protein kinase; ANOVA, analysis of
variance; CE, collision energy; CES, collision energy spread;
CFL, compact fluorescent lamp; pCI, confidence interval; c-
myc, myelocytomatosis; DMAP, 4-(dimethylamino)pyridine;
DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
EGFR, epidermal growth factor receptor; EICs, extracted ion
chromatograms; ERBB, erythroblastic oncogene B; ESI,
electrospray ionization; FAHFAs, fatty acid esters of hydroxy
fatty acids; 3HDA, 3-hydroxydecanoic acid; HER2, human
epidermal growth factor receptor 2; HFAs, hydroxy fatty acids;
HPAs, hydroxypalmitic acids; HRMS, high-resolution mass
spectrometry; HSAs, hydroxystearic acids; IDA, information-
dependent acquisition; mTOR, mammalian target of rapamy-
cin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-
lium bromide, thiazolyl blue; OFAs, oxo fatty acids; OPAs,
oxopalmitic acids; OSAs, oxostearic acids; p53, tumor protein
53; PI3K, phosphoinositide 3-kinase; PIK3CA, phosphatidyli-
nositol-4,5-biphosphate 3-kinase catalytic subunit α; p-STAT3,
phosphorylated-STAT3; PTEN, phosphatase and tensin
homologue; SHFAs, saturated hydroxy fatty acids; STAT3,
transcription regulator signal transducer and activator of
transcription 3; TBAF, tetra-n-butylammonium fluoride;
TBDMS, tert-butyldimethylsilyl; THF, tetrahydrofuran
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