Stereochemistry of Endogenous Palmitic Acid Ester of 9-Hydroxystearic Acid and Relevance of Absolute Configuration to Regulation

Article abstract of DOI:10.1021/jacs.7b01269

Lipids have fundamental roles in the structure, energetics, and signaling of cells and organisms. The recent discovery of fatty acid esters of hydroxy fatty acids (FAHFAs), lipids with potent antidiabetic and anti-inflammatory activities, indicates that our understanding of the composition of lipidome and the function of lipids is incomplete. The ability to synthesize and test FAHFAs was critical in elucidating the roles of these lipids, but these studies were performed with racemic mixtures, and the role of stereochemistry remains unexplored. Here, we synthesized the R- and S- palmitic acid ester of 9-hydroxystearic acid (R-9-PAHSA, S-9-PAHSA). Access to highly enantioenriched PAHSAs enabled the development of a liquid chromatography-mass spectrometry (LC-MS) method to separate and quantify R- and S-9-PAHSA, and this approach identified R-9-PAHSA as the predominant stereoisomer that accumulates in adipose tissues from transgenic mice where FAHFAs were first discovered. Furthermore, biochemical analysis of 9-PAHSA biosynthesis and degradation indicate that the enzymes and pathways for PAHSA production are stereospecific, with cell lines favoring the production of R-9-PAHSA and carboxyl ester lipase (CEL), a PAHSA degradative enzyme, selectively hydrolyzing S-9-PAHSA. These studies highlight the role of stereochemistry in the production and degradation of PAHSAs and define the endogenous stereochemistry of 9-PAHSA in adipose tissue. This information will be useful in the identification and characterization of the pathway responsible for PAHSA biosynthesis, and access to enantiopure PAHSAs will elucidate the role of stereochemistry in PAHSA activity and metabolism in vivo.

Full text of DOI:10.1021/jacs.7b01269

Article
pubs.acs.org/JACS
Stereochemistry of Endogenous Palmitic Acid Ester of
9‑Hydroxystearic Acid and Relevance of Absolute Configuration to
Regulation
Andrew T. Nelson,†,‡,§,# Matthew J. Kolar,∥,# Qian Chu,∥ Ismail Syed,⊥ Barbara B. Kahn,⊥
Alan Saghatelian,∥ and Dionicio Siegel*,†
†Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California-San Diego, 9500 Gilman Drive, La Jolla, California
92093-0934, United States
‡Department of Chemistry, University of Texas at Austin, 105 East 24th Street, A5300, Austin, Texas 78712-1224, United States
§School of Medicine, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555, United States
∥Clayton Foundation Laboratories for Peptide Biology, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla,
California 92037-1002, United States
⊥Division of Endocrinology, Diabetes, Metabolism, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical
School, Boston, Massachusetts 02215, United States
S
* Supporting Information
ABSTRACT: Lipids have fundamental roles in the structure, energetics,
and signaling of cells and organisms. The recent discovery of fatty acid
esters of hydroxy fatty acids (FAHFAs), lipids with potent antidiabetic
and anti-inflammatory activities, indicates that our understanding of the
composition of lipidome and the function of lipids is incomplete. The
ability to synthesize and test FAHFAs was critical in elucidating the roles
of these lipids, but these studies were performed with racemic mixtures,
and the role of stereochemistry remains unexplored. Here, we synthesized
the R- and S- palmitic acid ester of 9-hydroxystearic acid (R-9-PAHSA, S-
9-PAHSA). Access to highly enantioenriched PAHSAs enabled the
development of a liquid chromatography−mass spectrometry (LC−MS)
method to separate and quantify R- and S-9-PAHSA, and this approach
identified R-9-PAHSA as the predominant stereoisomer that accumulates
in adipose tissues from transgenic mice where FAHFAs were first
discovered. Furthermore, biochemical analysis of 9-PAHSA biosynthesis and degradation indicate that the enzymes and pathways
for PAHSA production are stereospecific, with cell lines favoring the production of R-9-PAHSA and carboxyl ester lipase (CEL),
a PAHSA degradative enzyme, selectively hydrolyzing S-9-PAHSA. These studies highlight the role of stereochemistry in the
production and degradation of PAHSAs and define the endogenous stereochemistry of 9-PAHSA in adipose tissue. This
information will be useful in the identification and characterization of the pathway responsible for PAHSA biosynthesis, and
access to enantiopure PAHSAs will elucidate the role of stereochemistry in PAHSA activity and metabolism in vivo.
were named Fatty Acid esters of Hydroxy Fatty Acids
(FAHFAs) to describe their structures (Figure 1).
INTRODUCTION
■
Mice engineered to overexpress the glucose transporter,
GLUT4, selectively in their adipose tissue (AG4OX mice)1−3
have provided insights into the link between obesity and Type
2 diabetes because they are obese but have lower fasting
glycemia and enhanced glucose tolerance. Analysis of adipose
tissues from AG4OX mice demonstrated marked increases in de
novo lipogenesis,2,3 and this activity is necessary for the
enhanced glucose tolerance in these animals. Lipidomics
analysis of adipose tissue from AG4OX mice to identify
specific lipids that might contribute to the insulin sensitive
phenotype led to the discovery of a novel class of lipids4 that
are highly elevated in AG4OX adipose tissues.4 These lipids
FAHFAs contain two acyl chains connected through a single
ester bond (Figure 1), and further targeted liquid chromatog-
raphy−mass spectrometry (LC−MS) determined that at least
16 different FAHFA families exist,4 which differ in their acyl
chain constituents (Figure 1), and additional FAHFA families
have been found since.5,6 For instance, palmitic acid esters of
hydroxy stearic acids (PAHSAs) and oleic acid esters of
hydroxy stearic acid (OAHSAs) are two of the most abundant
families that were identified.4 In addition, for each FAHFA
Received: February 6, 2017
© XXXX American Chemical Society
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Figure 2. S-9-PAHSA (1-S) and R-9-PAHSA (1-R).
Figure 1. Endogenous FAHFA structures. Analysis of AG4OX mice
revealed the existence of FAHFAs. Sixteen different FAHFA families,
which differ in the composition of their acyl chains (e.g., PAHSA and
OAHSA) were observed. And within each family, there exist multiple
regioisomers such as 9- and 5-PAHSA.
9-PAHSA from AG4OX mouse adipose tissue. This work
represents a critical step in understanding PAHSA biology by
defining endogenous FAHFA structure.
RESULTS AND DISCUSSION
■
Synthesis of Enantiopure R- and S-9-PAHSA and 9-
HSA. We developed multigram scale syntheses of enantiopure
R- and S-9-PAHSA and HSA to further understand the
influence of FAHFA structure on their pronounced biological
effects. Building upon previous work synthesizing related
compounds such as palmitic acid esters of hydroxy palmitic
acid (PAHPAs),8 and methods for accessing enantioenriched 9-
HSA (54% ee)9 we have developed a route that provides
material on large scale (>5 g batches), high enantiomeric excess
(>98% ee), and free from heavy metal impuritiesall enabling
extensive biological testing.
family that has been looked at in detail, different ester
regioisomers exist (Figure 1). In adipose tissue of wild-type
and AG4OX mice, eight distinct PAHSA regioisomers (5, 7, 8,
9, 10, 11, 12, and 13) were identified.4 Regioisomers
demonstrate differential regulation and biological activity. For
example, all the PAHSA regioisomers were markedly elevated
in adipose tissue and serum of AG4OX mice while the isomers
in liver were decreased. Furthermore, when wild type mice were
placed on a high-fat diet, only two PAHSA isomers were
changed in serum, while all isomers were reduced in
subcutaneous adipose tissue but some were elevated in
perigonadal adipose tissue.4 This suggests that PAHSAs can
be regulated as a family or individually under different
conditions and in different tissues. Furthermore, 5- and 9-
PAHSA have potent antidiabetic effects in vivo and enhance
glucose transport and insulin secretion in vitro, but only 9-
PAHSA has anti-inflammatory effects in dendritic cells,
highlighting regiospecific biological activity.4
Interest in the PAHSAs was piqued further by the analysis of
human sera that revealed that PAHSA levels are lower in
insulin-resistant humans relative to their insulin-sensitive
counterparts, suggesting that PAHSAs may play an important
role in maintaining normal blood sugar and insulin sensitivity in
people.4 Indeed, oral administration of 9-PAHSA or 5-PAHSA
to mice on a high-fat diet improved glucose tolerance through
multiple pathways that include increases in glucagon-like
peptide 1 (GLP-1) and insulin secretion.4
The anti-inflammatory properties of FAHFAs can also extend
to the treatment of inflammatory diseases as was demonstrated
by recent work showing that oral 9-PAHSA treatment reduces
the severity and impedes inflammation and damage associated
with colitis in a mouse model.7 And other novel FAHFA
families with anti-inflammatory activities have recently been
discovered. Omega-3 fatty acid FAHFAs, produced after the
addition of omega-3-fatty acids to cells, have potent anti-
inflammatory activity.5
Thus, FAHFAs, and in particular PAHSAs, are an exciting
class of novel lipids with a range of biological activities. One
important structural question that remains unanswered is the
natural configuration of the chiral center in PAHSAs. We
studied 9-PAHSA, the most abundant and a biologically active
PAHSA. Herein we report the first asymmetric synthesis of R-
and S-9-PAHSA (Figure 2) and R- and S-9-HSA, and utilize
these compounds to define the stereochemistry of endogenous
Using epichlorohydrin as both a readily available chiral
starting material and lynchpin that connects the three regions
of PAHSAs, the synthesis of both enantiomers was achieved.
The synthesis of R-9-PAHSA (1-R) began with the addition of
7-octenylmagnesium bromide into S-(+)-epichlorohydrin 4
using catalytic copper(I) iodide to generate chlorohydrin 5
(Scheme 1). Epoxide formation from chlorohydrin 5, yielding
epoxide 6, was followed by a second epoxide opening reaction
at the less substituted position using octylmagnesium chloride
in the presence of cuprous iodide to afford secondary alcohol 7
as a solid. The relay of enantiopurity, and retention of
configuration at the chiral center, was assessed by derivatization
of 7 as the S-O-acetylmandelate ester9,10 with the diastereo-
meric ratio found to be greater than 99:1 (Supporting
Information, SI). Palmitoylation of alcohol 7 with palmitoyl
chloride and pyridine provided ester 8. Reaction of 8 with
ozone in the presence of N-methylmorpholine N-oxide
(NMO)11 followed by Pinnick oxidation12 of the crude
intermediate provided R-9-PAHSA (1-R) in 55% yield.
Degradation of R-9-PAHSA (1-R) by saponification yielded
R-9-HSA (SI). S-9-PAHSA (1-S) and S-9-HSA were synthe-
sized in an analogous manner, substituting R-(+)-epichlorohy-
drin 9 at the start of the synthesis (in place of S-
(+)-epichlorohydrin 4) through the same 5 step sequence in
61% overall yield (Scheme 1).
Determination of Endogenous 9-PAHSA Stereochem-
istry. Fatty acid oxidation is a common event in the production
of bioactive lipids. Prostaglandins and leukotrienes, for example,
are generated from the oxidation of arachidonic acids.13 The
enzymatic introduction of oxygen occurs in a stereospecific
manner, and the stereochemistry in these lipids define their
activity.14 The PAHSAs also contain an oxidized fatty acid, the
HSA, which places a stereocenter in this lipid. Since
stereochemistry has an important role in bioactive lipid
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a
Scheme 1. Syntheses of R-9-PAHSA from S-(+)-Epichlorohydrin (4) and S-9-PAHSA from R-(+)-Epichlorohydrin (9)
a
Conditions: (a) 7-octenylmagnesium bromide, CuI, THF, −78 to 23 °C (1-R sequence 92%, 1-S sequence 91%); (b) NaOH, Et2O, 23 °C; (c)
octylmagnesium chloride, CuI, Et2O, −78 to 23 °C (1-R sequence 70%, 1-S sequence 90%; over two steps); (d) palmitoyl chloride, pyr., CH2Cl2, 0
to 23 °C(1-R sequence 91%, 1-S sequence 78%); and (e) O3, NMO, CH2Cl2, −10 °C then NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH, 23 °C
(1-R sequence 94%, 1-S sequence 96%).
function, and can provide information about the enzymes and
biochemical pathways for lipid metabolism, we sought to define
the endogenous stereochemistry of 9-PAHSA.
Under these conditions, R-9-PAHSA elutes from the column at
20.2 min, while S-9-PAHSA elutes at 17.4 min, affording 2.8
min of peak separation to resolve these stereoisomers. One
caveat with this method is that liquid chromatography
conditions required to separate the stereoisomers use formic
acid, a mobile phase modifier typically reserved for positive
mode ionization, but 9-PAHSA detection by mass spectrometry
requires negative mode ionization. In our initial tests we used a
mobile phase that was similar to our established FAHFA
measurement method, which includes 0.01% ammonium
hydroxide,15 however, this could not achieve baseline resolution
of the stereoisomers (Figure S2). It is important to note that
with the presence of 0.1% formic acid, we observed a 25-fold
reduction in the sensitivity of our detection due to the impaired
ionization of the 9-PAHSA (Figure S2). As a result, any
possibility of detecting endogenous 9-PAHSA requires a sample
with high concentrations of this lipid.
We have an established method to separate and analyze these
different families and isomers using reversed-phase chromatog-
raphy,15 however, this method is unable to resolve stereo-
isomers. The first step in determining the stereochemistry of
natural FAHFAs required us to develop a method capable of
separating the different isomers. We attempted several
approaches to separate PAHSAs. First, we used chiral GC,
where 1-R and 1-S were converted to the corresponding methyl
ester using TMS-diazomethane, but we were unable to separate
these stereoisomers. Next, we converted rac-1 to the
corresponding amide with S-1-phenylethan-1-amine to generate
diastereomers that we hoped to separate. These attempts were
also unsuccessful, likely owing to the large distance between to
two chiral centers.
Unable to separate the 9-PAHSA stereoisomers with our
resources, we decided to use Phenomenex’s (Torrance, CA)
chiral screening service. We provided enantiopure R-9-PAHSA
and rac-9-PAHSA. These compounds were tested on 6 of their
chiral columns (Cellulose-1, Cellulose-2, Cellulose-3, Cellulose-
4, Amyolse-2, and Amylose-1) using 3 different mobile phases
(water/acetonitrile, hexane/ethanol, and methanol/isopropa-
nol). The column and mobile phase that provided the best
resolution was the Lux 5 μm Cellulose-3 column with
methanol/isopropanol/trifluoroacetic acid (TFA) (90:10:0.1)
(Figure S1). Using these conditions and column as a starting
point, we continued to refine the method for our purposes.
Specifically, we needed to modify the method since TFA is not
compatible with LC-MS,16 and we needed the sensitivity of
mass spectrometry to be able to detect 9-PAHSA from
endogenous sources.
We have previously shown that FAHFA levels are
dramatically upregulated in AG4OX mice.3 Specifically, 9-
PAHSA levels were elevated ∼16−18 fold in the white adipose
tissue (WAT) of AG4OX and the final concentration of 9-
PAHSA is ∼1000 pmol/g in perigonadal white adipose tissue
(PGWAT). The high 9-PAHSA concentrations make the
PGWAT from AG4OX mice the best tissue to measure
endogenous 9-PAHSA under conditions of reduced sensitivity.
Additionally, by comparing WT and AG4OX samples by chiral
LC-MS we can readily identify the 9-PAHSA peak as the one
with the largest fold change between the two samples to
provide an unambiguous identification of this lipid in the
resulting chromatograms. LC-MS analysis of the samples
revealed that R-9-PAHSA was elevated in AG4OX samples
compared to WT PGWAT, with the R-9-PAHSA peak clearly
elevated in the LC-MS chromatogram (Figure 3A). There is
also a much smaller peak that overlaps with the S-9-PAHSA
standard. We cannot determine if this is S-9-PAHSA or a 10-
PAHSA stereoisomer because S-9-PAHSA and 10-PAHSA have
These efforts led to an optimized method that uses a Lux 3
μm Cellulose-3 column in combination with an isocratic
MeOH/H2O/formic acid (96:4:0.1) mobile phase (Figure 3A).
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The pancreas has the highest 9-PAHSA hydrolytic activity
compared to other tissues, and we showed that this FAHFA
degrading activity is due to CEL. To determine if CEL prefers
R- or S-9-PAHSA, we carried out a previously reported LC-MS
assay to measure hydrolytic activity.17,19 We expressed CEL in
HEK293T cells and isolated membrane lysates from these cells
to measure CEL-mediated 9-PAHSA hydrolysis. The data
clearly demonstrate that CEL prefers S-9-PAHSA to R-9-
PAHSA as a substrate (Figure 4A), indicating that the enzyme
Figure 4. CEL prefers S-9-PAHSA as a substrate. (a) R-, S-, and rac-9-
PAHSA hydrolysis activity for membrane lysates from CEL-transfected
and mock-transfected HEK293T cells. Data represent means and the
standard deviation for two biological replicates. (b) R-, S-, and rac-9-
PAHSA hydrolysis activity for mouse pancreatic membrane lysates.
Data represents mean and the standard error of the mean for three
biological replicates. **p < 0.01 by Student’s t test.
is stereoselective. In support of this finding, this same trend was
also observed when testing the hydrolytic activity of these
enantiomers in mouse pancreatic membrane lysate (Figure 4B).
This data supports the idea that PAHSA metabolism in vivo is
stereoselective, and at least in the pancreas, this activity favors
the degradation of S-9-PAHSA.
Figure 3. R-9-PAHSA is the prominent enantiomer in mouse adipose
tissue. (a) Representative PAHSA MS traces in PGWAT of WT and
AG4OX mice with rac-, R-, and S-9-PAHSA standards resolved on a
Lux 3 μm Cellulose-3 chiral column. (b) 9-PAHSA and R-9-PAHSA
fold change in AG40X PGWAT vs WT PGWAT analyzed on a
reversed-phase column and chiral column, respectively. PAHSA levels
were normalized to the endogenous lipid d18:1/16:0 ceramide. Data
represent means the standard error of the mean for three biological
replicates. ns, not significant.
Although the biosynthetic enzymes responsible for FAHFA
synthesis are unknown, it has been previously shown that the
addition of palmitoyl-CoA and 9-HSA to liver and WAT lysate
results in the production of 9-PAHSA.4 Here, we present a cell-
based assay where the addition of HSAs results in the
production of FAHFAs, such as 9-PAHSA. In this assay, we
treat HEK293T cells with either R- or S-9-HSA, incubate the
cells for 2 h, and then harvest the cells and extract the 9-
PAHSA for subsequent analysis by LC-MS. The R- or S-9-HSA
is acylated by endogenous free fatty acids such as palmitate and
oleate, as these were not added as acyl-donors. R-9-HSA-treated
cells exhibited a substantial increase of 9-PAHSA and 9-
OAHSA (∼80%) compared to S-9-HSA-treated cells (Figure
5). The stereospecificity of 9-PAHSA biosynthesis supports the
idea of that PAHSA biosynthesis is mediated by an unidentified
acyl transferase and is not the result of a nonenzymatic
acylation. Furthermore, these preliminary data indicate a
preference for the biosynthesis of R-9-PAHSA, which, if similar
in adipose tissue could explain the observation of R-9-PAHSA
as the preferred stereoisomer in vivo.
similar retention times (Figure S3). We analyzed these same
samples using our standard (i.e., nonchiral reverse phase)
method (Figure S4) and found an equivalent fold change for R-
9-PAHSA (chiral column) and total 9-PAHSA (reversed-phase
column) (Figure 3B) highlighting that R-9-PAHSA is the main
source of the elevated 9-PAHSA levels we observe in AG4OX
PGWAT.
Stereospecificity of 9-PAHSA Degradation and Bio-
synthesis. Having identified the predominant stereoisomer in
AG4OX PGWAT, we wanted to test whether the biochemical
pathways or enzymes that control 9-PAHSA biosynthesis and
degradation are stereospecific. We have recently identified the
enzyme carboxyl ester lipase (CEL) to be a FAHFA
hydrolase.17 CEL is an enzyme of the exocrine pancreas and
is secreted into the gut to assist with the digestion of lipids from
the diet.18 Though originally identified as a triglyceride and
cholesterol ester hydrolase, our recent work clearly shows that
this enzyme prefers FAHFAs, including 9-PAHSA.
CONCLUSIONS
■
The importance of absolute structure in the biochemistry and
biological activity of lipids prompted us to develop the first
stereoselective synthesis of PAHSAs, which enabled us to
identify the natural isomer in adipose tissue as R-9-PAHSA.
Starting from this important finding, this chemistry will enable
access to numerous enantiopure FAHFAs, necessary to better
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ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
Grants R01 DK043051 and P30DK57521 (to B.B.K.) and R01
DK106210 (to B.B.K. and A.S.), a grant from the JPB
Foundation (to B.B.K.), Leona M. and Harry B. Helmsley
Charitable Trust Grant 2012-PG-MED002 (to A.S.), F30
DK112622 (A.T.N.) and F30 DK112604 (M.J.K.), the NCI
Cancer Center Support Grant P30 (CA014195 MASS core,
A.S.), and Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals
(A.S.).
REFERENCES
■
(1) Shepherd, P. R.; Gnudi, L.; Tozzo, E.; Yang, H.; Leach, F.; Kahn,
B. B. J. Biol. Chem. 1993, 268 (30), 22243−2246.
(2) Tozzo, E.; Shepherd, P. R.; Gnudi, L.; Kahn, B. B. Am. J. Physiol.
1995, 268 (5), E956−E964.
Figure 5. R-9-HSA is the preferred 9-HSA enantiomer for FAHFA
biosynthesis. Biosynthesis of 9-PAHSA and 9-OAHSA in HEK293T
cells after incubation with 100 μM of R- or S-HSA for 2 h. *p < 0.05 by
two-sided Student’s t-test comparing R-9-HSA-treated versus S-9-
HSA-treated HEK293T cells.
(3) Herman, M. A.; Peroni, O. D.; Villoria, J.; Schon, M. R.;
Abumrad, N. A.; Bluher, M.; Klein, S.; Kahn, B. B. Nature 2012, 484
(7394), 333−8.
(4) Yore, M. M.; Syed, I.; Moraes-Vieira, P. M.; Zhang, T.; Herman,
M. A.; Homan, E. A.; Patel, R. T.; Lee, J.; Chen, S.; Peroni, O. D.;
Dhaneshwar, A. S.; Hammarstedt, A.; Smith, U.; McGraw, T. E.;
Saghatelian, A.; Kahn, B. B Cell 2014, 159 (2), 318−332.
(5) Kuda, O.; Brezinova, M.; Rombaldova, M.; Slavikova, B.; Posta,
M.; Beier, P.; Janovska, P.; Veleba, J.; Kopecky, J., Jr.; Kudova, E.;
Pelikanova, T.; Kopecky, J. Diabetes 2016, 65 (9), 2580−90.
(6) Ma, Y.; Kind, T.; Vaniya, A.; Gennity, I.; Fahrmann, J. F.; Fiehn,
O. J. Cheminf. 2015, 7, 53.
(7) Lee, J.; MoraeS-Vieira, P. M.; Castoldi, A.; Aryal, P.; Yee, E. U.;
Vickers, C.; Parnas, O.; Donaldson, C. J.; Saghatelian, A.; Kahn, B. B J.
Biol. Chem. 2016, 291 (42), 22207−22217.
(8) Balas, L.; Bertrand-Michel, J.; Viars, F.; Faugere, J.; Lefort, C.;
CaspaR-Bauguil, S.; Langin, D.; Durand, T. Org. Biomol. Chem. 2016,
14 (38), 9012−9020.
understand their chemistry and biology. As the synthetic
approach is modular, it is readily adaptable to the synthesis of
related compounds. With the improved sensitivity of our chiral
LC-MS method the measurement of additional FAHFA
stereoisomers in other tissues is possible.
An important insight from the current work is that FAHFAs
are products of enzymatic, not chemical production of the
hydroxylated acids. Chemical oxidation of lipids with reactive
oxygen species, such as peroxides, results in the production of
racemic fatty acids.20 By contrast, enzyme-mediated hydroxy
fatty acid production proceeds stereospecifically to produce
enantiopure hydroxy lipids.20,21 The discovery that R-9-
PAHSA, but not S-9-PAHSA, is increasing in the AG4OX
WAT supports the idea that this is an enzyme-mediated
pathway and chemical oxidation of lipids does not have a role in
PAHSA production in these mice. Additional work will
endeavor to identify the enzymes and pathways that mediate
PAHSA production in vivo, and small molecule activators or
inhibitors of these PAHSA biosynthetic enzymes might
someday allow control over endogenous PAHSA levels. The
continued interplay between chemical synthesis, biology, and
biochemistry will reveal the biological potential of FAHFAs and
identify new ways to regulate these molecules for therapeutic
benefit.
(9) Ebert, C.; Felluga, F.; Forzato, C.; Foscato, M.; Gardossi, L.;
Nitti, P.; Pitacco, G.; Boga, C.; Caruana, P.; Micheletti, G. J. Mol. Catal.
B: Enzym. 2012, 83, 38−45.
(10) Yang, W.; Dostal, L.; Rosazza, J. P. Appl. Environ. Micro. 1993,
59 (1), 281−284.
(11) Schwartz, C.; Raible, J.; Mott, K.; Dussault, P. H. Org. Lett.
2006, 8 (15), 3199−3201.
(12) Bal, B. S.; Childers, W. E.; Pinnick, H. W. Tetrahedron 1981, 37
(11), 2091−2096.
(13) Funk, C. D. Science 2001, 294 (5548), 1871−1875.
(14) Corey, E.; Marfat, A.; Goto, G.; Brion, F. J. Am. Chem. Soc. 1980,
102 (27), 7984−7985.
(15) Zhang, T.; Chen, S.; Syed, I.; Stahlman, M.; Kolar, M. J.;
̊
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b01269.
■
́
Homan, E. A.; Chu, Q.; Smith, U.; Boren, J.; Kahn, B. B. Nat. Protoc.
S
2016, 11 (4), 747−763.
́
(16) GarcIa, M.; Hogenboom, A.; Zappey, H.; Irth, H. J. Chrom. A
2002, 957 (2), 187−199.
(17) Kolar, M. J.; Kamat, S. S.; Parsons, W. H.; Homan, E. A.; Maher,
T.; Peroni, O. D.; Syed, I.; Fjeld, K.; Molven, A.; Kahn, B. B.
Biochemistry 2016, 55 (33), 4636−4641.
Detailed description of synthetic procedures for non-
racemic 9-PAHSA, characterization data, and spectra.
Analytical and biological assay procedures are provided
(PDF)
(18) Chen, Q.; Sternby, B.; Nilsson, Å. Biochim. Biophys. Acta, Lipids
Lipid Metab. 1989, 1004 (3), 372−385.
(19) Parsons, W. H.; Kolar, M. J.; Kamat, S. S.; Cognetta, A. B., III;
Hulce, J. J.; Saez, E.; Kahn, B. B.; Saghatelian, A.; Cravatt, B. F. Nat.
Chem. Biol. 2016, 12 (5), 367−372.
AUTHOR INFORMATION
Corresponding Author
*drsiegel@ucsd.edu
■
(20) Niki, E.; Yoshida, Y.; Saito, Y.; Noguchi, N. Biochem. Biophys.
Res. Commun. 2005, 338 (1), 668−676.
ORCID
(21) Schneider, C.; Pratt, D. A.; Porter, N. A.; Brash, A. R. Chem.
Biol. 2007, 14 (5), 473−488.
Dionicio Siegel: 0000-0003-4674-9554
Author Contributions
#A.T.N. and M.J.K. contributed equally.
Notes
The authors declare no competing financial interest.
E
DOI: 10.1021/jacs.7b01269
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX