The Properties and Role of O-Acyl-ω-hydroxy Fatty Acids and Type I-St and Type II Diesters in the Tear Film Lipid Layer Revealed by a Combined Chemistry and Biophysics Approach

Article abstract of DOI:10.1021/acs.joc.0c02882

The tear film lipid layer (TFLL) that covers the ocular surface contains several unique lipid classes, including O-acyl-ω-hydroxy fatty acids, type I-St diesters, and type II diesters. While the TFLL represents a unique biological barrier that plays a central role in stabilizing the entire tear film, little is known about the properties and roles of individual lipid species. This is because their isolation from tear samples in sufficient quantities is a tedious task. To provide access to these species in their pure form, and to shed light on their properties, we here report a general strategy for the synthesis and structural characterization of these lipid classes. In addition, we study the organization and behavior of the lipids at the air-tear interface. Through these studies, new insights on the relationship between structural features, such as number of double bonds and the chain length, and film properties, such as spreading and evaporation resistance, were uncovered.

Full text of DOI:10.1021/acs.joc.0c02882

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Article
The Properties and Role of O‑Acyl-ω-hydroxy Fatty Acids and Type
I‑St and Type II Diesters in the Tear Film Lipid Layer Revealed by a
Combined Chemistry and Biophysics Approach
Tuomo Viitaja,⊥ Jan-Erik Raitanen,⊥ Jukka Moilanen, Riku O. Paananen,* and Filip S. Ekholm*
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ABSTRACT: The tear film lipid layer (TFLL) that covers the ocular
surface contains several unique lipid classes, including O-acyl-ω-hydroxy
fatty acids, type I-St diesters, and type II diesters. While the TFLL
represents a unique biological barrier that plays a central role in
stabilizing the entire tear film, little is known about the properties and
roles of individual lipid species. This is because their isolation from tear
samples in sufficient quantities is a tedious task. To provide access to
these species in their pure form, and to shed light on their properties, we
here report a general strategy for the synthesis and structural
characterization of these lipid classes. In addition, we study the
organization and behavior of the lipids at the air−tear interface.
Through these studies, new insights on the relationship between
structural features, such as number of double bonds and the chain length, and film properties, such as spreading and evaporation
resistance, were uncovered.
Meibomian gland secretions, recent results have suggested that
they may be central to TFLL function.11−14 OAHFAs have
been proposed to be potential indicators of DED progression,
and a consistent decrease in the number of OAHFA species
has been correlated to DED severity in patient-derived
meibum samples.15 While a positive correlation between the
Schirmer I test and the tear film breakup time for the total
OAHFA levels has been noted,14 uncertainties still remain
since statistically significant decreases in total tear OAHFA
levels has not been confirmed in DED patients.15 Nevertheless,
reduced levels of type I-St and type II diesters have been
reported in patient-derived tear samples compared to those in
healthy controls.16 Moreover, animal studies have shown that
the knockout of the Cyp4f39-gene involved in the synthesis of
OAHFAs and their related diesters in the Meibomian glands
leads to the development of a severe dry eye phenotype in
mice.17 Therefore, there are indications that OAHFAs and
their diester derivatives may be central to proper TFLL
function. However, the physical properties and molecular
behavior of these lipid classes have not previously been studied
1. INTRODUCTION
The tear film lipid layer (TFLL) resides on top of the tear film,
where it covers the ocular surface and protects and lubricates
the surface of the eye (Figure 1). It is a specialized lipid
membrane secreted by the Meibomian glands, forming a
barrier that, according to the traditional view, prevents the
evaporation of aqueous tear fluid. However, the evaporation-
resistant function is still poorly understood.1 In recent years,
there has been a growing interest in understanding the
structure and function of the TFLL. This is due to an increased
awareness of the relationship between its intrinsic properties
and the maintenance of ocular surface health. Most notably, a
compromised TFLL function is closely related to ocular
surface diseases such as dry eye disease (DED), which affects
more than 500 million people on a global scale2 and
constitutes a significant public health concern and societal
economic burden.3,4 A common characteristic of DED is the
drying of the ocular surface due to the excess evaporation of
tear fluid,5 a condition that is presumably linked to structural
changes in the TFLL composition.6−9 Addressing the proper-
ties of individual TFLL lipids is thus important to understand
the molecular-level basis of DED. The TFLL contains several
unique classes of lipids that are characterized by ultralong
unsaturated hydrocarbon chains, including O-acyl-ω-hydroxy
fatty acids (OAHFAs) and type I-St (cholesteryl) and type II
(wax) diesters (Figure 1).10
Received: December 4, 2020
Published: March 17, 2021
Although each of these special lipid classes constitutes a
relatively small fraction (3−4 mol % each) of the total lipids in
© 2021 The Authors. Published by
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Figure 1. A schematic presentation of the ocular surface and the structure of the tear film with a further emphasis on the organization of the TFLL
and TFLL lipid classes, which are the focus of this study.
Figure 2. Retrosynthetic analysis on which the designed synthetic route to produce 20:1-OAHFA, 20:1-DiE, and 20:1-St-DiE was based. The three
starting fragments (A, B, and C), the core intermediate, and the products are shown with the key organic transformations highlighted.
in detail and thus open questions regarding their role in TFLL
function and organization remain.
majority of the unique TFLL lipids and their structural
analogues. In addition to the synthesis, the most detailed NMR
spectroscopic characterization data reported to date are
supplied. These methods were used to synthesize a model
OAHFA, a type I-St diester, and a type II diester. With the
pure TFLL model lipids in hand, we studied their biophysical
properties and evaporation resistance with Langmuir mono-
layer techniques. Our results indicate that the disordering
effect of the additional double bond in OAHFAs likely
balances the ultralong chain lengths in naturally occurring
OAHFAs to promote spreading on the tear film surface. In
addition, type I-St diesters were found to exhibit a liquid-
crystalline structure at the aqueous interface, suggesting their
involvement in the multilamellar organization of the TFLL.
Biophysical studies on OAHFAs and diesters have been
hindered by their limited availability. The isolation of these
lipid species, devoid of biological contaminations, from the
natural source has proved to be challenging due to variations in
the lipid composition between species and individuals, the
minute quantities of tear samples that are extractable from
humans, and the large number of lipid species present in the
TFLL. As a result, simplified structural analogues have been
used in previous biophysical studies.18,19 Our recent study
addressing the the biophysical properties of OAHFA and type
II diester analogues revealed that OAHFAs are capable of
forming evaporation-resistant monolayers at the air−tear
interface.19 While these results were promising, the structural
analogues studied deviated from the naturally occurring TFLL
lipids, which contain an additional cis-double bond in one of
the hydrocarbon chains.20
We therefore decided to build on our previous work and
investigate the effects of an additional double bond on the
biophysical properties of the lipids. This required a complete
revision of the synthetic strategy and the development of
synthetic routes for the synthesis of an OAHFA and its related
type I-St and type II diesters. To the best of our knowledge,
there are only a handful of reports18−22 on the synthesis of type
II diesters and OAHFAs in the literature and no reports on the
synthesis of type I-St diesters. Furthermore, the majority of the
previous studies that address the synthesis and characterization
of OAHFAs suffer from ambiguities concerning the synthetic
protocols applied and, even more surprisingly, the supplied
characterization data is often lacking. In this work, we have
therefore invested a great deal of effort into both these aspects
and provide a block approach applicable to the synthesis of the
2. RESULTS AND DISCUSSION
2.1. Total Synthesis of Tear Film Lipid Probes. The
successful construction of complex organic molecules, such as
the three TFLL lipid model compounds targeted in this study,
requires a sound and tailored synthetic strategy. We therefore
performed a detailed retrosynthetic analysis, which revealed
that the OAHFA and its type I-St and type II diesters can all be
constructed from one common core intermediate (see Figure
2). The core intermediate can be prepared from three separate
fragments, which are denoted A, B, and C.
It is important to note that this general strategy is applicable
to the preparation of a large number of naturally occurring
TFLL-lipids and their unnatural analogues by varying the chain
lengths and structural features of fragments A, B, and C. In this
work, we focused solely on the preparation of a short 20:1-
OAHFA and its type I-St cholesteryl and type II oleoyl
diesters. A shorter version of the naturally occurring OAHFAs
was targeted to examine the effect of the double bond, since we
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a
Scheme 1. Overview of the Reaction Routes and Isolated Yields
a
(i) (1) 48% aq HBr, cyclohexane, reflux, 18 h, 77%; (2) imidazole, TBDMSCl, CH2Cl2, rt, o/n, 94%. (ii) (1) PPh3, neat, 120 °C, 17 h, quant.; (2)
NaHMDS, dry THF, HMPA, −78 °C, 1 h, then 8-bromo-1-octanal, −78 °C, rt, 24 h, 34%. (iii) (1) KOAc, DMSO, 50 °C, 27 h, 66%; (2) NaOMe,
THF/MeOH (1:2), 22 h, rt, 78%. (iv) (1) Oleic acid, DMAP, EDC·HCl, CH2Cl2, rt, o/n, 93%; (2) TBAF, THF, 1 h, rt, 92%. (v) Jones reagent,
acetone/EtOAc (1:1), 0 °C, 45 min, 89%. (vi) Cholesterol, DMAP, EDC·HCl, CH2Cl2, rt, o/n, 67%. (vii) Oleic acid, DMAP, EDC·HCl, CH2Cl2,
rt, o/n, 70%.
have recently studied the properties of the corresponding 20:0-
OAHFA and found them to be interesting.19 Considering that
the naturally occurring lipids almost exclusively have a (Z)-
configuration around the double bond,20 we envisioned that
the highly (Z)-selective Wittig olefination reaction would be a
suitable protocol to generate the required stereochemistry in a
coupling reaction between fragments A and B. For the
construction of the core intermediate, we further chose to
use oleic acid as the representative fragment C since it is the
most abundant acyl chain found in these classes of TFLL-
lipids.11,12 From the core intermediate onward, we envisioned
that mild oxidation and esterification reactions would be
required to avoid unwanted transesterification side reactions
and the oxidation of the double bonds. With this retrosynthetic
analysis as a base, we set out to synthesize the OAHFA
analogue and the related diesters.
was used. These minor adjustments to the reaction protocol,
which resembles the protocol employed by Cateni et.al.,29 led
to the exclusive formation of 1 in an excellent 94% yield. To
access fragment B, 1 was reacted with 1 equiv of PPh3 at 120
°C under neat reaction conditions. While similar reactions are
commonly performed in refluxing acetonitrile30 or toluene, an
additional solvent was not required.29 To confirm that
fragment B had formed, i.e., the phosphonium bromide salt,
31P NMR spectra were recorded, and the disappearance of the
PPh3 signal at −5.4 ppm and the appearance of the signal at
24.4 ppm were monitored. When the conversion was complete,
the crude product was used as such in the Wittig olefination
reaction. The second fragment required for the Wittig reaction,
i.e., fragment A, was prepared by the selective oxidation of
commercial 8-bromo-1-octanol with PCC to afford 8-bromo-
octanal in an 84% yield. The successful formation of the
1
Our synthetic route began with the preparation of fragment
B, and the routes to all three target lipids are displayed in
Scheme 1. In the first step, commercially available 1,12-
dodecanediol was successfully monobrominated according to a
protocol reported earlier by Greaves et al.23 The monobromi-
nated 12-bromo-1-dodecanol could be isolated in a 77% yield
in a reaction using a large excess of HBr in cyclohexane. The
yield is well within the 65−92% range commonly reported in
the literature.24−26 To access compound 1, the temporary
protection of the hydroxyl group as a TBDMS-ether was
required. The TBDMS-ether was chosen because of its stability
toward basic conditions and the mild and selective installation
and deprotection protocols available in the literature.27
Surprisingly, the installation of the TBDMS-ether featuring a
small excess of TBDMSCl (1.3 equiv) led to a 3:4 mixture of
the desired product and an unknown side product. By the
careful characterization of the compound mixture by NMR
spectroscopy, the second product was determined to be 12-
chloro-1-tert-butyldimethylsilyloxydodecane based on the
chemical shift of C-12 (45 vs 34.1 ppm in 1). To the best of
our knowledge, this type of halide−halide displacement
reaction has not previously been reported to take place
under the employed reaction conditions, although the reaction
protocols applied by other groups suggest that similar
problems likely have been previously encountered.28 To
circumvent this side reaction, the reaction solvent was changed
from dry DMF to dry CH2Cl2 and the amount of TBDMSCl
was reduced from 1.3 to 0.8 equiv, while 2 equiv of imidazole
aldehyde was confirmed by H NMR spectroscopy, and all
signals were assigned. After workup and drying, fragments A
and B were immediately coupled in a (Z)-selective Wittig
reaction according to a modified procedure to give 2 in a 34%
yield.31 In more detail, the required phosphonium ylide was
formed in situ with NaHMDS at −78 °C before the coupling
with fragment A was performed. The low yield observed is due
to the use of crude starting materials and the sluggish reaction
in which a large number of side products were formed. As a
result, the chromatographic separation of 2 was challenging
and required multiple tedious column purifications before a
pure product could be isolated. The direct conversion of 2 to 3
with H2O in DMF at elevated temperatures was attempted but
proved unsuccessful, and a two-step protocol was devised
based on the earlier work of Lee et al.32 The bromide was first
displaced with an acetyl group in a 66% isolated yield using a
large excess of KOAc in DMSO. The purified compound was
then deacetylated under Zemplen33 conditions to afford 3 in a
́
77% yield. We initially attempted to perform this two-step
process according to the one-pot protocol reported by Lee
et.al.;32 however, satisfactory results with our substrate could
not be obtained. To access the core intermediate 4,
esterification with oleic acid was required. We chose to avoid
the use of Fischer esterification with sulfuric acid, which may
isomerize the double bonds,34 and sought inspiration from the
previously published work of Balas et al.22
We settled on the use of a Steglich-type esterification
protocol with the minor modification of replacing DCC with
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Figure 3. (Left) The numbering of the atoms in 20:1-OAHFA is displayed in the molecular structure along with the colors used for the
visualization of the different signals in the spectra (top) and the 5.3−1.5 ppm region of the 1H NMR spectrum that highlights the accuracy of the
spectral simulations with the PERCH software (the measured spectrum is in blue and the simulated spectrum is in red) (bottom). (Right)
Overlapping Ed-HSQC (CH and CH3 are in blue and CH2 in green) and HMBC (red) spectra, with the most important HMBC correlation
highlighted with green circles.
EDC·HCl. Under these conditions, the esterification pro-
ceeded smoothly in a 93% yield. The temporary silyl protective
group was deprotected with 3 equiv of TBAF in an excellent
yield. From this core intermediate, the 20:1-OAHFA (5) could
be prepared in an 89% yield by a mild oxidation with the Jones
reagent according to our previously established reaction
protocol.19 The Jones oxidation is an especially useful protocol
for these lipids because alkene functionalities are unaffected by
the reaction conditions.35 The use of EtOAc as a cosolvent is
rare in the literature, but we have found that it has a beneficial
effect on the reaction outcome and is somewhat superior to the
other cosolvents typically applied (water or THF). With the
20:1-OAHFA prepared, we completed the synthesis of the
related cholesteryl type I-St diester 6 (20:1-St-DiE) by a
Steglich-type esterification with cholesterol in a 67% yield
following the previously described protocol. The oleoyl type II
diester 7 (20:1-DiE) was prepared from 4 and oleic acid in a
similar fashion in a 70% yield.
In summary, the three TFLL model lipids 5−7 were
obtained in 10 or 11 synthetic steps in overall yields ranging
between 5−7.5%. All synthesized products, with the exception
of fragments A and B, were purified by conventional
techniques and characterized in detail by NMR spectroscopy
(1D and 2D techniques), which were further coupled with
quantum-mechanical spectral simulations with the PERCH
(PEak reseaRCH) software and HRMS. Owing to the lack of
characterization data reported for these compounds in the
literature, a further segment on the NMR spectroscopic
characterization is provided below.
2.2. Complete NMR Spectroscopic Characterization
of the Tear Film Lipid Probes. In the literature, many of the
previous synthetic routes to OAHFAs are accompanied by a
lack of structural characterization data to varying degrees. In
some cases, only TLC Rf values, FT-IR, and mass data are
provided.18,21 In other publications, NMR analysis was
performed but, for example, the 13C NMR data were not
assigned, and the determination of the stereochemistry in key
intermediates and end products leaves room for improve-
ment.20 In this work, great effort was invested in the structural
characterization of the end products by NMR spectroscopic
techniques, and a detailed review of the characterization flow is
warranted. While all molecules were characterized to the
greatest extent possible, the 20:1-OAHFA (5) will be used as
the sole example here. The numbering of the molecule and an
excerpt of the key methods used in the NMR spectroscopic
characterization are summarized in Figure 3.
The NMR spectroscopic characterization began with the
identification of the carbonyl carbons at 179.6 (C-1) and 174.2
ppm (C-1′). The observed HMBC correlations between C-1′−
H-20, C-1′−H-2′, and C-1−H-2 highlighted in Figure 3 were
used to ascertain that these two signals were appropriately
assigned and the acyl chain was located at the correct position.
With H-2 (t at 2.34 ppm), H-20 (t at 4.05 ppm), and H-2′ (t
at 2.29 ppm) identified and their corresponding carbons
assigned through the use of Ed-HSQC, H-3 and C-3 (t at 1.63
ppm and s at 24.9 ppm, respectively), C-18 (s at 26.1 ppm), H-
19 and C-19 (tt at 1.61 ppm and s at 28.8 ppm, respectively)
and H-3′ and C-3′ (t at 1.61 ppm and s at 25.2 ppm,
respectively) could be assigned by a combination of COSY,
Ed-HSQC, and HMBC.
The second starting point in the characterization was the
characteristic chemical shifts of the alkene functionalities
(5.35−5.34 ppm in 1H and 130.1−129.9 ppm in 13C) and the
1
signals in close proximity to them (2.06−1.96 ppm in H and
27.4−27.3 ppm in 13C). These signals could not be assigned by
conventional techniques due to their severe overlap, and
moreover the coupling patterns were found to be complex and
higher-order in their nature. Yet, coupling constants for H-12−
H-13 and H-9′−H-10′ were required to ensure a (Z)-
configuration in the double bonds. Therefore, we used the
quantum-mechanical spectral simulation software PERCH,36
which can be used to extract chemical shifts and coupling
constants in the presence of overlapping signals and higher
order effects.37−40 With the aid of spectral simulations, we
were able to extract the chemical shifts of the alkenes and,
more importantly, all the coupling constants in the complex dtt
patterns could be defined. The coupling constants for the
vicinal protons in the alkenes were determined to be J9′,10′
=
11.1 Hz and J12,13 = 11.6 Hz, which are in agreement with the
literature values given for the (Z)-configuration,41 thus
confirming the correct stereochemistry of the alkene moieties.
The third starting point used in the spectral characterization
was at the H-18′ and C-18′ signals, which appear at
characteristic chemical shifts (t at 0.88 ppm and s at 14.3
ppm, respectively). By applying the conventional techniques
listed above, the C-16′ and H-17′ and C-17′ signals could be
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Figure 4. Organization of 20:1-OAHFA at the aqueous interface. (A) A representative surface-pressure compression−expansion cycle and the
mean surface potential ( SD, n = 3) of the 20:1-OAHFA monolayers at 35 °C. (B) The corresponding BAM images. In the BAM images, the pure
PBS surface appears black, and the local image intensity increases according to the thickness of the lipid film on the surface (also shown in Figure 5
and 6). The scale bar depicts 500 μm. (C) Schematic representation of the molecular organization of the 20:1-OAHFA films during compression,
with red and blue circles representing the carboxylic and ester groups, respectively, and lines representing the hydrocarbon chains.
assigned. All in all, we were able to solve all the well-resolved
evaporation resistance. Briefly, the lipids were spread on the
surface of a phosphate-buffered saline (PBS) subphase
maintained at 35 °C to mimic the conditions of the ocular
surface. The lipid film was compressed and expanded using
Delrin barriers to simulate the compression occurring during
blinking, and the properties of the lipid film were measured at
different compression states. Reversible compression−expan-
sion cycles were observed for all the synthesized lipids (Figure
S26).
2.3.1. 20:1-OAHFA. 20:1-OAHFA (5) spread readily and
formed a monolayer on the PBS subphase. Upon compression,
the surface pressure started to increase at a mean molecular
area of around 130 Å2/molecule and the film appeared
homogeneous in BAM images (Figure 4A and Bi), thereby
implying a disordered monolayer organization. The isotherm
lift-off area was similar to our previously reported results with
the analogous 20:0-OAHFA.19 The results suggest that 20:1-
OAHFA adopts a disordered conformation at low surface
pressures in which it lies flat on the aqueous interface with the
carboxylic acid and ester groups interacting with the aqueous
sublayer (Figure 4Ci).
When compressed to smaller areas per molecule, the
molecules gradually adopted an upright orientation (Figure
4Cii) as previously described.18,19 This change in orientation
can be observed as a decrease in the surface potential (Figure
4A). Despite this change in orientation, the 20:1-OAHFA
monolayer remained in a disordered phase in contrast to
saturated OAHFAs of a corresponding length, which under-
went a transition to a condensed monolayer phase at a surface
pressure of 2 mN/m.19 This finding is supported by the
homogeneous appearance of the films in the BAM images,
although the reflected intensity increased with the increasing
surface pressure, indicating a gradual increase in the thickness
of the film (Figure 4Bii and Biii). When the film was
compressed to 40 Å2/molecule, a second-order phase
transition was observed in the 20:1-OAHFA surface-pressure
isotherm that manifested as a steep increase in the slope
(Figure 4A). This phase transition potentially reflects a change
in the conformation of the 20:1-OAHFA from a tilted
alignment to a horizontal alignment (Figure 4Ciii), which is
in line with previous reports on fatty acid monolayers.47
However, the 20:1-OAHFA monolayer remained disordered
even in this phase since the formation of solid domains was not
1
signals in the H and 13C NMR spectra and a number of the
overlapping signals with complex coupling patterns, as
displayed in Figure 3 and further reported in the Experimental
Section in more detail. The regions where multiple signals
overlapped, i.e., 1.37−1.23 and 29.9−29.3 ppm, could not be
solved in detail, but all signals that occurred in these regions
could nevertheless be identified. The accurate reference
chemical shifts for the OAHFA and its diesters reported
herein provide a valuable foundation for future NMR
spectroscopic characterization studies of lipids isolated from
Meibomian gland secretion or the synthesized analogues of
these unique TFLL lipids. In addition, the chemical shifts
reported provide a base for the development of NMR-based
lipidomic profiling tools.42−45 With the synthesis and accurate
characterization studies completed, we turned our attention to
the investigation of the biophysical properties of these lipids to
shed light on their role in the TFLL.
2.3. Biophysical Properties of OAHFAs and Related
Diesters. The TFLL can be divided into an amphiphilic
sublayer, which resides at the interface between the aqueous
tear film and the TFLL, and an overlying nonpolar sublayer
(see Figure 1). Due to their hydrophobic nature, diesters are
expected to exist in the nonpolar sublayer, while amphiphilic
OAHFAs are expected to reside in the amphiphilic sublayer.
However, as outlined in the introduction, the detailed
biophysical properties of tear film OAHFAs and diesters
have been poorly characterized, and several open questions
regarding their role in TFLL organization and function remain.
Understanding the biophysical properties and molecular
behavior of the species represent an important and hitherto
missing link in refining our view of the factors that influence
the structure and function of the TFLL.
The synthesized OAHFA and diesters were designed to
function as excellent molecular probes of TFLL lipids. We
were therefore expecting to uncover new insights on the role
and behavior of these lipid classes in the TFLL while
simultaneously being able to correlate the results to previous
reports with simplified structural analogues.18,19 To this end,
we studied their organization at the aqueous interface using a
Langmuir trough system equipped with surface-potential and
surface-pressure sensors, a Brewster angle microscope (BAM),
and a custom Langmuir−Schaefer-type46 setup to measure the
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Figure 5. Organization of 20:1-DiE at the aqueous interface. (A) A representative surface-pressure compression−expansion cycle and the mean
surface potential ( SD, n = 3) of the 20:1-DiE monolayer at 35 °C. (B) Corresponding BAM images. The scale bar depicts 500 μm. (C) Schematic
representation of the molecular organization of the 20:1-DiE films during compression.
observed in the BAM images (Figure 4Biii). In addition, the
film collapse occurred at 30 Å2/molecule, which is significantly
larger than the mean molecular area of most condensed
monolayers (20 Å2/molecule).19,47
isotherms of 20:1-DiE closely resembled previously studied
type II diesters featuring a saturated interconnecting diol
moiety.19 Therefore, the likely configuration of 20:1-DiE
molecules in the monolayer involves both ester groups facing
the water interface and the oleoyl chains oriented toward the
air in a disordered fashion (Figure 5Ci). Such a disordered
low-density monolayer did not result in a detectable
evaporation resistance (see the Supporting Information).
When compressed, plateaus in the surface pressure and surface
potential were observed that were accompanied by the
appearance of high-intensity aggregates in the BAM images
(Figure 5Bii and Biii), indicating that the film collapsed at a
surface pressure of 2 mN/m. Although the collapse surface
pressure was low, a stable monolayer was formed, indicating
that the presence of a cis-double bond in the diol moiety of
20:1-DiE facilitated spreading on the aqueous surface. The
presence of an additional double bond thus changes the
behavior of the DiE to a certain extent, since the corresponding
saturated 20:0-DiE was incapable of forming a stable
monolayer even at 43 °C as reported in our previous work.19
The collapse pressure of 20:1-DiE was close to that of a
saturated 15:0-DiE,19 indicating that the addition of a cis-
double bond in the diol moiety had a similar effect on the
diester spreading properties as a five-carbon decrease in the
diol chain length. Based on these observations and previous
results, the spreading properties of the most-abundant
naturally occurring type II diesters in tear fluid with 30−34
carbons in the interconnecting diol chain12,49 can be estimated.
In our series of synthetic probes, the collapse surface pressure
of the diesters was found to decrease by 0.7 mN/m for each
one-carbon increase in the diol chain length.19 Therefore, an
extension of the 20:1-DiE diol chain with 10−14 carbon atoms
(representing naturally occurring type II diesters) would not
result in a type II diester with monolayer-forming capabilities
due to the predicted zero equilibrium spreading pressure.
2.3.3. Type I 20:1-St Diester. While type I-St diesters
have been known to exist in the TFLL for decades,50 little is
known about their properties due to the lack of molecular
probes from this lipid class. Here we studied 20:1-St-DiE,
which is a shorter analogue of the TFLL type I-St diesters. In
contrast to 20:1-DiE, 20:1-St-DiE only partially spread on the
PBS surface at 35 °C, as indicated by the lack of surface
pressure (Figure 6A) and the high-intensity aggregates
dispersed throughout the monolayer in the BAM images
The presence of an additional double bond in 20:1-OAHFA
therefore prevented the formation of a condensed monolayer
phase at high surface pressures similar to those in natural tears
(27−31 mN/m).48 The absence of a solid monolayer phase
was accompanied by a lack of evaporation resistance (see the
Supporting Information), which is in line with our previous
study.19 The disordered structure presented here describes the
behavior of the shortest OAHFAs detected in Meibomian
gland secretions and tears.11,15 Despite the lack of evaporation
resistance, these OAHFAs may contribute to stabilizing and
spreading the nonpolar TFLL lipids through their surfactant
properties.
In addition, the organization of the most abundant OAHFAs
in the TFLL with even longer hydroxy fatty acid (HFA) chains
(30:1 to 34:1)11,15,20 can be estimated by comparing the
results to our earlier work.19 The overall organization of the
20:1-OAHFA closely resembled that of the shorter 12:0-
OAHFA;19 therefore, the disordering effect caused by the
presence of a second cis-double bond is similar in magnitude to
an eight-carbon decrease of the HFA chain length. Therefore,
the ultralong OAHFAs with unsaturated HFA chains in the
TFLL would be expected to organize similarly to the 20:0-
OAHFA, and we consider it to be likely that they are capable
of forming a solid monolayer with evaporation-resistant
properties;19 however, this needs to be confirmed by future
work.
2.3.2. Type II 20:1-Diester. Type II diesters belong to a
hydrophobic lipid class and are therefore expected to reside in
the nonpolar sublayer of the TFLL. Interestingly, they have
been found to appear in reduced amounts in DED patients.16
Their role in the TFLL function is not clear, but they have
been suggested to stabilize the TFLL by connecting the
amphiphilic and nonpolar sublayers.17 Therefore, under-
standing their surface behavior and properties is important.
The oleoyl type II diester (7, 20:1-DiE) was found to spread
on the aqueous interface in a similar fashion to the 20:1-
OAHFA (Figure 5A). The surface-pressure isotherm lift-off
was observed at 130 Å2/molecule, and BAM images indicated
the formation of a homogeneous monolayer (Figure 5Bi). On
a general level, the surface-pressure and surface-potential
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Figure 6. (A) Surface pressure and surface potential (mean SD, n = 3) of 20:1-St-DiE with (B) the corresponding BAM images at 35 °C and
room temperature. The scale bar depicts 500 μm. (C) Schematic representation of the molecular organization of the 20:1-St-DiE films during
compression at 35 °C and room temperature.
(Figure 6Bi−Biii). The reduced surface activity compared to
that of 20:1-DiE can be explained by the bulkier nature of the
nonpolar cholesteryl moiety, which leads to stronger intra-
molecular van der Waals interactions between the diesters and
thus favors aggregation. The 20:1-St-DiE films lacked
evaporation-resistant properties at 35 °C (see the Supporting
Information), which can be attributed to the incomplete
spreading and disordered structure of the film (Figure 6Ci−
Ciii).
structural features of these type I-St-DiEs in more detail. At
this stage, it is sufficient to state that 20:1-St-DiE appeared to
form a liquid-crystalline phase similar to the multilayers formed
by cholesteryl esters and the results suggest that the type I-St
diester may be involved in the formation of multilamellar
structures in the TFLL.
3. CONCLUSION
Herein we described the chemical synthesis, detailed NMR-
spectroscopic characterization, and investigation of the
biophysical properties of a model tear fluid OAHFA, a type
I-St diester, and a type II diester species. We have devised a
functioning synthetic strategy that permits the preparation of
these lipid classes from one core intermediate. The most
important benefits of the chosen synthetic strategy are that the
block approach can be applied to the synthesis of a variety of
TFLL lipids and structural analogues thereof by varying the
chemical structures of fragments A, B, and C (see Figure 2).
When this information is further coupled with the most-
detailed NMR spectroscopic characterization data reported for
these compounds to date, a critical tool for the future synthesis
work and NMR spectroscopic lipidomic profiling of the tear-
film emerges.
The biophysical characterization described provides a
general description of the surface behavior of these unique
lipid species, offering insights on their role in the TFLL under
states of health and disease. Monounsaturated OAHFA
analogues were recently demonstrated to form an evapo-
ration-resistant solid monolayer at the aqueous interface, and
the evaporation resistance was found to improve with an
increase in the HFA chain length.19 However, as increasing the
chain length also markedly decreases the spreading rate of the
surfactants,52,53 a compromise between chain length and
spreadability is likely essential to achieve effective evaporation
resistance. Herein, we have showed that an additional double
bond in the HFA chain was accompanied by a more disordered
molecular organization of the OAHFAs, which led to the loss
of the evaporation resistance. Therefore, it seems likely that the
ultralong chain lengths observed in naturally occurring
OAHFAs require the presence of an additional double bond
to achieve an appropriate balance between spreadability and
evaporation resistance.
We therefore decided to further investigate whether the
surface organization of 20:1-St-DiE is affected by the
temperature. Interestingly, 20:1-St-DiE spread to form a
monolayer at room temperature but not at 40 °C (see Figure
S25). This was evidenced by a shift of the surface-pressure and
surface-potential isotherms to larger areas per molecule (Figure
6A and B). In addition, a homogeneous monolayer was
observed in the BAM images at room temperature (Figure
6Biv). A surface potential of approximately 300 mV was
observed at the surface-pressure lift-off similar to 20:1-DiE,
thus suggesting that the conformation of 20:1-St-DiE at the
aqueous interface may be similar with both ester groups facing
the water interface (Figure 6Civ). Upon the compression of
the monolayer, the surface pressure started to increase at
approximately 150 Å2/molecule, reflecting the larger size of
20:1-St-DiE compared to that of 20:1-DiE. The formation of
circular higher-intensity domains with a uniform intensity
appeared during compression (Figure 6Bv). This was
interesting because phase transitions were not apparent from
the recorded isotherm data. Moreover, the appearance of these
domains resembled the formation of multilamellar structures
reported to occur in mixed films containing polar lipids and
cholesteryl nervonate, where layers of the cholesteryl ester
form above the polar lipid monolayer.51 Therefore, we propose
a putative organization for 20:1-St-DiE in which the diesters
adopt an extended conformation similar to that of cholesteryl
ester multilayers but with the second ester groups oriented
toward the water (Figure 6Cv). Unfortunately, the details of
their organization could not be determined based on these
results alone. When the films were compressed to even smaller
areas (<80 Å2/molecule), the observed domains were
compressed into bright droplets, which is suggestive of a
collapse of the lipid film (Figure 6Bvi). The absence of a
plateau in the surface-pressure isotherm poses a challenge for a
more detailed analysis of the structural features of the film.
Further work is currently under planning to address the
Diesters have been suggested to reside between the polar
OAHFAs and the nonpolar wax and cholesteryl esters due to
their intermediate polarity.17 However, based on our results
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(C-11, C-2), 29.7−28.9 (C-4−C-9), 28.3 (C-10), and 25.9 (C-3).
HRMS (EI): m/z calculated for C12H25BrONa [M + Na]+ 287.0989,
found 287.1001.
both diester classes display very limited surface activity, and
even the shorter-chain analogues studied here collapsed at low
surface pressures. Therefore, it appears unlikely that the
naturally occurring ultralong diesters would partition to the
aqueous interface instead of the bulk of the nonpolar sublayer.
Instead, they may have a role in multilamellar ordering and the
organization of the nonpolar lipid layer. The synthesized type
I-St diester in particular displayed a tendency to form
multilamellar structures, suggesting that diesters of this subtype
may be involved in forming the multilamellar lipid structures
that have been proposed to exist in the TFLL.54,55
In addition to the biophysical properties of the OAHFAs
and diesters described here, their interactions with other tear
film lipid components, such as wax esters, cholesteryl esters,
and phospholipids, is likely essential to the overall function of
the tear film. Our study provides a solid and hitherto missing
foundation for addressing the role of these lipid classes in the
organization and function of the TFLL in addition to multiple
new tools that are applicable to ocular surface research and
beyond.
4.1.2. 12-Bromo-1-tert-butyldimethylsilyloxydodecane (1). To a
solution containing 12-bromo-1-dodecanol (0.500 g, 1.2 equiv) in dry
CH2Cl2 (3 mL) was added imidazole (0.215 g, 2 equiv). The reaction
mixture was stirred under an argon atmosphere, and TBDSMCl
(0.238 g, 1 equiv) was added after complete dissolution. The reaction
mixture was stirred at rt for 18 h, then poured onto a cold saturated
aq. NaHCO3 solution (20 mL) and extracted with CH2Cl2 (3 × 20
mL). The combined organic phase was washed with H2O (50 mL),
dried over Na2SO4, filtered, and concentrated. The crude oil was
purified by column chromatography using hexane/EtOAc (97:3) as
the eluent and dried on the vacuum line to give the title compound as
a thick colorless oil (0.564 g, 94% yield): Rf= 0.81 (hexane/EtOAc
1
95:5). H NMR (500.13 MHz, CDCl3, 25 °C): δ 3.60 (t, 2H, J1,2
6.6 Hz, H-1), 3.40 (t, 2H, J12,11 = 6.9 Hz, H-12), 1.85 (tt, 2H, J11,10
7.5 Hz, H-11), 1.50 (tt, 2H, J1,2 = 6.6 Hz, H-2), 1.42 (tt, 2H, J10,9
=
=
=
6.6 Hz, H-10), 1.35−1.22 (m, 14H, H-3−H-9), 0.89 (s, 9H, 1-
OSi(CH2)2C(CH3)3), and 0.04 (s, 6H, 1-OSi(CH3)2C(CH3)3).
13C{1H} NMR (125.68 MHz, CDCl3, 25 °C): δ 63.5 (C-1), 34.1
(C-12), 33.0 (C-11, C-2), 29.8−28.9 (C-4−C-9), 28.3 (C-10), 26.1
(1-OSi(CH3)2C(CH3)3), 26.0 (C-3), 18.5 (1-OSi(CH3)2C(CH3)3),
and −5.1 (1-OSi(CH3)2C(CH3)3). HRMS (EI): m/z calculated for
C18H39BrOSiNa [M + Na]+ 401.1854, found 401.1852.
4. EXPERIMENTAL SECTION
4.1.3. 1-tert-butyldimethylsilyloxydodecane, Triphenyl-
phosphonium Bromide. A mixture containing 1 (2.0 g, 1 equiv)
and PPh3 (1.39 g, 1 equiv) was stirred under an argon atmosphere at
120 °C o/n and cooled to rt. The formation of the triphenylphos-
phonium bromide salt was confirmed by 31P NMR analysis, and the
thick resinous product was used in the subsequent Wittig reaction as
such.31P NMR (202.4 MHz, CDCl3, 25 °C): δ 24.4 (Ph3P+).
4.1.4. 8-Bromo-1-octanal. 8-Bromo-1-octanol (1.01 g, 1 equiv)
was dissolved in CH2Cl2 (80 mL), and PCC (1.563 g, 1.5 equiv) was
added. The reaction mixture was stirred at rt for 3 h and then Et2O
(80 mL) was added, followed by filtration through Celite to remove
the remnants of PCC. The flask was washed with Et2O (2 × 80 mL),
and the combined filtrates were filtered through Celite before
concentration. H2O (50 mL) and Et2O (50 mL) were added to this
residue. The light green organic phase was separated, and the aqueous
phase was extracted with CH2Cl2 (2 × 50 mL). The combined
organic phase was washed with H2O (100 mL), dried over Na2SO4,
filtered, and concentrated to give the title compound as an oil (0.84 g,
84% yield). The crude product was used in the subsequent Wittig
All reagents were purchased from commercial sources. Dry solvents
were purified by the VAC vacuum solvent purification system prior to
use when dry solvents were needed. All reactions containing moisture-
or air-sensitive reagents were carried out under an argon atmosphere.
All reactions requiring heating were performed using an oil bath. TLC
was performed on aluminum sheets precoated with silica gel 60 F254
(Merck). Flash chromatography was carried out using silica gel 40.
Spots were visualized by UV, then sprayed with a 1:4 H2SO4/MeOH
solution and heated. HRMS spectra were recorded using a Bruker
Micro Q-TOF with ESI (electrospray ionization) operated in the
positive mode. NMR spectra were recorded with a Bruker Avance III
NMR spectrometer operating at 500.13 or 499.82 (1H), 125.68 (13C,)
and 202.40 MHz (31P). All products were characterized by a
combination of 1D (1H, 13C{1H}, and 31P) and 2D techniques (DQF-
COSY, TOCSY, Ed-HSQC, and HMBC) with pulse sequences
provided by the instrument manufacturer. The probe temperature was
kept at 25 °C unless otherwise stated. The chemical shifts are
expressed on the δ scale (ppm) using TMS (tetramethylsilane) or
residual chloroform as the internal standards. The coupling constants
are given in Hertz and provided only once when first encountered.
The coupling patterns are given as s (singlet), d (doublet), t (triplet),
or m (multiplet). Details on the numbering of the molecules are
provided in the Supporting Information. The computational analysis
of the 1H NMR spectra of all compounds was achieved using of
PERCH (PEak reseaRCH) NMR software, and starting values and
spectral parameters were obtained from the various NMR techniques
1
reaction as such. H NMR (500.13 MHz, CDCl3, 25 °C): δ 9.74 (t,
1H, J1,2 = 1.6 Hz, H-1), 3.38 (t, 2H, J8,7 = 6.8 Hz, H-8), 2.41 (dt, 2H,
J
2,3 = 7.3 Hz, H-2), 1.83 (tt, 2H, J7,6 = 7.0 Hz, H-7), 1.61 (tt, 2H, J3,4 =
7.4 Hz, H-3), 1.42 (tt, 2H, J6,5 = 7.0 Hz, H-6), and 1.35−1.29 (m, 4H,
H-4, H-5).
4.1.5. (12Z)-20-Bromo-1-tert-butyldimethylsilyloxyeicos-12-ene
(2). A solution containing 1-tert-butyldimethylsilyloxydodecane,
triphenylphosphonium bromide (3.164 g, 2 equiv) in dry THF (30
mL), and HMPA (8.9 mL) under an argon atmosphere was cooled to
−78 °C. After 10 min, NaHMDS (8.2 mL, 0.6 M in toluene, 2 equiv)
was slowly added, and the resulting mixture was stirred for 1 h. A
solution of freshly prepared 8-bromo-1-octanal (0.536 g, 1.05 equiv)
dissolved in dry THF (8 mL) was slowly added at −78 °C, and the
reaction mixture was allowed to warm to rt over 24 h before it was
quenched with an aq phosphate buffer (freshly prepared, pH 7.2, 80
mL). Extraction with Et2O (3 × 80 mL) was performed, and the
combined organic phase was dried over Na2SO4, filtered, and
concentrated. The crude product was purified by column chromatog-
raphy using a hexane/Et3N (100:0.1) to hexane/EtOAc/Et3N
(399:1:0.1 → 98.7:1.3:0.1) gradient as the eluent and further dried
on the vacuum line to give the title compound as a thick yellowish oil
used.56 Melting point analysis was performed using a Bu
̈
chi B-545
̈
melting point instrument (BUCHI Labortechnik AG) when possible.
4.1. Synthetic Protocols and Substrate Specific Character-
ization Data. 4.1.1. 12-Bromo-1-dodecanol. To a solution
containing 1,12-dodecanediol (2 g, 1 equiv) in cyclohexane (26.2
mL) was added HBr (26.2 mL, 23.6 equiv, 48% sol. in H2O), and the
biphasic system was refluxed for 18 h. The reaction mixture was then
cooled to rt, and the organic layer was separated. The aqueous phase
was extracted with CH2Cl2 (5 × 25 mL). The combined organic
phase was washed with a saturated aq NaHCO3 solution (5 × 25 mL)
and brine (50 mL), dried over Na2SO4, filtered, and concentrated.
The crude product was purified by column chromatography using
hexane/EtOAc (7:3) and dried on the vacuum line to give the title
compound as a white solid (2.03 g, 77% yield): Rf = 0.34 (hexane/
1
1
EtOAc 7:3). H NMR (500.13 MHz, CDCl3, 25 °C): δ 3.64 (t, 2H,
(0.427 g, 34% yield): Rf = 0.31 (hexane/EtOAc/Et3N 98:1:1). H
J1,2 = 6.5 Hz, H-1), 3.41 (t, 2H, J12,11= 6.9 Hz, H-12), 1.85 (tt, 2H,
J11,10 = 7.5 Hz, H-11), 1.56 (tt, 2H, J2,3 = 7.5 Hz, H-2), 1.42 (tt, 2H,
J10,9 = 6.4 Hz, H-10), and 1.38−1.22 (m, 14H, H-3−H-9). 13C{1H}
NMR (125.68 MHz, CDCl3, 25 °C): δ 63.2 (C-1), 34.2 (C-12), 33.0
NMR (499.82 MHz, CDCl3, 25 °C): δ 5.35 (dtt, 1H, J12,14 = −1.5,
J
12,11 = 7.1, J12,13 = 11.0 Hz, H-12), 5.34 (dtt, 1H, J13,11 = −1.5, J13,14 =
7.0 Hz, H-13), 3.60 (t, 2H, J1,2 = 6.6 Hz, H-1), 3.40 (t, 2H, J20,19 = 6.9
Hz, H-20), 2.02 (ddt, 2H, J14,15 = 6.7 Hz, H-14), 2.01 (ddt, 2H, J11,10
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= 7.4 Hz, H-11), 1.85 (tt, 2H, J19,18 = 7.5 Hz, H-19), 1.50 (tt, 2H, J2,3
= 6.7 Hz, H-2), 1.43 (tt, 2H, J18,17 = 6.7 Hz, H-18), 1.38−1.22 (m,
22H, H-3−H-10, H-15−H-17), 0.89 (s, 9H, 1-OSi(CH2)2C(CH3)3),
and 0.05 (s, 6H, 1-OSi(CH3)2C(CH3)3). 13C{1H} NMR (125.68
MHz, CDCl3, 25 °C): δ 130.3 (C-12), 129.9 (C-13), 63.5 (C-1), 34.2
(C-20), 33.1 (C-2), 33.0 (C-19), 29.9−28.8 (C-4−C-10, C-15−C-
17), 28.3 (C-18), 27.4−27.3 (C-11, C-14), 26.2 (1-OSi(CH3)2C-
(CH3)3), 26.0 (C-3), 18.6 (1-OSi(CH3)2C(CH3)3), and −5.1 (1-
OSi(CH3)2 C(CH3)3). HRMS (EI): m/z calculated for
C26H53BrOSiNa [M + Na]+ 511.2949, found 511.2995.
extracted with CH2Cl2 (2 × 10 mL). The combined organic phase
was washed with H2O (2 × 15 mL), dried over Na2SO4, filtered, and
concentrated. The crude product was purified by column chromatog-
raphy using hexane/EtOAc/Et3N (95:5:0.1) as the eluent and dried
under vacuum to give the title compound as a white solid (0.061 g,
93% yield): Rf = 0.46 (hexane/EtOAc/Et3N 95:5:0.1). 1H NMR
(500.13 MHz, CDCl3, 25 °C): δ 5.35 (dtt, 1H, J9′,11′ = −1.1, J9′,8′
7.1, J9′,10′ = 11.6 Hz, H-9′), 5.35 (dtt, 1H, J12,14 = −1.2, J12,11 = 7.1,
12,13 = 10.5 Hz, H-12), 5.34 (dtt, 1H, J10′,8′ = −1.2, J10′,11′ = 6.8 Hz, H-
=
J
10′), 5.34 (dtt, 1H, J13,11 = −1.5, J13,14 = 6.6 Hz, H-13), 4.05 (t, 2H,
J20,19 = 6.7 Hz, H-20), 3.60 (t, 2H, J1,2 = 6.7 Hz, H-1), 2.29 (t, 2H,
J2′,3′ = 7.3 Hz, H-2′), 2.06−1.96 (m, 8H, H-11, H-14, H-8′, H-11′),
1.62 (tt, 2H, J3′,4′ = 6.7 Hz, H-3′), 1.61 (tt, 2H, J19,18 = 6.6 Hz, H-19),
1.50 (tt, 2H, J2,3 = 6.7 Hz, H-2), 1.37−1.23 (m, 44H, H-3−H-10, H-
15−H-18, H-4′−H-7′, H-12′−H-17′), 0.89 (s, 9H, 1-OSi(CH2)2C-
(CH3)3), 0.88 (t, 3H, J18′,17′ = 7.1 Hz, H-18′), and 0.05 (s, 6H, 1-
OSi(CH3)2C(CH3)3). 13C{1H} NMR (125.68 MHz, CDCl3, 25 °C):
δ 174.1 (C-1′), 130.2−129.9 (C-12, C-13, C-9′, C-10′), 64.5 (C-20),
63.5 (C-1), 34.5 (C-2′), 33.0 (C-2), 32.1 (C-16′), 29.9−29.3 (C-4−
C-10, C-15−C-17, C-4′−C-7′, C-12′−C-15′), 28.8 (C-19), 27.4−
27.3 (C-11, C-14, C-8′, C-11′), 26.1 (1-OSi(CH2)2C(CH3)3), 26.0
(C-3, C-18), 25.2 (C-3′), 22.8 (C-17′),18.6 (1-OSi(CH3)2C(CH3)3),
14.3 (C-18′), and −5.1 (1-OSi(CH3)2C(CH3)3). HRMS (EI): m/z
calculated for C46H86O3SiNa [M + Na]+ 713.6246, found 713.6214.
4.1.9. (12Z)-20-Oleoyloxyeicos-12-enol (4). A solution containing
(12Z)-20-oleoyloxy-1-tert-butyldimethylsilyloxyeicos-12-ene (0.031 g,
1 equiv) in dry THF (0.5 mL) under an argon atmosphere was cooled
to 0 °C on an ice-bath, and TBAF (0.140 mL, 1 M in THF, 3 equiv)
was added. After 5 min, the ice-bath was removed, and the reaction
mixture was stirred at rt for 1 h before being quenched with H2O (2
mL) and extracted with EtOAc (3 mL). The organic phase was
washed with H2O (2 × 5 mL), and the aqueous phase was re-
extracted with EtOAc (2 × 5 mL). The combined organic phase was
dried over Na2SO4, filtered, and concentrated. The crude product was
purified by column chromatography using hexane/EtOAc/Et3N
(7:3:0.1) as the eluent and dried under vacuum to give the title
compound as a white solid (0.023 g, 92% yield): Rf = 0.58 (hexane/
EtOAc/Et3N 7:3:0.1). 1H NMR (500.13 MHz, CDCl3, 25 °C): δ 5.35
(dtt, 1H, J9′,11′ = −2.1, J9′,8′ = 6.4, J9′,10′ = 10.9 Hz, H-9′), 5.35 (dtt,
1H, J12,14 = −1.8, J12,11 = 7.3, J12,13 = 11.5 Hz, H-12), 5.34 (dtt, 1H,
4.1.6. (12Z)-20-Acetoxy-1-tert-butyldimethylsilyloxyeicos-12-
ene. To a solution containing 2 (0.265 g, 1 equiv) in DMSO (12
mL) was added KOAc (0.266 g, 5 equiv), and the suspension was
stirred at rt o/n. After 24 h, additional KOAc (0.159 g, 3 equiv) was
added, and the temperature was raised to 50 °C. After 27 h, the
reaction mixture was brought to rt, and H2O (25 mL) was added.
Extraction with Et2O (3 × 25 mL) was performed, and the combined
organic phase was washed with brine (30 mL), dried over Na2SO4,
filtered, and concentrated. The crude product was purified by column
chromatography using hexane/EtOAc/Et3N (98:2:0.1 → 95.5:0.1) as
the eluent and dried on the vacuum line to give the title compound as
a yellowish oil (0.169 g, 66% yield): Rf = 0.51 (hexane/EtOAc/Et3N
1
95:5:0.1). H NMR (500.13 MHz, CDCl3, 25 °C): δ 5.35 (dtt, 1H,
J12,14 = −1.5, J12,11 = 7.1, J12,13 = 11.1 Hz, H-12), 5.34 (dtt, 1H, J13,11 =
−1.5, J13,14 = 7.2 Hz, H-13), 4.05 (t, 2H, J20,19 = 6.8 Hz, H-20), 3.59
(t, 2H, J1,2 = 6.7 Hz, H-1), 2.04 (s, 3H, 20−OCOCH3), 2.01 (ddt,
2H, J14,15 = 6.0 Hz, H-14), 2.00 (ddt, 2H, J11,10 = 7.0 Hz, H-11), 1.62
(tt, 2H, J19,18 = 7.0 Hz, H-19), 1.50 (tt, 2H, J2,3 = 6.6 Hz, H-2), 1.39−
1.22 (m, 24H, H-3−H-10, H-15−H-18), 0.89 (s, 9H, 1-OSi(CH2)2C-
(CH3)3), and 0.04 (s, 6H, 1-OSi(CH3)2C(CH3)3). 13C{1H} NMR
(125.68 MHz, CDCl3, 25 °C): δ 171.4 (20-OCOCH3), 130.2 (C-12),
129.9 (C-13), 64.8 (C-20), 63.5 (C-1), 33.1 (C-2), 29.9−29.3 (C-4−
C-10, C-15−C-17), 28.8 (C-19), 27.4−27.3 (C-11, C-14), 26.2 (1-
OSi(CH3)2C(CH3)3), 26.0 (C-3, C-18), 21.2 (20−OCOCH3), 18.6
(1-OSi(CH3)2C(CH3)3), and −5.1 (1-OSi(CH3)2C(CH3)3). HRMS
(EI): m/z calculated for C28H56O3SiNa (M + Na+) 491.3899, found
491.3879.
4.1.7. (12Z)-20-Hydroxy-1-tert-butyldimethylsilyloxyeicos-12-ene
(3). To a solution containing (12Z)-20-acetoxy-1-tert-butyldimethyl-
silyloxyeicos-12-ene (0.022 g, 1 equiv) in MeOH (1 mL) and THF
(0.5 mL) under argon atmosphere was added NaOMe (0.003 g, 1
equiv), and the resulting mixture was stirred at rt. After 22 h, the
reaction was quenched by the addition of aq HCl (10% sol. v/v) and
H2O (15 mL). The resulting mixture was extracted with Et2O (3 × 15
mL), and the combined organic phase was dried over Na2SO4,
filtered, and concentrated. Drying under vacuum gave the title
compound as a white solid (0.015 g, 78% yield): Rf = 0.56 (hexane/
EtOAc/Et3N 7:3:0.1). 1H NMR (500.13 MHz, CDCl3, 25 °C): δ 5.34
(dtt, 1H, J12,14 = −1.0, J12,11 = 7.0, J12,13 = 11.3 Hz, H-12), 5.34 (dtt,
1H, J13,11 = −1.0, J13,14 = 7.0 Hz, H-13), 3.64 (t, 2H, J1,2 = 6.7 Hz, H-
1), 3.59 (t, 2H, J20,19 = 6.7 Hz, H-20), 2.01 (ddt, 2H, J14,15 = 6.3 Hz,
H-14), 2.00 (ddt, 2H, J11,10 = 7.4 Hz, H-11), 1.57 (tt, 2H, J2,3 = 7.4
Hz, H-2), 1.50 (tt, 2H, J19,18 = 6.6 Hz, H-19), 1.40−1.22 (m, 24H, H-
3−H-10, H-15−H-18), 0.89 (s, 9H, 1-OSi(CH2)2C(CH3)3), and 0.04
(s, 6H, 1-OSi(CH3)2C(CH3)3). 13C{1H} NMR (125.68 MHz,
CDCl3, 25 °C): δ 130.2 (C-12), 129.9 (C-13), 63.5 (C-20), 63.3
(C-1), 33.0 (C-19), 32.9 (C-2), 29.9−29.4 (C-4−C-10, C-15−C-17),
27.4−27.3 (C-11, C-14), 26.1 (1-OSi(CH3)2C(CH3)3), 26.0−25.9
(C-3, C-18), 18.6 (1-OSi(CH3)2C(CH3)3), and −5.1 (1-OSi-
(CH3)2C(CH3)3). HRMS (EI): m/z calculated for C26H54O2SiNa
[M + Na]+ 449.3793, found 449.3832.
J
10′,8′ = −1.0, J10′,11′ = 7.2 Hz, H-10′), 5.34 (dtt, 1H, J13,11 = −2.0, J13,14
= 6.6 Hz, H-13), 4.05 (t, 2H, J20,19 = 6.7 Hz, H-20), 3.64 (t, 2H, J1,2
=
6.6 Hz, H-1), 2.29 (t, 2H, J2′,3′ = 7.3 Hz, H-2′), 2.06−1.96 (m, 8H, H-
11, H-14, H-8′, H-11′), 1.62 (tt, 2H, J3′,4′ = 6.7 Hz, H-3′), 1.61 (tt,
2H, J19,18 = 6.6 Hz, H-19), 1.56 (tt, 2H, J2,3 = 6.7 Hz, H-2), 1.37−1.23
(m, 44H, H-3−H-10, H-15−H-18, H-4′−H-7′, H-12′−H-17′), and
0.88 (t, 3H, J18′,17′ = 7.1 Hz, H-18′). 13C{1H} NMR (125.68 MHz,
CDCl3, 25 °C): δ 174.2 (C-1′), 130.2−129.9 (C-12, C-13, C-9′, C-
10′), 64.5 (C-20), 63.2 (C-1), 34.5 (C-2′), 33.0 (C-2), 32.1 (C-16′),
29.9−29.3 (C-4−C-10, C-15−C-17, C-4′−C-7′, C-12′−C-15′), 28.8
(C-19), 27.4−27.3 (C-11, C-14, C-8′, C-11′), 26.0−25.9 (C-3, C-18),
25.2 (C-3′), 22.8 (C-17′), and 14.3 (C-18′). HRMS (EI): m/z
calculated for C38H72O3Na [M + Na]+ 599.5381, found 599.5342.
4.1.10. (12Z)-20-Oleoyloxyeicos-12-enoic acid (5). A solution
containing 4 (0.026 g, 1 equiv) in acetone (2 mL) and EtOAc (2 mL)
was cooled to 0 °C on an ice-bath, and the Jones reagent (0.050 mL,
2.2 equiv) was added. The resulting mixture was stirred at 0 °C for 45
min. H2O (5 mL) was added ,and the reaction mixture was then
extracted with Et2O (3 × 15 mL). The combined organic phase was
washed with brine (15 mL), dried over Na2SO4, filtered, and
concentrated. The crude product was purified by column chromatog-
raphy using hexane/EtOAc/AcOH (7:3:0.1) as the eluent and dried
on the vacuum line to give the title compound as a white solid (0.024
4.1.8. (12Z)-20-Oleoyloxy-1-tert-butyldimethylsilyloxyeicos-12-
ene. To a solution of 3 (0.040 g, 1 equiv) in dry CH2Cl2 (2 mL)
under an argon atmosphere were added DMAP (0.012 g, 1 equiv) and
EDC·HCl (0.046 g, 2.5 equiv), and the resulting mixture was cooled
to 0 °C on an ice-bath. Oleic acid (0.032 g dissolved in 0.5 mL of dry
CH2Cl2, 1.2 equiv) was added, and the reaction mixture was stirred at
0 °C for 10 min and then at rt o/n. The reaction mixture was
quenched after 20 h with H2O (2 mL) and diluted with CH2Cl2 (10
mL). The organic phase was separated, and the aqueous phase was
1
g, 89% yield): Rf = 0.43 (hexane/EtOAc/AcOH 7:3:0.1). H NMR
(500.13 MHz, CDCl3, 25 °C): δ 5.35 (dtt, 1H, J9′,11′ = −1.3, J9′,8′
=
6.9, J9′,10′ = 11.1 Hz, H-9′), 5.35 (dtt, 1H, J12,14 = −1.8, J12,11 = 7.3,
J
12,13 = 11.6 Hz, H-12), 5.34 (dtt, 1H, J10′,8′ = −0.8, J10′,11′ = 7.2 Hz, H-
10′), 5.34 (dtt, 1H, J13,11 = −2.2, J13,14 = 6.8 Hz, H-13), 4.05 (t, 2H,
J20,19 = 6.8 Hz, H-20), 2.34 (t, 2H, J2,3 = 7.1 Hz, H-2), 2.29 (t, 2H,
J2′,3′ = 7.3 Hz, H-2′), 2.06−1.96 (m, 8H, H-11, H-14, H-8′, H-11′),
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1.63 (tt, 2H, J3,4 = 6.4 Hz, H-3), 1.61 (tt, 2H, J3′,4′ = 6.6 Hz, H-3′),
1.61 (tt, 2H, J19,18 = 6.6 Hz, H-19), 1.37−1.23 (m, 42H, H-4−H-10,
H-15−H-18, H-4′−H-7′, H-12′−H-17′), and 0.88 (t, 3H, J18′,17′ = 7.1
Hz, H-18′). 13C{1H} NMR (125.68 MHz, CDCl3, 25 °C): δ 179.6
(C-1), 174.2 (C-1′), 130.1−129.9 (C-12, C-13, C-9′, C-10′), 64.6 (C-
20), 34.5 (C-2′), 34.4 (C-2), 32.0 (C-16′), 29.9−29.3 (C-4−C-10, C-
15−C-17, C-4′−C-7′, C-12′−C-15′), 28.8 (C-19), 27.4−27.3 (C-11,
C-14, C-8′, C-11′), 26.1 (C-18), 25.2 (C-3′), 24.9 (C-3), 22.8 (C-
17′), and 14.3 (C-18′). HRMS (EI): m/z calculated for C38H70O4Na
[M + Na]+ 613.5174, found 613.5175. mp: 29.5−30.7 °C.
and the aqueous layer was extracted with CH2Cl2 (2 × 10 mL). The
combined organic layers were washed with H2O (2 × 15 mL), dried
over Na2SO4, filtered, and concentrated. The crude product was
purified by column chromatography using hexane/EtOAc/Et3N
(95:5:0.1) as the eluent and dried on the vacuum line to give the
title compound as an oil (0.032 g, 70% yield): Rf = 0.59 (hexane/
1
EtOAc/Et3N 95:5:0.1). H NMR (499.82 MHz, CDCl3, 25 °C): δ
5.39−5.30 (m, 6H, H-9, H-10, H-12′, H-13′, H-9″, H-10′′), 4.05 (t,
2H, J1′,2′ = 6.7 Hz, H-1′), 4.05 (t, 2H, J20′,19′ = 6.7 Hz, H-20′), 2.28 (t,
2H, J2,3 = 7.5 Hz, H-2), 2.28 (t, 2H, J2″,3′′ = 7.5 Hz, H-2″), 2.05−1.96
4.1.11. Cholesteryl-(12Z)-20-oleoyloxyeicos-12-enoate (6). Cho-
lesterol (0.019 g, 1.2 equiv) was dissolved in dry CH2Cl2 (1.5 mL)
under an argon atmosphere, then DMAP (0.005 g, 1 equiv) and EDC·
HCl (0.020 g, 2.5 equiv) were added. The reaction mixture was
cooled on an ice bath and then 5 (0.024 g in 1 mL of dry CH2Cl2; 1
equiv) was added. The resulting mixture was stirred at 0 °C for 10
min and then at rt o/n. The reaction mixture was quenched after 20 h
with H2O (5 mL) and diluted with CH2Cl2 (10 mL). The organic
phase was separated, and the aqueous phase was extracted with
CH2Cl2 (2 × 10 mL). The combined organic layers were washed with
H2O (2 × 15 mL), dried over Na2SO4, filtered, and concentrated. The
crude product was purified by column chromatography using hexane/
EtOAc/Et3N (100:0:0.1−95:5:0.1) as the eluent and dried on the
vacuum line to give the title compound as a yellow oil (0.026 g, 67%
(m, 12H, H-8, H-11, H-11′, H-14′, H-8′′, H-11′′), 1.61 (tt, 2H, J3,4
6.7 Hz, H-3), 1.61 (tt, 2H, J2′,3′ = 7.0 Hz, H-2′), 1.61 (tt, 2 H, J19′,18′
=
=
7.1 Hz, H-19′), 1.61 (tt, 2H, J3″,4′′ = 6.6 Hz, H-3″), 1.37−1.20 (m,
64H, H-4−H-7, H-12−H-17, H-4′−H-10′, H-15′−H-18′, H-4′′−H-
7′′, H-12′′−H-17′′), 0.88 (t, 3H, J18,17 = 7.1 Hz, H-18), and 0.88 (t,
3H, J18″,17′′ = 7.1 Hz, H-18″). 13C{1H} NMR (125.68 MHz, CDCl3,
25 °C): δ 174.1 (C-1, C-1″), 130.1−129.9 (C-9′′, C-10′′, C-12′, C-
13′, C-9, C-10), 64.5 (C-1′, C-20′), 34.6 (C.2, C-2′′), 32.1 (C-16, C-
16′′), 29.9−29.3 (C-4−C-7, C-12−C-15, C-4′−C-10′, C-15′−C-17′,
C-4′′−C-7′′, C-12′′−C-15′′), 28.8 (C-2′, C-19′), 27.4−27.3 (C-8, C-
11, C-11′, C-14′, C-8′′, C-11′′), 26.1 (C-3′, C-18′), 25.2 (C-3, C-
3′′), 22.8 (C-17, C-17′′), and 14.3 (C-18, C-18′′). HRMS (EI): m/z
calculated for C56H104O4Na [M + Na]+ 863.7835, found 863.7818.
4.2. Langmuir Monolayer Experiments. Lipids were spread to
the air-buffer interface of a KSV NIMA Langmuir large trough (Biolin
Scientific, Espoo, Finland; dimensions 580 × 145 mm) filled with PBS
buffer in 5 (20:1-OAHFA), 4.87 (20:1-DiE) and 6.41 mM (20:1-St-
DiE) chloroform solutions. The measurement setup was contained in
an acrylic enclosure (volume 290 L), and dry air was continuously
passed through an ozone solutions ODS-3P ozone destruct unit
(Hull, Iowa) and into the enclosure at a rate of 76 L/min to maintain
a low-ozone atmosphere and prevent the oxidation of the studied
lipids during the measurements. Before starting the measurements,
chloroform was allowed to evaporate for 3 min. The films were
compressed at a constant rate of 10 mm/min (3.7%/min from the
initial area) in measurements with BAM or surface potential and a rate
of 60 mm/min (22.2%/min from the initial area) in compression−
relaxation cycles (Figure S26). The surface pressure was measured
using a Wilhelmy plate, and the surface potential was measured using
KSV SPOT (Espoo, Finland). Brewster angle microscopy images were
captured using a KSV NIMA microBAM camera (Espoo, Finland). A
circulating water bath (LAUDA ECO E4) was used to control the
temperature of the subphase. Measurements were performed at room
temperature, 35 1, and 40 1 °C for all lipids. All measurements
were repeated at least three times to ensure repeatability.
1
yield): Rf = 0.27 (hexane/EtOAc/Et3N 95:5:0.1). H NMR (499.82
MHz, CDCl3): δ 5.37 (dddd, 1H, J6,4a = −0.3, J6,4b = −1.9, J6,7a = 2.0,
J
6,7b = 5.2 Hz, H-6), 5.35 (dtt, 1H, J9″,11′′ = −1.1, J9″,8′′ = 6.7, J9″,10′′ =
11.3 Hz, H-9″), 5.35 (dtt, 1H, J12′,14′ = −1.5, J12′,11′ = 6.7, J12′,13′ = 11.6
Hz, H-12′), 5.34 (dtt, 1H, J10″,8′′ = −1.5, J10″,11′′ = 7.2 Hz, H-10″), 5.34
(dtt, 1H, J13′,11′ = −2.0, J13′,14′ = 6.8 Hz, H-13′), 4.61 (dddd, 1H, J3,2a
= 4.7, J3,4a = 4.8, J3,4b = 11.3, J3,2b = 11.4 Hz, H-3), 4.05 (t, 2H, J20′,19′
=
6.7 Hz, H-20′), 2.31 (dddd, 1H, J4a,2a = −2.5, J4a,4b = −13.0 Hz, H-
4a), 2.30 (ddddd, 1H, J4b,7b = −2.7, J4b,7a = −3.3 Hz, H-4b), 2.29 (t,
2H, J2′,3′ = 7.7 Hz, H-2′), 2.26 (t, 2H, J2″,3′′ = 7.6 Hz, H-2″), 2.05−
1.98 (m, 9H, H-12a, H-11′, H-14′, H-8′′, H-11′′), 1.97 (dddd, 1H,
J
7a,8 = 4.9, J7a,7b = −17.6 Hz, H-7a), 1.85 (ddd, 1H, J1a,2b = 3.3, J1a,2a
=
=
=
3.4, J1a,1b = −13.6 Hz, H-1a), 1.84 (dddd, 1H, J2a,1b = 3.7, J2a,2b
−12.2 Hz, H-2a), 1.82 (dddd, 1H, J16a,15b = 6.0, J16a,17 = 9.1, J16a,15a
9.6, J16a,16b = −13.4 Hz, H-16a), 1.65−1.55 (m, 8H, H-3′, H-19′, H-
3″, H-2b, H-15a), 1.55−1.45 (m, 4H, H-7b, H-25, H-11a, H-11b),
1.44 (dddd, 1H, J8,14 = 10.3, J8,9 = 11.7 Hz, H-8), 1.40−1.20 (m, 46H,
H-4′−H-10′, H-15′−H-18′, H-4″−H-7′′, H-12′′−H-17′′, H-16b, H-
20, H-22a, H-23a), 1.16 (ddd, 1H, J12b,11a = 3.2, J12b,12a = −12.8,
J
12b,11b = 13.9 Hz, H-12b), 1.15−1.04 (m, 6H, H-1b, H-15b, H-17, H-
23b, H-24a, H-24b), 1.02 (s, 3H, H-19), 1.00 (dddd, 1H, J22b,23a = 4.8,
J
J
22b,23b = 9.0, J22b,20 = 9.0, J22b,22a = −13.6 Hz, H-22b), 0.99 (ddd, 1H,
14,15a = 7.6, J14,15b = 12.6 Hz, H-14), 0.95 (ddd, 1H, J9,11a = 3.8, J9,11b
4.3. Evaporation Measurements. Evaporation measurements
were conducted using a modified Langmuir−Schaefer method46 as
described previously.51 In short, the lipids were spread to the air-
buffer interface of a KSV minitrough (Helsinki, Finland), the lipid film
was compressed to a desired mean molecular area (3.5−40 Å2/
molecule), and the evaporation rate through the film was measured by
placing a box filled with silica gel above the film surface and measuring
the amount of water absorbed by the desiccant. The measurement
time was 5 min. Disposable desiccant cartridges (SP Industries,
Warminster, PA) with silica gel were used, but the membrane was
replaced with a Millipore Immobilon-P PVDF membrane with a 450
nm pore size (Bedford, MA). Evaporation measurements were
repeated three times for each lipid.
=
12.3 Hz, H-9), 0.91 (d, 3H, H-21), 0.88 (t, 3H, J18″,17′′ = 6.8 Hz, H-
18″), 0.87, 0.86 (each d, each 3H, each J26/27,25 = 6.6 Hz, H-26, and
H-27), and 0.67 (s, 3H, H-18). 13C{1H} NMR (125.68 MHz,
CDCl3): δ 174.1(C-1′), 173.5 (C-1″), 139.9 (C-5), 130.2−129.9 (C-
9′′, C-10′′, C-12′, C-13′), 122.7 (C-6), 73.8 (C-3), 64.5 (C-20′), 56.8
(C-14), 56.3 (C-17), 50.2 (C-9), 42.5 (C-13), 39.9 (C-12), 39.7 (C-
24), 38.3 (C-4), 37.2 (C-1), 36.8 (C-10), 36.3 (C-22), 35.9 (C-20),
34.9 (C-2′′), 34.6 (C-2′), 32.1−32.0 (C-7, C-8, C-16′′), 29.9−29.3
(C-4′−C-10′, C-15′−C-17′, C-4′′−C-7′′, C-12′′−C-15′′), 28.8 (C-
19′), 28.4 (C-16), 28.2 (C-25), 28.0 (C-2), 27.4−27.3 (C-11′, C-14′,
C-8′′, C-11′′), 26.0 (C-18′), 25.2−25.1 (C-3′, C-3′′), 24.4 (C-15),
24.0 (C-23), 23.0 and 22.7 (C-26, C-27), 22.8 (C-17′′), 21.2 (C-11),
19.5 (C-19), 18.9 (C-21), 14.3 (C-18′′) and 12.0 (C-18). HRMS
(EI): m/z calculated for C65H114O4Na [M + Na]+ 981.8617, found
981.8614.
4.1.12. (12Z)-1,20-dioleoyloxyeicos-12-ene (7). To a solution of 4
(0.031 g, 1 equiv) in dry CH2Cl2 (1.5 mL) under an argon
atmosphere were added DMAP (0.007 g, 1 equiv) and EDC·HCl
(0.027 g, 2.5 equiv). The reaction mixture was cooled on an ice bath,
and oleic acid (0.019 g in 0.5 mL dry CH2Cl2; 1.2 equiv) was added.
The resulting mixture was stirred for 10 min at 0 °C and then at rt o/
n. The reaction mixture was quenched after 20 h with H2O (2 mL)
and diluted with CH2Cl2 (10 mL). The organic phase was separated,
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02882.
Numbering of the molecules studied in this work, NMR
spectra of synthesized compounds, and supporting
figures related to biophysical experiments (PDF)
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(7) King-Smith, P. E.; Hinel, E. A.; Nichols, J. J. Application of a
Novel Interferometric Method to Investigate the Relation between
Lipid Layer Thickness and Tear Film Thinning. Invest. Ophthalmol.
Visual Sci. 2010, 51 (5), 2418−2423.
AUTHOR INFORMATION
■
Corresponding Authors
Riku O. Paananen − Department of Chemistry, University of
Helsinki, FI-00014 Helsinki, Finland; Ophthalmology,
University of Helsinki and Helsinki University Hospital, FI-
00290 Helsinki, Finland; Email: riku.o.paananen@
helsinki.fi
Filip S. Ekholm − Department of Chemistry, University of
Helsinki, FI-00014 Helsinki, Finland; orcid.org/0000-
0002-4461-2215; Email: filip.ekholm@helsinki.fi
(8) King-Smith, P. E.; Reuter, K. S.; Braun, R. J.; Nichols, J. J.;
Nichols, K. K. Tear Film Breakup and Structure Studied by
Simultaneous Video Recording of Fluorescence and Tear Film
Lipid Layer Images. Invest. Ophthalmol. Visual Sci. 2013, 54 (7), 4900.
(9) Dursch, T. J.; Li, W.; Taraz, B.; Lin, M. C.; Radke, C. J. Tear-
Film Evaporation Rate from Simultaneous Ocular-Surface Temper-
ature and Tear-Breakup Area. Opt Vis Sci. 2018, 95 (1), 5−12.
(10) Butovich, I. A. Tear Film Lipids. Exp. Eye Res. 2013, 117, 4−27.
(11) Brown, S. H. J.; Kunnen, C. M. E.; Duchoslav, E.; Dolla, N. K.;
Kelso, M. J.; Papas, E. B.; Lazon de la Jara, P.; Willcox, M. D. P.;
Blanksby, S. J.; Mitchell, T. W. A Comparison of Patient Matched
Meibum and Tear Lipidomes. Invest. Ophthalmol. Visual Sci. 2013, 54
(12), 7417−7424.
(12) Chen, J.; Green, K. B.; Nichols, K. K. Quantitative Profiling of
Major Neutral Lipid Classes in Human Meibum by Direct Infusion
Electrospray Ionization Mass Spectrometry. Invest. Ophthalmol. Visual
Sci. 2013, 54 (8), 5730−5753.
(13) Kunnen, C. M. E.; Brown, S. H. J.; de la Jara, P. L.; Holden, B.
A.; Blanksby, S. J.; Mitchell, T. W.; Papas, E. B. Influence of
Meibomian Gland Expression Methods on Human Lipid Analysis
Results. Ocul Surf. 2016, 14 (1), 49−55.
Authors
Tuomo Viitaja − Department of Chemistry, University of
Helsinki, FI-00014 Helsinki, Finland; Ophthalmology,
University of Helsinki and Helsinki University Hospital, FI-
00290 Helsinki, Finland
Jan-Erik Raitanen − Department of Chemistry, University of
Helsinki, FI-00014 Helsinki, Finland; orcid.org/0000-
0002-2818-7570
Jukka Moilanen − Ophthalmology, University of Helsinki and
Helsinki University Hospital, FI-00290 Helsinki, Finland
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02882
(14) Lam, S. M.; Tong, L.; Reux, B.; Duan, X.; Petznick, A.; Yong, S.
S.; Khee, C. B. S.; Lear, M. J.; Wenk, M. R.; Shui, G. Lipidomic
Analysis of Human Tear Fluid Reveals Structure-Specific Lipid
Alterations in Dry Eye Syndrome. J. Lipid Res. 2014, 55 (2), 299−
306.
(15) Lam, S. M.; Tong, L.; Yong, S. S.; Li, B.; Chaurasia, S. S.; Shui,
G.; Wenk, M. R. Meibum Lipid Composition in Asians with Dry Eye
Disease. PLoS One 2011, 6 (10), e24339.
Author Contributions
⊥These authors contributed equally.
Notes
The authors declare no competing financial interest.
(16) Chen, J.; Keirsey, J. K.; Green, K. B.; Nichols, K. K. Expression
Profiling of Nonpolar Lipids in Meibum From Patients With Dry Eye:
A Pilot Study. Invest. Ophthalmol. Visual Sci. 2017, 58 (4), 2266−
2274.
(17) Miyamoto, M.; Sassa, T.; Sawai, M.; Kihara, A. Lipid Polarity
Gradient Formed by ω-Hydroxy Lipids in Tear Film Prevents Dry
Eye Disease. eLife 2020, 9, e53582.
(18) Schuett, B. S.; Millar, T. J. An Investigation of the Likely Role
of (O-Acyl) ω-Hydroxy Fatty Acids in Meibomian Lipid Films Using
(O-Oleyl) ω-Hydroxy Palmitic Acid as a Model. Exp. Eye Res. 2013,
115, 57−64.
ACKNOWLEDGMENTS
■
Financial support from the Eye and Tissue Bank Foundation,
the Mary and Georg C. Ehrnrooth Foundation, the Ruth and
Nils-Erik Stenbäck Foundation, the Swedish Cultural Founda-
tion, the Evald and Hilda Nissi Foundation, the Biomedicum
Helsinki Foundation, and the Finnish Eye Foundation is
gratefully acknowledged. The authors further thank BSc.
student Tobias Svärd (University of Helsinki) for laboratory
assistance.
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