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Regiocontrolled syntheses of FAHFAs and

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Cite this: Org. Biomol. Chem., 2016,

LC-MS/MS differentiation of regioisomers†
14, 9012
Laurence Balas,*a Justine Bertrand-Michel,b,c,d Fanny Viars,b,c,d Julien Faugere,b,c,d
Corinne Lefort,b,c Sylvie Caspar-Bauguil,b,d,e Dominique Langinb,d,e and
Thierry Duranda

Received 26th July 2016,
Accepted 26th August 2016
DOI: 10.1039/c6ob01597b
www.rsc.org/obc
An efficient regiospecific total synthesis of several branched fatty acyl hydroxyl-fatty acids (FAHFA) has
been achieved from available terminal alkenes and alkynes. The key steps feature a boron trifluoride
mediated epoxide ring opening with acetylide carbanions, followed by hydrogenation of the alkyne func-
tion. The carboxylic acid of the hydroxylated chains is introduced at the last step of the synthesis to allow
the esterification of the branched hydroxyl group by fatty acids beforehand. The chemical syntheses of a
“linear” FAHFA and a branched FAHFA analog containing a Z-olefin in the hydroxyl-fatty acid chain are
also reported. A LC-MS/MS method has been developed. Several reversed phase columns were
compared. Regioisomers were separated.

Introduction and insulin-stimulated glucose transport in adipocytes.3,4

FAHFAs are a class of endogenous lipids which are esters of
fatty acids (FA) and hydroxy-fatty acids (HFA).
(O-Acyl)-omega-hydroxy fatty acids have been detected at
relatively high concentrations (5% w/w) in meibum.1,2 Very
recently, Yore et al. identified branched FAHFAs in mamma-
lian adipose tissues and blood plasma and also in rodent and
human foods.3 Multiple combinations of FAs and HFAs exist.
Among the FAs found in branched FAHFAs, oleic acid (OA,
C18:1 n-9), palmitic acid (PA, C16:0 n-9) and palmitoleic acid
(PO, C16:1 n-7) are the most abundant FAs while the HFA
subunit is predominantly a saturated C18 chain or a C16 chain
i.e. hydroxystearic acid (HSA) or hydroxypalmitic acid (HPA)
respectively. A great variety of regioisomers were detected. The
ester bond may be located at carbon positions 5, 7, 8, 9, 10, 11,
12 and 13.
Among these newly uncovered FAHFAs, 5- and 9-PAHPA
improve glucose tolerance while directly stimulating insulin
secretion in pancreas, incretin secretion by intestinal cells,
a
Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université
Montpellier, ENSCM, Faculté de Pharmacie, 15 av. Charles Flahault, 34093
Montpellier Cedex 5, France. E-mail: laurence.balas@umontpellier.fr
b
INSERM, UMR 1048, Institute of Metabolic and Cardiovascular Diseases, Toulouse,
France
c
Metatoul-Lipidomic Facility, MetaboHUB, France
d
University of Toulouse, Paul Sabatier University, France
e
Toulouse University Hospitals, Laboratory of Clinical Biochemistry, Toulouse,
France
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6ob01597b
9-PAHPA was also shown to possess anti-inflammatory effects.3
Data in mice and humans therefore suggest that FAHFAs act
as endocrine mediators of insulin sensitivity and may exert
anti-diabetic effects. Each regioisomer and family type may be
differently expressed and regulated, depending on the studied
fluids, tissues and cells.3,5 These observations are striking for
at least two reasons. First, unlike mediators of insulin resist-
ance, the number of adipose tissue-derived factors with
positive effects on insulin sensitivity is limited. Second, FAs,
especially the saturated forms such as palmitate which are
parts of FAHFA, have been established as exerting detrimental
effects on insulin action in liver and skeletal muscle.
The diversity of FAHFAs prompted us to synthesize internal
standards for lipidomic analyses in different tissues and
fluids. A few of them may be purchased but some regioisomers
are not yet commercially available. Very recently, an in silico
MS/MS library was established for automatic annotation.6
However, authentic or synthetic standards are still needed for
quantitation. Besides, according to results from Kahn’s labora-
tory,3 branched esters of HPA (not yet commercially available)
may also serve as biomarkers of insulin sensitivity in obese
people since several FAHPAs are also relatively abundant in
sera of mice overexpressing the insulin-sensitive glucose trans-
porter GLUT4 in adipose tissue (about 8 mmol L−1) while their
concentration is rather low in sera of wild type mice.
The diversity of FAHFAs prompted us to seek a regio-con-
trolled and versatile synthesis of various branched FAHFAs (1).
The chemical strategy is exemplified with the preparation of
regioisomers having the ester bond at carbon atom 5, 7, 9 or
10. In nature, many HFAs show an unsaturated lipid chain.7

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Thus, a total synthesis of a branched FAHFA (2) having a
(Z)-double bond in the HFA chain is also presented herein,
using the same retrosynthesis analysis as for the above-
mentioned fully saturated branched FAHFAs. In addition, a
novel synthesis of a linear FAHFA i.e. omega-OAHPA (3) was
also developed in order to compare its bioactivities and HPLC
behaviour with its branched regioisomers.
To monitor the presence of these very promising
biomarkers in biological samples, an easy and rapid method
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of quantitation is needed. Liquid chromatography (LC) seems
currently the most appropriate technique to analyze mixtures
of these structurally close molecules, with optimal peak resolu-
tion. Mass spectrometry with the triple quadrupole technology
is expected to provide the most sensitive mode of detection.
Thus, having several synthetic FAHFAs in hand, including
several regioisomers, we investigated different LC conditions
to obtain an efficient separation of this class of compounds in
a short time.
Results and discussion
Retrosynthesis
FAHFAs are comprised of both an ester bond and a free car-
boxylic acid group. The presence of the free carboxylic acid
group is expected to prevent a regio-selective direct esteri-
fication of HFA by FA. Thus, the synthesis of branched FAHFAs
1 was envisioned via the esterification of a secondary hydroxyl
function in fatty chains 4, whose carboxylic acid function is
“masked” as a silylated primary alcohol. The deprotection and
oxidation to carboxylic acid were performed in the last steps of
the synthesis.
The retrosynthetic analysis is outlined in Scheme 1.
Two routes were developed depending on the location of
the branched ester function. Both routes are based on an
epoxide ring opening with lithium acetylides, leading to
propargylic alcohols 5 or 5′ at position y in the fatty chain.
Scheme 1 Retrosynthesis for branched FAHFAs 1 (y = 5, 7, 9 or 10).
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Reduction of the alkyne functions 5 or 5′ should provide the
desired fully saturated carbon chains 4. The terminal epoxides 6
or 6′ may come from the epoxidation of terminal olefins 7 or 7′.
Branched FAHFAs with the ester bond at position 5, 9 or 10
were prepared from a non-functionalized terminal alkynes 8
and 1-hydroxy y-alkenes (y = 5, 9 or 10) 7. In the case of
branched FAHFAs with the ester bond at position 7, the
required 1-hydroxy-7-octene (compound 7 with z = 3) was not
commercially available. However, an alternative route may be
performed using a similar retrosynthetic analysis from
non-functionalized terminal alkenes 7′ and 4-pentyn-1-ol 8′.
Synthesis of branched FAHFAs in the 5-, 9- or 10-series
(compounds 1a–1e)
Fatty acids8 and hydroxyl fatty acids are ubiquitous in
nature.7,9
The choice of the FA and HFA in branched FAHFAs was
motivated by the high difference of concentration observed3 in
sera of adipose GLUT4 overexpressing mice compared to the
levels detected in wild type mice. Thus, we focused our atten-
tion towards HPA and HSA derivatives. However, the strategy
may be applied to many other FAs and HFAs.
The synthesis is depicted in Scheme 2.
Upon silylation of the commercially available hydroxyl-
alkenes 7a–c, MCPBA epoxidation provided the expected
epoxide 6a–c in excellent yields. Inversion of these two first
steps, i.e. starting from MCPBA epoxidation, then silylation of
alcohols 6d–e, was less convenient and less efficient.
Alkynylation10 of the resulting epoxides 6a–c with lithium
acetylide of terminal alkynes 8a–c proceeds smoothly in the
presence of boron trifluoride diethyl etherate.
Surprisingly, hydrogenation of the resulting alkynes 5a–c
over palladium on carbon (10 wt% loading) was disappointing.
Many side-products were formed along with the expected
desired saturated alcohols 4a–c. Isolated yields did not exceed
more than 32% or 54% in ethanol or ethyl acetate respectively.
Hydrogenation over the Rosenmund catalyst,11,12 without
adding any amine, allowed the conversion of alkynes 5a–c to
the expected fully saturated carbon chains 4a–c in excellent
yields. Although extremely slow (from 48 h to 72 h at 28 °C),
esterification with oleic acid (OA) or palmitic acid (PA) in the
presence of DMAP and a slight excess of 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (EDCI) afforded the expected
branched FAHFA precursors 9a–c and 9d–e. Esterification
under the Steglich conditions (dicyclohexylcarbodiimide,
DMAP)13 provided the fatty esters 9a–e in similar yields but at
shorter reaction times (15 h to 24 h). Subsequently,
deprotection of the tert-butyldimethylsilyl group in the
presence of fluoride anions followed by Jones oxidation
afforded the desired branched FAHFAs 1a–e.
The strategy was applied to the synthesis of five new
FAHFAs (1a–e) that, according to Yore et al. publication,3 are
present in sera of mice overexpressing GLUT4 in adipose
tissue.
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Scheme 2 Synthesis of compounds 1a–e, FAHFAs in the 5-, 9- and 10-series.
Synthesis of branched FAHFAs in the 7-series
(compounds 1f–1i)
As above-mentioned, the FAHFAs having the ester bond at
position 7 could not be obtained from 1-hydroxy-7-octene
(compound 7 with z = 3, Scheme 1) since it was not commer-
cially available. None of these family members are commer-
cially available to date. We thus focused our efforts on the
preparation of palmitate and oleate esters of HPA or HSA to
compare their biological activities with their corresponding
regioisomers (commercially available or described above in
Scheme 2).
The synthesis of these 7-FAHFAs is presented in Scheme 3.
It is closely related to the synthesis described in Scheme 2,
except that the starting terminal olefin 7′a–b is not function-
alized (non-oxygenated). Inversely, the non-oxygenated terminal
alkyne 8 in Scheme 2 is replaced in Scheme 3 with 4-pentyn-1-ol
8′. All the steps were carried out with excellent yields, using the
same reaction conditions as described in Scheme 2. Thus,
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starting from the same terminal alkyne 11, readily prepared
from 4-pentyn-1-ol 8′, four new targets 7-PAHPA, 7-PAHSA,
7-OAHPA and 7-OAHSA were successfully obtained in six steps
from the terminal olefins 7′a and 7′b.
Branched FAHFAs having an unsaturated hydroxy-fatty acid
chain (target 2)
Many unsaturated HFAs have been reported as endogenous
metabolites.14–18
For example, 10-hydroxy-(12Z)-octadecenoic acid 12 has
been detected18 in the colon, small intestine, and plasma of
mice. This HFA is a gut microbial metabolite of linoleic acid
that improves inflammation-associated impairment of the
intestinal epithelial barrier partly via a GPR40 pathway.19 The
unsaturated hydroxyl-fatty acids may be esterified as well as
their saturated analogs and they may play important biological
roles. Thus, we carried out the total synthesis of an oleate ester
of 10-hydroxy-(12Z)-octadecenoic acid 12. This unsaturated
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Scheme 3 Synthesis of compounds 1f–1i, FAHFAs of the 7-series.

branched 10-OAHFA 2 is an unsaturated analog of 10-OAHSA

1c having a (Z)-double bond at carbon atom 12.
As summarized in Scheme 4, the unsaturated branched
10-OAHFA 2 was prepared from the same alkyne precursor 5c
as its saturated analog 10-OAHSA 1c (Scheme 2).
Semi-hydrogenation of the alkyne function 5c using the
Brown catalyst12 provided a (12Z)-unsaturated C18 fatty diol
13 in excellent yield. The monitoring of the reaction by TLC is
difficult as both the starting alkyne 5c and the targeted olefin
13 are coeluted. The reaction was highly stereo- and chemo-
selective. No over-reduction was observed by 1D, 2D-NMR and
ESI-MS analyses.
Subsequently, Steglich esterification13 with OA, deprotect-
ion of silyl ether 14 followed by Jones oxidation of the resulting
primary alcohol 15 afforded the desired unsaturated target 2 in
excellent yields.
This convenient and efficient synthesis of (12Z)-unsaturated
branched OAHFA 2 opens the doors to the synthesis of many
other unsaturated branched FAHFAs.
To date, there is no piece of information in the literature Scheme 4 Synthesis of FAHFA 2, the oleate ester of 10-hydroxy-12(Z)-
about the chirality of the branched FAHFAs3,5,20 or their parent octadecenoic acid 12.

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Scheme 5 Synthesis of the “linear” FAHFA 3.
HFA precursors.15,18,19 When the configuration of the HFA is
reported, it can be R or S, depending on the lipids and their
biosynthesis.14,16 It is noteworthy that the synthetic route
suggested in the present paper offers the possibility to obtain
FAHFAs in a chiral pure form starting from chiral epoxides
(readily obtained by changing the epoxidation conditions).
OA-omegaHPA (3): a linear FAHFA
OA-omegaHPA 3 has been synthesised by Schuett et al.2 in
2013 from 16-hydroxy palmitic acid and oleoyl chloride. This
one-step synthesis is however hampered by long and tedious
purification steps since two column chromatographies (silica
then Sephadex gels) are needed. In addition, yields, rf, IR, MS
and NMR data were not given. Thus, we planned a novel retro-
synthesis from 1,16-hexadecadiol 16 and oleic acid. This two
step synthesis is depicted in Scheme 5.
Esterification of 1,16-hexadecadiol 16 by oleic acid using
EDCI in CH2Cl2 at 28 °C provided the expected mono-ester 17.
However, the conversion was slow and yields were poor
(ca. 20–30%), even with a prolonged reaction time (>60 h) and
large excess of EDCI and diol 16. The reaction was poorly selec-
tive, leading to diester (about 8–10%). Replacement of CH2Cl2
with pyridine dramatically improved the selectivity and
reactivity, affording the desired mono-ester 17 in 95% yield.
Contrary to the corresponding branched OAHPA precursors,
primary alcohol 17 was poorly soluble in most organic
solvents. Thus, its oxidation to carboxylic acid 3 was firstly
performed in a highly polar solvent (DMF) in the presence of
pyridinium dichromate (PDC). The desired carboxylic acid
target 3 was obtained in 43% yield only (contaminated by
several non-identified by-products) and the reaction stopped at
the aldehyde stage (isolated, ca. 36%), even after a prolonged
reaction time (>24 h) and warm temperatures (28 °C–30 °C).
Although the primary alcohol 17 was poorly soluble in
acetone, the Jones oxidation provided a much better alterna-
tive since the expected target OA-omegaHPA 3 was obtained in
excellent yield (83%) within a few hours.
Optimization of MS detection
To quantify FAHFAs in biological samples a rapid and robust
LC-MS method is needed. Due to their chemical structures,
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liquid chromatographic separation is the most adapted
system. It was coupled to a triple quadrupole to obtain the
best sensitivity. Four commercial FAHFAs and ten synthetic
compounds (1 and 3) were used to optimize the LC-MS ana-
lysis. The mass detection was optimized to achieve a suitable
selective and sensitive method. Thanks to the carboxylic acid
group, the FAHFA detection was performed by ESI-MS in the
negative ion mode as [M − H]− ions. First, the fragmentor
voltage (F) was optimized for each compound. This parameter
promotes the transmission of the ions between the ionization
source and the first quadrupole. Low voltage values lead to
poor transmission efficiency whereas very high voltage values
lead to excessive fragmentation. The optimum F values, corres-
ponding to the maximum of intensity for the transmission of
the [M − H]− ions without fragmentation, are summarized in
Table 1. As reported by Yore et al.,3 the main fragmentation of
all the tested FAHFAs occurred at the ester bond. The most
abundant transition (quantifier transition, Quanti) corres-
ponded to the fatty acid moiety, and it was used for the quan-
titation of the molecule. The other transition (qualifier
transition, Quali) corresponding to the dehydrated hydroxy
fatty acid moiety confirmed the assignment of the family
member. The collision energy (CE) was optimized for the two
main transitions of each compound (Table 1).
Optimization of the chromatographic separation
According to Yore et al.3 biological tissues or fluids may
contain diverse families of branched FAHFAs and, for each of
these FAHFAs, many regioisomers are expected since the ester
bond may be at diverse positions (5, 7, 9, 11, 12 or 13). Since
the multiple reaction monitoring (MRM) transitions of regio-
isomers are identical, it is necessary to develop LC conditions
that separate them, otherwise overlapping will hamper the
assignment and quantitation of the corresponding isomers. To
be conveniently applied to detection in large collections of
samples, the LC analysis time must be short.
During the time we were investigating LC conditions with
our synthetic FAHFAs, Zhang et al. reported20 a nice separation
using a Luna C18 column (10 cm, 3 mm, 100 Å), running a
basic eluent with 0.01% ammonium hydroxide. However, the
time of analysis was very long (100 min). The challenge was to
reduce the retention times, without altering the efficiency of
the separation. Improvement of the LC method was investi-
gated with three synthetic PAHPA regioisomers i.e. 9-PAHPA
(1e), 7-PAHPA (1h) and 5-PAHPA (1d). All the conditions are
summarized in Table 2.
Like Zhang et al.20 we firstly chose the most popular reverse
phase column i.e. a C18-bonded silica column too (10 cm,
similar quality). However elution was carried out under acidic
conditions (eluent 1 instead of eluent 2, Table 2). Elution was
performed isocratically. A shorter time of analysis was
obtained but the peaks were broad and overlapped (Fig. 1,
graph a).
Next, the C18-bonded silica stationary phase was replaced
by a C30-bonded silica phase already used for hydrophobic
lipids of such triacylglycerols,21 expecting that this longer
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Table 1 Multiple reaction monitoring (MRM) of FAHFAs used for this study. RT: retention time, Quanti: quantifier transition, Quali: qualifier tran-
sition. F: fragmentor voltage, CE: collision energy

Compounds RT (min) Precursor ion (m/z) Product ion (m/z) F (V) CE (V)

9-PAHPA (1e) 4.02 Quanti 509.5 255.3 150 20

Quali 509.5 253.2 18
7-PAHPA (1h) 4.24 Quanti 509.5 255.3 150 18
Quali 509.5 253.2 16
5-PAHPA (1d) 4.46 Quanti 509.5 255.3 150 18
Quali 509.5 255.3 12
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9-POHSA Quanti 535.5 299.3 110 20

4.13 Quali 535.5 291.3 20
9-OAHPA (1b) 4.14 Quanti 535.8 281.4 110 22
Quali 535.8 253.3 22
7-OAHPA (1f) 4.43 Quanti 535.8 281.4 110 27
Quali 535.8 253.3 20
5-OAHPA (1a) 4.63 Quanti 535.8 281.4 110 12
Quali 535.8 253.3 12
9-PAHSA 5.86 Quanti 537.5 255.0 150 11
Quali 537.5 281.3 18
7-PAHSA (1i) 6.25 Quanti 537.5 255.0 150 25
Quali 537.5 281.3 22
9-PAHSAd-31 5.51 Quanti 568.5 286.5 150 25
Quali 568.5 281.3 24
13-OAHSA 5.49 Quanti 563.5 281.3 110 18
Quali 563.5 299.4 18
10-OAHSA (1c) 5.92 Quanti 563.5 281.3 110 25
Quali 563.5 299.4 23
9-OAHSA 6.04 Quanti 563.5 281.3 110 24
Quali 563.5 299.4 24
7-OAHSA (1g) 6.47 Quanti 563.5 281.3 110 25
Quali 563.5 299.4 25
OAwHPA (3) 4.65 Quanti 535.8 281.0 110 24
Quali 535.8 271.0 20

carbon arm improved the hydrophobic interactions with
FAHFAs. Two lengths of column (5 cm, entry 2 and 15 cm,
entry 3, Table 2) were tested with acidic solvent 1. The
separation was nicely improved with the longer column (Fig. 1,
graph c compared to b and a), although the valley between two
of the three regioisomers was not completely resolved.
The best separation was obtained with the basic buffered
mobile phase recently reported by Zhang et al.20 The pH was
slightly modified (7.5 instead of 8.8) compared to Zhang
et al.20 because of the pH stability range of the C30 column
(maximum pH = 8). The three regioisomers 9-PAHPA, 7-PAHPA
and 5-PAHPA were eluted in less than 6 min with a full
separation (Fig. 1, graph d).
It is noteworthy that a much better efficiency of ionisation
(Fig. 2) was obtained under the basic conditions (Table 2,
entry 4) compared to conditions with the acidic mobile phase
(Table 2, entry 3).
Subsequently we applied these conditions (Table 2, entry 4) to
the mixture of fourteen FAHFAs and obtained a satisfactory profile
in less than 10 minutes (Fig. 3) with separation of all species.
The chromatogram of the fourteen FAHFAs is complex to
analyze as all the FAHFAs were eluted within a few minutes
interval, with overlapping (Fig. 3, graph a). However, extracted
chromatograms for each FAHFA family allowed the detection
and identification of each FAHFA of the mixture (Fig. 3, graph
b to f ).
Sensitivity of the method
Based on the internal standard (ISTD) method, each
calibration curve was established with ten concentrations for

Table 2 Liquid chromatographic conditions

Mobile phases (isocratic mode)

Entry Column A B (%B)

1 C18, 10 cm (1.72 µm) Acquity UPLC BEH, Waters 1 H2O/MeOH: 60/40, (v/v), 0.1% HCOOH, iPrOH/MeOH: 90/10 (v/v), 0.1% 60
10 mM HCOONH4 HCOOH, 10 mM HCOONH4
2 C30, 5 cm (2.6 µm) Accucore, Thermo Fisher 60
3 C30, 15 cm (2.6 µm) Accucore, Thermo Fisher 60
4 C30, 15 cm (2.6 µm) Accucore, Thermo Fisher 2 H2O, 0.01% NH4OH, MeOH, 0.01% NH4OH, 93
5 mM AcONH4 5 mM AcONH4

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Fig. 1 Chromatographic separation of PAHPA isomers: 9-PAHPA (1e),

7-PAHPA (1h) and 5-PAHPA (1d) using different LC conditions. The
mobile phases are described in Table 2.

fourteen FAHFAs, ranging from 500 to 0.48 ng mL−1 in the

presence of 12.5 ng of ISTD (9-PAHSA-d31). The curves were
fitted using a linear regression model with different weight
factors. The linearity of the method was assessed for each
FAHFA by evaluating the correlation coefficient (ESI,
Table S2†). The limit of quantitation (LOQ) and the limit of
detection (LOD) were evaluated with LOQ corresponding to the
lowest concentration leading to a signal to noise ratio over 10,
and LOD corresponding to the lowest concentration leading to
a signal to noise ratio over 3. The LOQ values ranged from 2 ng
mL−1 to 7 ng mL−1 and the LOD values ranged from
Fig. 3 LC analysis of a mixture of fourteen FAHFAs using the LC con-
ditions described in Table 2, entry 4. Graph a shows the chromatogram
of the total ionic current (TIC) of the fourteen FAHFAs. Graphs b to f
show the extracted chromatograms from TIC of the selected reaction
monitoring for each FAHFA family.

1.3 ng mL−1 to 3.8 ng mL−1 (ESI, Table S2†). These values are
classical for triple quadrupole technology.

Optimization of FAHFA extraction

Fig. 2 Effect of the pH of the mobile phase on the efficiency of the ion-
isation in LC-MS/MS analyses. The numbers on the peaks correspond to
the location of the ester bond in each regioisomer.
Prior to quantitation of FAHFAs in biological samples, it was
necessary to develop a rapid and efficient protocol for their
selective extraction.
Thus, FAHFA extraction was optimized by addition of
fourteen synthetic FAHFAs into human plasma samples and
measuring the quantity of collected FAHFAs. The first step of
the sample preparation is a classical Bligh & Dyer liquid–liquid
extraction22 under acidic conditions. Acidification with acetic

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acid was compared with acidification with citric acid (reported
by Zhang et al.20). Yields of extraction were slightly better with
citric acid (data not shown). These latter conditions were thus
adopted for the final protocol. Subsequently, a purification
process is needed to reduce the matrix effect and to increase
the sensitivity of the quantitation by LC-MS/MS analysis. SPE
cartridges of pure silica were used to separate FAHFAs from
neutral lipids. In the published method20 the conditioning of
the SPE was performed with hexane (15 mL), then the neutral
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lipids were eluted with a large amount of hexane/AcOEt (95/5;
v/v; 16 mL) and the FAHFAs were eluted with AcOEt (16 mL).
This method seems efficient. However, due to the large volume
of solvent used for each step the method is time consuming.
We optimized the SPE step by changing the nature of the
solvent and minimizing the volume of solvents. Thus,
conditioning was done with CH2Cl2 (4 mL) and neutral lipids
were eliminated by elution with CH2Cl2 (2 mL). Subsequently,
FAHFAs were eluted with MeOH/CH2Cl2 (10/90; v/v). With this
method of preparation (liquid liquid extraction followed by
SPE pre-concentration) yields ranged from 60 to 89% (ESI,
Table S1†).
Conclusions
A seven step strategy was developed to synthesize branched
FAHFAs that may constitute biomarkers of insulin resistance
and type 2-diabetes risk.
The location of the secondary hydroxyl group is readily
tunable by changing the length of the alkyl chain of the two
starting materials i.e. the terminal alkyne 8 and the terminal
olefin 7. Application to the preparation of regioisomers of the
5-, 9- and 10-series showed the versatility of the strategy
(Scheme 2). When alkenes 8 and alkynes 7 were not commer-
cially available with a suitable length, the synthesis was started
from a non-hydroxylated alkene 7′ and an omega-hydroxylated
alkyne 8′. This alternative route was exemplified with the total
synthesis of the regioisomers of the 7-series (Scheme 3).
The great flexibility of this strategy allows us to reach an
analog (2) containing a (Z)-double bond in the HFA chain by
simply changing the conditions of the hydrogenation step.
The Brown catalyst yielded a (Z)-selective semi-reduction of the
propargylic alcohol 5c into (Z)-allylic alcohol 13 whereas hydro-
genation over Pd/BaSO4 of the alkyne precursor 5c provided a
saturated fatty chain 4c. Taken together, ten novel branched
FAHFAs have been synthesized i.e. 5-, 7- and 9-PAHPAs; 5-, 7-,
and 9-OAHPAs; 7- and 10-OAHSAs and its 12(Z)-unsaturated
OAHC18 analog 2.
In the present paper, we exemplified our strategy using the
most abundant FAs according to Yore’s results3 i.e. OA and PA.
However, many other FAHFAs may be obtained by simply
replacing the FA in the esterification step.
In addition, a new synthesis has also been developed for
the preparation of OA-omega-HPA 3, a “linear” endogenous
FAHFA (79% yield over 2 steps, without any mono-protection
of 1,16-diol 16).
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Paper
The procedure for FAHFA extraction from healthy human
plasma using liquid–liquid extraction followed by SPE
concentration, was optimized (60–89% yield, detection at the
nanomolar range; LOQ = 2–7 ng mL−1; LOD = 1.3–3.8 ng mL−1).
Subsequently, although tricky due to the highly non-polar
and closely related chemical structures of these FAHFAs,
we finally obtained suitable LC conditions with short retention
times, allowing a good separation of various FAHFAs.
Application of this method is currently in progress to quantify
these promising biomarkers in a large series of plasma
samples.
Experimental section
Experimental details are provided in the ESI.†
Acknowledgements
This work was supported by the Centre National de la
Recherche scientifique (CNRS), University of Montpellier,
Agence Nationale de la Recherche (OBELIP ANR-12-BSV1-
0025), AstraZeneca and Région Midi-Pyrénées. Lipidomic ana-
lyses were performed on the Toulouse INSERM Metatoul-
Lipidomique Core Facility-MetaboHub ANR-11-INBS-010. DL is
a member of Institut Universitaire de France.
Notes and references
1 I. A. Butovich, Prog. Retinal Eye Res., 2009, 28, 483–498.
2 B. S. Schuett and T. J. Millar, Exp. Eye Res., 2013, 115,
57–64.
3 M. M. Yore, I. Syed, P. M. Moraes-Vieira, T. Zhang,
M. A. Herman, E. A. Homan, R. T. Patel, J. Lee, S. Chen,
O. D. Peroni, A. S. Dhaneshwar, A. Hammarstedt, U. Smith,
T. E. McGraw, A. Saghatelian and B. B. Kahn, Cell, 2014,
159, 318–332.
4 M. Masoodi, O. Kuda, M. Rossmeisl, P. Flachs and
J. Kopecky, Biochim. Biophys. Acta, 2015, 1851, 503–518.
5 W. H. Parsons, M. J. Kolar, S. S. Kamat, A. B. Iii, J. J. Hulce,
E. Saez, B. B. Kahn, A. Saghatelian and B. F. Cravatt, Nat.
Chem. Biol., 2016, 1–8.
6 Y. Ma, T. Kind, A. Vaniya, I. Gennity, J. F. Fahrmann and
O. Fiehn, J. Cheminf., 2015, 7, 53.
7 C. T. Hou, New Biotechnol., 2009, 26, 2–10.
8 J. Beld, D. J. Lee and M. D. Burkart, Mol. BioSyst., 2015, 11,
38–59.
9 D. L. Chance, K. O. Gerhardt and T. P. Mawhinney,
J. Chromatogr., A, 1998, 793, 91–98.
10 M. Yamaguchi and I. Hirao, Tetrahedron Lett., 1983, 24,
391–394.
11 K. W. Rosenmund, Eur. J. Inorg. Chem., 1918, 51, 585–593,
DOI: 510.1002/cber.19180510170.
12 C. Oger, L. Balas, T. Durand and J. M. Galano, Chem. Rev.,
2013, 113, 1313–1350.
Org. Biomol. Chem., 2016, 14, 9012–9020 | 9019

Paper
13 B. Neises and W. Steglich, Angew. Chem., Int. Ed. Engl.,
1978, 17, 522–524.
14 S. Tunaru, T. F. Althoff, R. M. Nusing, M. Diener and
S. Offermanns, Proc. Natl. Acad. Sci. U. S. A., 2012, 109,
9179–9184.
15 H. Yokoi, H. Mizukami, A. Nagatsu, H. Tanabe and
M. Inoue, Biol. Pharm. Bull., 2010, 33, 854–861.
16 K. R. Kim and D. K. Oh, Biotechnol. Adv., 2013, 31, 1473–1485.
17 R. L. Schild, W. T. Schaiff, M. G. Carlson, E. J. Cronbach,
Published on 30 August 2016. Downloaded by Northern Illinois University on 26/09/2016 16:56:40.
D. M. Nelson and Y. Sadovsky, J. Clin. Endocrinol. Metab.,
2002, 87, 1105–1110.
18 S. Kishino, M. Takeuchi, S. B. Park, A. Hirata, N. Kitamura,
J. Kunisawa, H. Kiyono, R. Iwamoto, Y. Isobe, M. Arita,
H. Arai, K. Ueda, J. Shima, S. Takahashi, K. Yokozeki,
9020 | Org. Biomol. Chem., 2016, 14, 9012–9020
View Article Online
Organic & Biomolecular Chemistry
S. Shimizu and J. Ogawa, Proc. Natl. Acad. Sci. U. S. A.,
2013, 110, 17808–17813.
19 J. Miyamoto, T. Mizukure, S. B. Park, S. Kishino, I. Kimura,
K. Hirano, P. Bergamo, M. Rossi, T. Suzuki, M. Arita,
J. Ogawa and S. Tanabe, J. Biol. Chem., 2015, 290, 2902–
2918.
20 T. Zhang, S. Chen, I. Syed, M. Stahlman, M. J. Kolar,
E. A. Homan, Q. Chu, U. Smith, J. Boren, B. B. Kahn and
A. Saghatelian, Nat. Protoc., 2016, 11, 747–763.
21 J. W. Lee, T. Nagai, N. Gotoh, E. Fukusaki and T. Bamba,
J. Chromatogr., B: Anal. Technol. Biomed. Life Sci., 2014, 966,
193–199.
22 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol., 1959,
37, 911–917.
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