Lipidomics characterization of biosynthetic and remodeling pathways of cardiolipins in genetically and nutritionally manipulated yeast cells

Article abstract of DOI:10.1021/acschembio.6b00995

Cardioipins (CLs) are unique tetra-acylated phospholipids of mitochondria and define the bioenergetics and regulatory functions of these organelles. An unresolved paradox is the high uniformity of CL molecular species (tetra-linoleoyl-CL) in the heart, liver, and skeletal muscles-in contrast to their high diversification in the brain. Here, we combined liquid chromatography-mass-spectrometry-based phospholipidomics with genetic and nutritional manipulations to explore CLs' biosynthetic vs postsynthetic remodeling processes in S. cerevisiae yeast cells. By applying the differential phospholipidomics analysis, we evaluated the contribution of Cld1 (CL-specific phospholipase A) and Taz1 (acyl-transferase) as the major regulatory mechanisms of the remodeling process. We further established that nutritional "pressure" by high levels of free fatty acids triggered a massive synthesis of homoacylated molecular species in all classes of phospholipids, resulting in the preponderance of the respective homoacylated CLs. We found that changes in molecular speciation of CLs induced by exogenous C18-fatty acids (C18:1 and C18:2) in wild-type (wt) cells did not occur in any of the remodeling mutant cells, including cld1Δ, taz1Δ, and cld1Δtaz1Δ. Interestingly, molecular speciation of CLs in wt and double mutant cells cld1Δtaz1Δ was markedly different. Given that the bioenergetics functions are preserved in the double mutant, this suggests that the accumulated MLCL-rather than the changed CL speciation-are the likely major contributors to the mitochondrial dysfunction in taz1Δ mutant cells (also characteristic of Barth syndrome). Biochemical studies of Cld1 specificity and computer modeling confirmed the hydrolytic selectivity of the enzyme toward C16-CL substrates and the preservation of C18:1-containing CL species.

Full text of DOI:10.1021/acschembio.6b00995

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Article
Lipidomics Characterization of Biosynthetic and Remodeling Pathways
of Cardiolipins in Genetically and Nutritionally Manipulated Yeast Cells.
Yulia Y Tyurina, Wenjia Lou, Feng Qu, Vladimir A Tyurin, Dariush Mohamedyani, Jenney Liu, Maik
Huttemann, Michael A. Frasso, Peter Wipf, H. Bayir, Miriam L. Greenberg, and Valerian E. Kagan
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00995 • Publication Date (Web): 05 Dec 2016
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Lipidomics Characterization of Biosynthetic and Remodeling Pathways of Cardiolipins in
Genetically and Nutritionally Manipulated Yeast Cells.
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Yulia Y. Tyurina#*1, Wenjia Lou*2, Feng Qu*1, Vladimir A Tyurin1, Dariush Mohammadyani1,7,
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Jenney Liu3, Maik Hüttemann3, Michael A. Frasso3, Peter Wipf4, H. Bayir#1,5
Miriam. L. Greenberg#2, Valerian E. Kagan#1,4,6
Departments of 1Environmental Health and Center for Free Radical and Antioxidant Health,
4Chemistry, 5Critical Care Medicine, 6Pharmacology and Chemical Biology,
University of Pittsburgh
,
2Department of Biological Sciences, Wayne State University
3Center for Molecular Medicine and Genetics, Wayne State University
7Thomas C. Jenkins Department of Biophysics, Johns Hopkins University.
* equal contribution
# Corresponding authors:
Tyurina YY (yyt1@pitt.edu)
Greenberg ML (mgreenberg@wayne.edu)
Bayir H (bayihx@ccm.upmc.edu)
Kagan VE (kagan@pitt.edu)
Key words: yeast, cardiolipin, Cld1, taffazin, Barth syndrome, cardiolipin remodeling
Abbreviations: cardiolipin, CL; monolysoꢀcardiolipin, MLCL; phosphatidylcholine, PC;
phosphatidylethanolamine, PE; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic
acid, PA; fatty acid, FA; polyunsaturated fatty acids, PUFA; myristic acid, C14:0; palmitic acid,
16:0; palmitoleic acid, 16:1; oleic acid, C18:1; linoleic acid, C18:2; arachidonic acid, C20:4;
docosahexaenoic acid, C22:6; oxidative phosphorylation, OxPhos; oxygen consumption rate,
ORC; phospholipase A2, PLA2; tafazzin, TAZ.
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ABSTRACT
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Cardioipins (CLs) are unique tetraꢀacylated phospholipids of mitochondria and define the
bioenergetics and regulatory functions of these organelles. An unresolved paradox is the high
uniformity of CLs molecular species (tetraꢀlinoleoylꢀCL) in the heart, liver and skeletal muscles –
in contrast to their high diversification in the brain. Here, we combined liquid chromatographyꢀ
massꢀspectrometry based phospholipidomics with genetic and nutritional manipulations to
explore CLs biosynthetic vs postꢀsynthetic remodeling processes in S. cerevisiae yeast cells. By
applying the differential phospholipidomics analysis, we evaluated the contribution of Cld1 (CL–
specific phospholipase A) and Taz1 (acylꢀtransferase) as the major regulatory mechanisms of
the remodeling process. We further established that nutritional “pressure” by high levels of free
fatty acids triggered a massive synthesis of homoꢀacylated molecular species in all classes of
phospholipids, resulting in the preponderance of the respective homoꢀacylated CLs. We found
that changes in molecular speciation of CLs induced by exogenous C18ꢀfatty acids (C18:1 and
C18:2) in wt cells did not occur in any of the remodeling mutant cells, including cld1∆, taz1∆,
and cld1∆taz1∆. Interestingly, molecular speciation of CLs in wt and double mutant cells
cld1∆taz1∆ was markedly different. Given that the bioenergetics functions are preserved in the
double mutant, this suggests that the accumulated MLCL – rather than the changed CL
speciation – are the likely major contributors to the mitochondrial dysfunction in taz1∆ mutant
cells (also characteristic of Barth syndrome). Biochemical studies of Cld1 specificity and
computer modeling confirmed the hydrolytic selectivity of the enzyme towards C16ꢀCL
substrates and the preservation of C18:1ꢀcontaining CL species.
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INTRODUCTION
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The wellꢀestablished tissue specificity of cardiolipins (CL)s1 has led to the paradigm that the
molecular speciation of CLs – achieved through the process of remodeling of the nascent
species ꢀ is defined by the unique mitochondrial demands of a particular tissue. This commonly
accepted paradigm has been recently challenged by a clear demonstration that unremodeled
CLs are functionally indistinguishable from remodeled CLs for yeast mitochondrial morphology
and major bioenergetic functions, such as in oxidative phosphorylation (OxPhos)2, where it is a
required structural component of the individual OxPhos complexes as well as supercomplexes3.
Recent findings have demonstrated that respiratory supercomplexes are destabilized by
disturbed CL remodeling in heart mitochondria from tafazzin knockdown mice, resulting in
disruption of interactions between the electron transporting complexes and the fatty acid (FA)
oxidation enzymes4.
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The two major reactions in the remodeling process are a phospholipaseꢀdriven production of
monolysoꢀCL (MLCL) intermediates followed by an acyltransferase (tafazzin) dependent
“rebuilding” of the molecule that reacylates MLCL via the acylꢀCoAꢀindependent transfer of an
acyl group from a donor phospholipid 2, 5
.
Recent developments, elucidating the role of mitochondria as a platform coordinating many
metabolic processes as well as life and death decisions, imply that essential functions not
associated with bioenergetics may dictate the necessity for the highly diversified molecular
speciation of CLs in tissues with signaling functions (e.g., in the brain). Not only has it been
shown that mitochondria have functions other than bioenergetics, but CL in particular has extraꢀ
bioenergetic functions. The first demonstration of a role for CL in cell viability apart from
bioenergetics was the demonstration that the crd1∆ (CL synthase) mutant has a temperatureꢀ
sensitive growth defect, unlike yeast mitochondrial bioenergetics mutants which do not exhibit
temperature sensitivity 6. Perturbation of CL synthesis has been shown to affect MAPK signaling
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and vacuolar function in yeast . This is in sharp contrast to such tissues as cardiac and
skeletal muscles in which mitochondrial energy production dictates the dominance of homoꢀ
acylated tetraoleoylꢀCL ((C18:1)4ꢀCL) or tetralinoleoylꢀCL ((C18:2)4ꢀCL) species over a rich
assortment of heteroꢀacylated CLs that may be required for signaling purposes9.
CL signaling may be realized through the production of oxygenated lipid mediators9b, thus
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requiring CL species containing polyunsaturated fatty acids (PUFA). Therefore, the plasticity
that is fineꢀtuned by adaptive changes in the microenvironment – rather than a rigid constitutive
program – is likely to define the functions and mechanisms of CL remodeling. The machineries
realizing these adaptive responses may include the availability of polyunsaturated fatty acids as
well as enzymatic mechanisms involved in CL remodeling – including phospholipases A and
acylꢀtransferases10.
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Yeast cells may be ideal organisms for the exploration of these pathways because they: i) can
be grown on media completely devoid of PUFA or supplemented with PUFA, ii) rely on only one
CLꢀspecific phospholipase A, Cld1, iii) utilize Taz1 as the major tool for reacylation of MLCL,
and iv) employ only one – Cld1/Taz1ꢀdependent – pathway for CL remodeling. With this in mind,
in the current work we employed detailed phospholipidomics analysis to explore the effects of
PUFA supplementation as well as cld1∆ and taz1∆ mutants on the molecular speciation of CLs
in S. cerevisiae. Herein, we report the results of differential phospholipidomics studies of S.
cerevisiae that provide new insights into CL remodeling and biosynthesis. Our findings identified
the roles of Cld1 and Taz1 as the major regulatory mechanisms, which are governed by the
availability of PUFA esterified into phospholipids that act as donors for the Taz1ꢀcatalyzed
reaction.
RESULTS
Exogenous FAs define the molecular speciation of CLs in wt and mutant S. cerevisiae cells. In
wt S. cerevisiae cells grown without FA supplementation, there is an assortment of CL
molecular species with different FA residues ranging from C10 to C18. Among those, only
singlyꢀunsaturated CL species were present whereas PUFA were not detected (Figure 1A,
Supporting Information Figure S1). To gain insight into the mechanisms of CL remodeling and
biosynthesis, we chose to expose yeast cells to FAs with different chain lengths as well as
unsaturation. In particular, we employed oleic (18:1), linoleic (18:2), arachidonic (20:4) and
docosahexaenoic (22:6) acids as components of the growth medium and established their
effects on the phospholipidomes of S. cerevisiae cells.
Supplementation of the growth medium with either 18:1 or 18:2 FAs caused dramatic changes
in CLs composition (Figure 1B, Supporting Information Figures S2 and S3). Only CL molecular
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species containing from one to four 18:1 or C18:2 were detectable, while essentially all other
molecular species of CLs with shorter chains and devoid of C18ꢀcontaining species were not
found (Figure 1). Notably, a huge preponderance of homoꢀacylated – (18:1)4ꢀCLꢀ and (18:2)4ꢀCL
species – was observed (Figure 1B). The second largest cluster of CLs included heteroꢀ
acylated ꢀ triꢀC18:1 and triꢀC18:2ꢀspecies, respectively (Figure 1). These robust effects of
exogenously added C18 FAs in wt cells were much less pronounced when C20:4 was used
(Figure 1). In fact, only singlyꢀ and doublyꢀ 20:4ꢀacylated CL species were detectable; no triꢀ or
tetraꢀ20:4ꢀCL species were observed (Figure 1B, Supporting Information Figure S4). Upon
treatment with 22:6, CLs speciation remained unchanged vs untreated wt cells (Figure 1B);
22:6ꢀcontaning CL species were not detectable. Overall, these data indicate that the remodeling
and biosynthetic pathways for CLs are very specific and favor the synthesis of C18ꢀcontaining
molecular species. The balance of natural processes of CL hydrolysis and reꢀacylation yields
only very low concentrations of MLCLs – as intermediates of these reactions. Supplementation
with FAs did not significantly affect the MLCL content in wt cells (Figure 2).
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CL remodeling from MLCL in yeast cells occurs via a single metabolic reaction catalyzed by
Taz1 11:
PLꢀ2FA + MLCLꢀ3FA ꢀ LPLꢀ1FA + CLꢀ4FA
where PLꢀ2FA is a donor diꢀacylated phospholipid, MLCLꢀ3FA is an acceptor MLCL, LPL is
lysoꢀphospholipid formed, and CLꢀ4FA is a remodeled CL. For CL reacylation, the enzyme
utilizes donor phospholipids, particularly PC and PE. Therefore, we also studied the effects of
C18:1 and C18:2 supplementations on the phospholipidome of these cells. We found no
changes in the total content of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in
supplemented cells. However, supplementation with 18:1 resulted in the accumulation of PE
and PC species containing 18:1 FA residues (Figure 3, Supporting Information Figure S5).
Similarly, addition to the medium of 18:2 was associated with higher levels of 18:2ꢀcontaining
PE and PC (Figure 3, Supporting Information Figure S5). The enrichment with C18 FA residues
was at the expense of the decreased contents of PE and PC species containing 16:0 and 16:1
(Figure 3). Further, we found that supplementation of cells with either 18:1 or 18:2 resulted in
the accumulation of homoꢀacylated PC and PE molecular species (containing either 18:1 or
18:2 in both snꢀ1 and snꢀ2 positions). Subsequently, we detected an increased content of 18:1ꢀ
lysoꢀPC(LPC)/lysoꢀPE(LPE) and 18:2ꢀLPC/LPE after supplementation of cells with these FAs
(Supporting Information Figure S6). This suggests that homoꢀacylated species of PC and PE
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were effectively utilized by Taz1 as preferred substrates for the reꢀacylation of MLCL. It is also
possible that these FAs were readily incorporated via de novo biosynthesis (presumably of
phosphatidic acid (PA)). Direct experimental assessment of the contribution of these alternative
pathways can be deduced from our lipidomics data obtained from wt and mutant cells
supplemented with FA, as shown below.
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Both Cld1 and Taz1 modify the cardiolipinome. Cld1 is a critical participant of the CL remodeling
process, as it catalyzes phospholipid hydrolysis with a significant selectivity towards 16:0 and
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16:1ꢀcontaining molecular species . This suggests that disruption of this process in the cld1
ꢀ
mutant should result in an altered molecular speciation of CLs, particularly with regards to
longerꢀchain (C16 and C18) species. However, according to a recently reported shotgun
lipidomics study, the lipid profile of cld1
ꢀ
was similar to that of wt, whereas taz1ꢀ had a
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reduction of CL and an accumulation of MLCL . To resolve this apparent conundrum, we
performed detailed LCꢀMS based characterization of cardiolipinomes in wt and mutant S.
cerevisiae cells. We found that the composition of CL from wt cells was different from that
detected in either cld1∆, taz1∆ or cld1∆taz1∆ mutants (Supporting Information Figure S1,
Tables S1 and S2). In addition, cld1∆ contained slightly (but significantly) higher levels of CLs
compared to wt cells (data not shown). In line with published data, the changes in the total
content of CL and MLCL found in taz1∆ mutants were no longer detectable in cld1∆taz1∆
mutants. Indeed, a dramatic accumulation of MLCL was detected in taz1∆ but not in cld1∆ or
cld1∆taz1∆ mutants (Figure 2). However, molecular speciation of CLs in wt cells vs doubleꢀ
mutant cells was markedly different. Similar results were obtained with wt cells and mutants
supplemented with OA or LA (Figure 2, Supplemental Figures 3 and 4). These data are
compatible with the role of Cld1 in “preparing” CLs for remodeling via hydrolysis/reacylation, and
suggest that elimination of the hydrolytic pathway by deletion of CLD1 would lead to
“conservation” of CLs. Consistent with this prediction, cld1∆ mutant cells were essentially
unable to produce remodeled tetraꢀ and triꢀ18:1 species of CLs (Figure 1B, Supporting
Information Table S2). Instead, their CLs were enriched with 16:0 and 16:1 containing molecular
species (Figure1B, Supporting Information Table S1).
Assuming that Cld1 is a participant in the only remodeling process in S. cerevisiae cells, one
can distinguish between the contribution of de novo biosynthesis vs remodeling processes in
the overall diversification of CLs. In wt cells, both the biosynthesis de novo and remodeling
reactions contribute to the overall diversity and content of CLs. Deletion of CLD1 would
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eliminate the remodeling process and reveal the nascent CL species generated via its de novo
biosynthesis. Thus, two categories of CLs can be determined by comparison of LC/MS spectra
of CLs from wt and cld1∆ cells (Figure 4), including: i) those produced by the biosynthetic
pathway, revealed in the cld1∆ mutant, ii) those generated as products (increased) or utilized as
substrates by the remodeling pathway (decreased). These latter respective categories of CLs
can be identified in the LC/MS spectra of CLs in ±(wt minus cld1∆) by the differential analysis.
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By applying these differential (subtraction) protocols1 , we found significant differences in
molecular speciation of CLs in wt and mutant cells. In cld1∆ cells (lacking the remodeling
pathway), CLs formed in the reaction catalyzed by CL synthase were mainly represented by
molecular species containing 16:0, 16:1 and 18:1 FA residues (Figure 4, Supporting Information
Figure S7). This was supported by LC/MS analysis of the CL biosynthesis precursor, –
phosphatidylglycerol (PG). We found that PGs were predominantly represented by 16:1/18:1,
16:1/18:1 and 16:0/16:1 molecular species in wt cells (Figure 5). On the other hand, during the
remodeling, esterified 16:0 was substituted with the 16:1 fatty acid residue (Figure 5). These
data suggest that in nonꢀFAꢀsupplemented wt cells, Cld1 predominantly hydrolyzed 16:0ꢀ
containing CLs that were then remodeled with 16:1 by Taz1ꢀcatalyzed reꢀacylation.
We further applied the same approach to distinguish between the CL species formed through
either biosynthetic or remodeling pathways in wt cells supplemented with either 18:1 or 18:2.
This analysis revealed that the remodeling route was entirely accountable for the replacement of
fatty acid residues in CLs yielding predominantly homoꢀacylated tetraꢀ18:1 or tetraꢀ18:2
molecular species of CLs, respectively (Figure 1B, Figure 4B, C, Supporting Information Figure
S7). Based on these newly developed protocols for the assessment of biosynthetic vs
remodeling origins of the CL species, we compared the relative contributions of these two
pathways to all CLs in determining the pool of homoꢀacylated species. We found that
biosynthesis was the major (wt cells, 18:2ꢀtreated cells) or significant (C18:1ꢀtreated cells)
contributor to the entire pool of CLs. Interestingly, the remodeling process was, by far, the most
important contributor to the pool of homoꢀacylated CLs. Specifically, we found that
CL(18:1/18:1/18:1/18:1) was the major product (Figure 4B, wtꢀcld1∆), while
CL(16:1_18:1_16:1_18:1) and CL(16:0_18:1_18:1_18:1) were the major substrates of the
remodeling process (Figure 4B, ꢀ(wtꢀcld1∆)). Based on the cld1∆ spectra, we determined that
1
Please note that the specific feature of the software utilized for the subtraction disallows plotting of the
negative spectra; therefore ꢀ(WTꢀ cld1∆) plots were used as explained in detail in Materials and Methods.
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both major substrates of the remodeling originated from CL biosynthesis. For LAꢀsupplemented
cells, we found that CL(18:2/18:2/18:2/18:2) was the major product (Figure 4C, wtꢀcld1∆), while
CL(16:0_18:2_16:0_18:2) and CL(16:0_18:2_18:2_18:2) were the major substrates of the
remodeling process (Figure 4C, ꢀ(wtꢀcld1∆)). Similar to OAꢀ supplementation, both major
remodeling substrates represented the products of CL biosynthesis. Further, the differential
analysis demonstrated that after OAꢀ or LAꢀ supplementation, Cld1 had preferentially
hydrolyzed C16:0ꢀ and C16:1ꢀfatty acid residues over C18:1ꢀ and C18:2ꢀcontaining CL species.
As a result, the remodeling process tended to convert heteroꢀacylated CL to homoꢀacylated
CLs, provided C18:1 or C18:2 were available.
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As all major phospholipids can serve as acyl sources for Taz1ꢀcatalyzed remodeling, we further
looked at the content of several phospholipids as well as their monolysoꢀforms in wt and taz1∆
mutant cells. Included in our phospholipidomics analyses were several major classes of
phospholipids: i) a biosynthetic precursor of CL, phosphatidylglycerol (PG); ii) two major
phospholipids present in both mitochondria and extraꢀmitochondrial compartments, PE and PC,
and iii) an extraꢀmitochondrial phospholipid, phosphatidylserine (PS). We found that the effects
of TAZ1 deletion were CL specific. Other phospholipids such as PC, PG, PS were not sensitive
to TAZ1 deletion.
Exogenous FAs do not affect molecular speciation of CL in remodeling of mutant S. cerevisiae
cells. While Cld1 and Taz1 represent the two major components of the machinery involved in
the CL remodeling process, the immediate motive force(s) triggering the process remain
unknown. As exogenously added C18 FAs caused efficient remodeling of CL to generate mostly
homoꢀacylated tetraꢀ18:1 and tetraꢀ18:2 molecular species in wt cells, we further explored the
CL composition in mutant cells. The dramatic changes in molecular speciation of CLs induced
by exogenous C18ꢀFAs in wt cells were essentially not found in any of the remodeling mutant
cells, including cld1∆, taz1∆, and cld1∆taz1∆ (Figure 1).
Because Cld1 is specific for the hydrolysis and remodeling of CLs but Taz1 can participate in
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remodeling of other phospholipids , we were interested in assessing the effects of deletion of
CLD1 as well as TAZ1 on the phospholipidomes of the mutant cells grown in the presence of
18:1 and 18:2 FAs. Our findings clearly demonstrate that deletion of CLD1 did not cause any
changes in PE and PC molecular species in either 18:1ꢀ or 18:2ꢀsupplemented cells. The
absence of Taz1 also did not substantially affect the content or molecular speciation of PC and
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PE (except for the accumulation of 16:0/16:1 and 16:1/16:1 PC molecular species but in cells
supplemented with C18:1).
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Specificity of Cld1 for different molecular species of CLs. Because of the apparent preference of
CL remodeling triggered by exogenous fatty acids for C18 species, we further investigated the
specificity of Cld1 in the hydrolysis of different molecular species of CLs. We used commercially
available homoꢀacylated CLs ꢀ (14:0)4ꢀCL, (18:1)4ꢀCL, (18:2)4ꢀCL and also three chemically
synthesized CLs ꢀ (16:0)4ꢀCL, (18:0)4ꢀCL, and (16:1)(16:0)3ꢀCL. The multistep preparations
followed a modular approach that allowed the incorporation of each glycerol unit sequentially.
Selectively protected glycerol building blocks were coupled to the desired fatty acid in the
presence of 1ꢀethylꢀ3ꢀ(3ꢀdimethylꢀaminopropyl)carbodiimide (EDCI) and N,Nꢀdimethylꢀ4ꢀ
aminopyridine (DMAP). Mild acidic deprotection using a resinꢀbound sulfonic acid gave the
desired 1,2ꢀdiacylglycerols. Following a sequence of phosphoramidite coupling/oxidation,
selective deprotection and a second phosphoramidite coupling/oxidation, the fully protected CL
precursor could be obtained. Successive removal of the phosphate and secondary alcohol
protecting groups, followed by treatment with ammonium hydroxide gave the CL diammonium
salts that were used for biological analysis.
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We found that (14:0)4ꢀCL, (18:1)4ꢀCL, (18:2)4ꢀCL were very poor substrates for CLD1 (Figure 6).
Both (16:0)4ꢀCL and (16:1)(16:0)3ꢀCL and also (18:0)4ꢀCL were readily hydrolyzed by Cld1
(Figure 6). To verify the selectivity of Cld1 for toward CL containing palmitic acid we performed
experiments using several types of mixtures of different CLs. In particular, we were interested in
testing Cld1 substrate specificity towards different CL species most commonly present in S.
cerevisae cells, as well as longꢀchain polyunsaturated CL species “unusual” for these cells. To
this end, we prepared two types of liposomes, diꢀoleoylꢀPC liposomes containing either CLs
isolated from cld1∆ yeast mutant (to obtain the most diversified set of yeast CLs in conditions of
suppressed remodeling process) or long chain polyunsaturated CLs isolated from fish heart
(Northern Red Snapper, Lutjanus campechanus) (Figure 7). These experiments provided
interesting details regarding the specificity of Cld1 towards different molecular species of CLs as
substrates. We found that hydrolysis of yeast CLs by Cld1 resulted in the release of
predominantly palmitic acid (16:0) and the formation of four major MLCL species such as
12:0_14:0_18:1; 14:0_14:0__18:1; 16:0_16:1_18:1 and 18:1/18:1/18:1. We did not observe
significant accumulation of MLCL and free fatty acids or decreased CL content when liposomes
containing fish heart CLs (with highly diversified CLs containing C22:5 and C22:6 fatty acid
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residues) were treated with Cld1. Thus, Cld1 selectively hydrolyzed CL species containing
palmitic acid.
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Computational studies of the interactions of homoꢀacylatedꢀCL with Cld1. To get insights into
specifics of Cld1 hydrolysis of CL and its molecular species, a homology model of fullꢀlength
structure of the protein was generated using Hsad, a steroidꢀdegrading hydrolase, from
Mycobacterium tuberculosis as a template (Figue 8 A)13. This choice was based on the
sequence analysis using the Protein BLAST tool14 and resulted in five templates with the
highest levels of identities to Cld1. Accordingly, five homology models of Cld1 were built using
the IꢀTASSER15 webserver. The model based on Hsad, a steroidꢀdegrading hydrolase from
Mycobacterium Tuberculosis (PDB ID: 2VF213), demonstrated the greatest Cꢀscore (Cꢀscore
was equal to ꢀ1.78). Therefore, it was selected for molecular docking modeling. The known
lipase motif (228AHSLG232) and acyl transferase motif (424HHLYLD429) are shown in Figure 8 BꢀC,
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respectively. According to Baile et al. , S230, H424 and D329 are the catalytic triad of Cld1
(Figure 8 D). Intriguingly, the overlay of cavity and cartoon representation of the model structure
revealed a channel toward the catalytic site. Subsequently, molecular docking method was
utilized to investigate the interactions of three homoꢀacylatedꢀCL species ꢀ (14:0)4ꢀCL, (16:0)4ꢀ
CL and (18:2)4ꢀCL. Notably, a palmitoyl residue (16:0) fit perfectly in this cavity (Figure 9).
However, neither chains of tetraꢀmyristoyl CL, nor chains of tetraꢀlinoleoyl CL penetrated into
the active site channel. This indicates that the channel can introduce a CL acyl chain to the
active site. Moreover, the length of the channel may function as a “ruler” defining the specificity
of Cld1 towards particular molecular species of CLs – in reasonable agreement with the
experimentally tested homoꢀ and heteroꢀacylated CLs.
Effects of C18:1, C18:2 and C20:4 FAs supplementation on mitochondrial bioenergetic function
in S. cerevisiae cells.
As CL is an essential structural component of the OxPhos machinery, we tested one possible
effect of fatty acid supplementation on intact cell respiration of yeast cells. Interestingly, yeast
cells analyzed in stationary phase in YPD media showed 45% and 21% increased respiration
rates when the media was supplemented with linoleic acid and arachidonic acid, respectively,
but not with oleic acid, where respiration was decreased by ꢀ26% compared to control (Figure
10). These data suggest that CL composition plays an important role for mitochondrial function
at the level of the oxidative phosphorylation system, and that linoleic acid supplementation
produces highest respiration rates.
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DISCUSSION
Predominantly localized in mitochondria, CLs interact with numerous mitochondrial proteins3
thus defining their role and significance in mitochondrial structural organization and
metabolic/signaling functions. Similar to other phospholipids, CLs are subject to continuous
turnover, resulting in changes in their molecular species that may be associated with their
modified interactions with proteins. The molecular mechanisms and the significance of these
changes – also called remodeling – in normal physiology and disease conditions are the subject
of intensive investigation16. We discovered that nutritional “pressure” by high levels of free FAs
in the growth medium triggered massive synthesis of unusual homoꢀacylated diꢀoleoylꢀ and diꢀ
linoleoylꢀ molecular species in all major classes of phospholipids, including direct CL precursors
as well as donors of acylꢀgroups for the remodeling reactions. Not surprisingly, this resulted in
the preponderant accumulation of the respective homoꢀacylated CL species.
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By employing detailed phospholipidomics analysis, this work established that the availability of
specific FA precursors is the major factor defining the diversity and molecular speciation of CLs
in S. cerevisiae. Supplementation with C18 FAs resulted in the predominant accumulation of
homoꢀtetraꢀacylated 18:1 and 18:2 species of CL. Based on the engagement of only one
biosynthetic and one remodeling pathway for CLs in S. cerevisiae, the specific contributions of
these respective mechanisms to the overall molecular diversification/speciation of CLs in
different growth conditions as well as in mutant forms can be established. For example, we
established that the remodeling process is the major contributor to the pool of homoꢀacylated
(C18:1)4ꢀCL and (C18:2)4ꢀCL in cells supplemented with these FAs.
The CL hydrolysis is a major part of the remodeling pathway. To convert nascent CL, containing
short saturated FA, to homoꢀacylated CL with long chain polyunsaturated FAs, CL first has to
undergo deacylation. In yeast, Cld1 fulfills this function and has been identified as a CLꢀspecific
hydrolase enzyme. Modeling data revealed the presence of a channel adjacent to the active site
of Cld1, which may introduce a FA of CL to the active site to be hydrolyzed. In line with LC/MS
data demonstrating preferable hydrolysis of 16:0 FA by Cld1, and modeling results revealing
most likely interactions of 16:0 FA with the channel, it is tempting to speculate that the channel
acts as a “molecular ruler” defining the specificity of Cld1 to particular FA residues in CLs.
By appropriately designing genetic manipulations (e.g., creating and producing appropriate
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deletion mutants such as cld1∆ and targeted transfection with mammalian remodeling genes
such as iPLA2γ or iPLA2D), yeast cells supplemented with desired PUFA can be used as
effective models in studies of mammalian CL metabolism. Similarly, the taz1∆ mutant
transfected with acyltransferases alcat1 or mlcat1 may lead to the elucidation of their specific
contributions to CL metabolic processes. In all these cases, the approach based on differential
(subtractive) lipidomics analysis may be particularly powerful in examination of remodeling and
biosynthesis.
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Supplementation with exogenous C18 FAs stimulated CL remodeling. As FAs can be
integrated into yeast CLs only via MLCL/Taz1ꢀdriven reactions, the acylꢀCoAꢀdependent
pathways are not utilized to remodel CL. However, it has been reported that acylꢀCoAs are
essential for reacylation of other phospholipids17. Thus, detailed lipidomic analyses of other
phospholipids in addition to CL are essential for comprehensive analysis of CL remodeling
pathways.
It has been well established that taz1
Δ cells are characterized by lower levels of CLs and
markedly increased content of MLCL relative to wt cells 12. Our results are in full agreement with
these earlier studies. In terms of molecular speciation of CLs, the same altered features of CLs
molecular speciation were also characteristic of taz1Δ mutant and double mutant cld1∆taz1∆
cells, suggesting that the selectivity of Cld1 for C16ꢀcontaining CLs is the major factor defining
the overall molecular speciation and remodeling of CLs in S. cerevisiae. This is clearly
illustrated by the analysis of MLCL molecular species that accumulate in taz1
16:1_16:1_16:0, 16:1_16:0_18:1, and 16:1_18:1_18:1 individual species dominated. Only
minimal amounts of MLCLs were detected in cld1 and cld1∆taz1∆ mutant cells, reconfirming
Δ cells, in which
Δ
that the Taz1ꢀcatalyzed reaction was the major contributor to the reꢀacylation/remodeling of CLs
in S. cerevisiae.
A recent study demonstrated that simultaneous deletion of cld1∆ and taz1∆ provided protection
against mitochondrial injury triggered by single deletion of taz1∆ 12. However, molecular
speciation of CLs in wt and double mutant cells cld1∆taz1∆ was markedly different. This
suggests that the accumulated MLCL – rather than the changed CL speciation – are the major
contributors to mitochondrial dysfunction. Another recent study demonstrated that unremodeled
CLs are functionally indistinguishable from remodeled CLs for yeast major bioenergetic
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functions2. In contrast, we found that mitochondrial respiration rates were altered in the
presence of different FA. Surprisingly, monounsaturated oleic acid had a small inhibitory effect
on intact cell respiration whereas the PUFAs ꢀ linoleic acid and arachidonic acid ꢀ significantly
increased mitochondrial respiration. This may give yeast cells a survival advantage by shifting
metabolism from glycolysis to the much more efficient OxPhos process under nonꢀproliferating
conditions. Interestingly, CL with four linoleic acid chains is the primary CL species in some
mammalian tissues including cow heart18, a tissue that fully depends on aerobic energy
production, suggesting that linoleic acid is the evolutionarily preferred CL side chain to support
optimal high levels of oxidative phosphorylation. It should be noted that the effect of the studied
FAs on mitochondrial function can be the result of several independent mechanisms. One
primary mechanism is likely through changes in CL FA composition (i.e., length and saturation
versus unsaturation state), which will affect the structure of the OxPhos complexes. Another
mechanism may be through cell signaling. For example, it is known that arachidonic acid, a
wellꢀstudied signaling molecule in higher organisms, also affects cell signaling in yeast, where it
leads to changes in the phosphorylation state of many proteins and in particular protein kinase
C (PKC) signaling19. PKC has been shown to regulate mitochondrial respiration by acting on
OxPhos complexes both in mammals and yeast20, suggesting that cell signaling may contribute
to the observed changes in the presence of arachidonic acid.
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Interestingly, these general considerations can be applied to specific pathogenic pathways in
disease conditions such as, for example, the one likely operating in patients suffering from the
Xꢀlinked disease Barth syndrome (BTHS) leading to a rare childhood cardiomyopathy with cyclic
neutropenia16. Recent detailed studies have identified seven functional classes of BTHS
mutations with several distinct lossꢀofꢀfunction mechanisms leading to a partial or total loss of
catalytic activity21. It has also been demonstrated that accumulation of MLCL (decreased
CL/MLCL ratio) rather than deficiency of “correct” unsaturated CL species is the major reason of
mitochondrial dysfunction in yeast cells12. Based on our data on the effective remodeling of CLs
supplemented with PUFA, one may wonder whether the supplementation with C18:2 may be of
therapeutic value, particularly in cases with a partial loss of TAZ catalytic activity22.
It has been long believed that remodeling of PUFA phospholipids is driven, at least in part, by
their peroxidation with subsequent hydrolysis of oxidatively modified FA residues and reꢀ
acylation23. The results of this work clearly indicate that the remodeling process occurs even in
the absence of oxidizable PUFA. This suggests that different mechanisms, independent of
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peroxidation, may drive the CL recycling process yielding the molecular species of CL optimized
in response to specific cell/tissue demands. Our results are compatible with the dominant role of
Taz in reꢀacylation of Cld1ꢀhydrolyzed CLs and explain the high selectivity of the overall
remodeling process towards homoꢀacylated 18:2 species of CLs and the role of the taz∆
mutations in the pathogenesis of Barth syndrome. However, further detailed studies of the
specificity and the role of PLAs in the reꢀacylation of oxidized vs nonꢀoxidized polyunsaturated
CL species may be necessary to better understand these intriguing and important general
mechanisms of biological adaptation.
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METHODS
Yeast strains, media, and growth conditions — The Saccharomyces cerevisiae strains used in
this work are listed in Table 1. Single deletion mutants were obtained from the yeast knockꢀout
deletion collection (Invitrogen). Double mutants were obtained by tetrad dissection. Complex
media contained yeast extract (1%), peptone (2%), and glucose (2%) (YPD) or glycerol (3%)
and ethanol (1%) (YPGE). The effect of fatty acids was determined in YPD or YPGE medium
supplemented with 0.2% oleic, arachidonic, linoleic, or docosahexaenoic acid solubilized in the
media with tergitol (1%). Unsupplemented controls contained only tergitol. Synthetic complete
(SC) medium contained all the essential components of Difco yeast nitrogen base, 2% glucose,
0.2% ammonium sulfate, vitamins, adenine (20.25 mg/L), arginine (20 mg/L), histidine (20
mg/liter), leucine (60 mg/L), lysine (200 mg/liter), methionine (20 mg/L), threonine (300 mg/L),
tryptophan (20 mg/L), uracil (20 mg/L), and inositol (75 ꢁM). For solid medium, 2% agar was
added. For selection of cells carrying plasmids, appropriate amino acids were omitted from the
media. Yeast strains were grown at 30°C. E. coli strain DH5 was used for plasmid maintenance
and amplification. Bacteria were grown at 37°C in LB medium (0.5% yeast extract, 1% tryptone,
1% NaCl), supplemented with ampicillin (100 ꢁg/mL) for selection purposes. Solid medium
contained 1.5% agar. Growth in liquid cultures was monitored spectrophotometrically. Cells
o
were harvested and centrifuged at 4 C (4,300 rpm, 5 min). Supernatant was discarded and
glass beads (0.3g) were added to the centrifuge tubes. Samples were vortexed (5 X 1min
intervals) and frozen at ꢀ80 oC for lipidomics analysis.
Construction of plasmids and expression of His6ꢀtagged CLD1 in S. cerevisiae — To construct a
His6ꢀtagged CLD1 overexpression plasmid, a 1373ꢀbp sequence containing the entire open
reading frame without stop codon of CLD1 was amplified from yeast genomic DNA using an
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EcoRIꢀtagged
forward
primer
CLD1_EcoRI_F3
(5’ꢀ
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TGATTAATAAGAATTCAACACAATGTTCAAGTCAACTTTAAACTCꢀ3’) and an XbaIꢀtagged
reverse primer CLD1_XbaI_R3 (5’ꢀATTTTGAGATTCTAGATATTTTTTGCATTTCTTTCGꢀ3’).
The PCR products were purified using the Wizard SV gel and PCR cleanꢀup system (Promega).
The purified DNA fragments were ligated into pYES2/CT cut with EcoRI and XbaI downstream
of the GAL1 promoter. All the plasmids were amplified and extracted using standard protocols.
The plasmids were transformed into yeast strains using a oneꢀstep transformation protocol.
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Purification of His6ꢀtagged Cld1 — S. cerevisiae strain BY4741 was used to express the
recombinant His6ꢀtagged Cld1. Cells were grown at 30°C in synthetic complete raffinose
medium lacking uracil. Galactose (2%) was added to induce expression of the recombinant
protein. Cell extracts were prepared by disrupting cells with glass beads (0.5 mm diameter),
vortexing for 5 min intermittently in disruption buffer containing 50 mM TrisꢀCl (pH 8.0) and 300
mM NaCl. Protease inhibitor (cOmplete EDTAꢀfree, Roche) was added to the disruption buffer
before breaking the cells. His6ꢀtagged Cld1 was purified with Millipore® PureProteome nickel
magnetic beads following the manufacturer's instructions. Millipore® UFC503024 Amicon®
Ultraꢀ0.5 centrifugal filter concentrator (30 KD) was used to concentrate the protein and remove
imidazole. Protein concentration was determined with a BIOꢀRAD DC protein assay kit using
bovine serum albumin as the standard.
LC/MS assessment of phospholipids — Lipids were extracted from yeast using the Folch
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procedure
and lipid phosphorus was determined by a microꢀmethod 25. LC/MS of
phospholipids was performed in negative mode using a Dionex UltimateTM 3000 RSLCnano
system coupled online QꢀExactive hybrid quadrupoleꢀorbitrap mass spectrometer
(ThermoFisher Scientific, San Jose, CA). Total lipids were separated on a normal phase column
(Silica Luna 3 µm, 100A, 150x2 mm, (Phenomenex, Torrance CA)) with flow rate 0.2 mL/min
using gradient solvents containing 5 mM CH3COONH4 (A – nꢀhexane:2ꢀpropanol:water, 43:57:1
(v/v/v) and B ꢀ nꢀhexane:2ꢀpropanol:water, 43:57:10 (v/v/v). The gradient conditions (all linear)
were as follows: 0ꢀ23 min (10%B to 32%B); 23ꢀ32 min (32%B to 65%B); 32ꢀ35 min (65%B to
100%B) 35ꢀ62 min (hold at 100%B); 62ꢀ64 min (100%B to 10%B); 64ꢀ80 min (10%B). Flow rate
was maintained at 200 microliters/min except for the 35ꢀ62 min time frame where the flow rate
was increase to 225 microliters/min. MS analysis was performed in negative ion mode (profile)
at a resolution of 140,000 for the full MS scan and 17,500 for the MS2 scan in a dataꢀdependent
mode with appropriate inclusion and exclusion lists. The scan range for MS analysis was 400ꢀ
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1800 m/z with a maximum injection time of 128 ms using 1 microscan. A maximum injection
time of 500 ms was used for MS2 (high energy collisional dissociation (HCD)) analysis with
collision energy set to 24. An isolation window of 1.0 Da was set for the MS2 scans. Capillary
spray voltage was set at 3.5 kV, and capillary temperature was 320 oC. The Sꢀlens Rf level was
set to 60 with a sheath gas flow of 8. To quantitatively assess phospholipids, PEꢀ(17:0/17:0);
PCꢀ(17:0/17:0); CLꢀ(14:0/14:0/14:0/14:0); LPEꢀ(17:1/0:0); LPCꢀ(17:1/0:0) from Avanti Polar
Lipids have been used as internal standards. Reference standards PEꢀ(18:1/18:1); PCꢀ
(18:1/18:1); CLꢀ(18:2/18:2/18:2/18:2); LPEꢀ(18:0/0:0); LPCꢀ(18:0/0:0) were used for calibrations
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and were also from AvantiPolar Lipids. Both Internal and reference standards of MLCLs were
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prepared from CLꢀ(14:0/14:0/14:0/14:0) CLꢀ(18:2/18:2/18:2/18:2), respectively as described
.
Precursor ions [MꢀH]ꢀ were identified with mass accuracy less than 5ppm. Therefore,
phospholipids, including CL, were identified by accurate mass and MS2 fragmentation analysis.
The fragmentation pattern provided information on fatty acids but not on their position.
Assuming that snꢀ1 position in phospholipids is commonly occupied by saturated and
monounsaturated acids we used this information to identify PE and PC molecular species.
Clearly, this is a more difficult task for CL molecular species containing four acyl chains.
Therefore, we presented CL molecular species based on the information provided in the paper
by Liebisch et al.,27. Spectra subtraction was done by Background Subtraction Module in
Xcalibur (Thermo Fisher Scientific). By defining one file as background (subtractor) and another
file as foreground (minuend), the difference can be evaluated from the differential spectra.
Since negative spectra cannot be calculated and exported from Xcalibur, we performed
swapping of the subtractor and the minuend which afforded the calculations and presentation of
the negative spectra.
Respiration measurements. Oxygen consumption rate (ORC) of intact yeast cells was analyzed
via the polarographic method in a closed 500 ꢁL chamber equipped with a micro Clarkꢀtype
oxygen electrode (Oxygraph Plus System, Hansatech Instruments) at 30°C. Yeast cells were
grown in synthetic YPD media supplemented with oleic acid, linoleic acid, or arachidonic acid.
Cells were harvested during the stationary phase. Cells were mixed in fresh media using a final
protein concentration of 0.5 mg/mL following measurements of respiration. KCN (0.2 mM) was
added at the end of the experiment to inhibit cytochrome c oxidase to correct for cytochrome c
oxidaseꢀindependent oxygen utilization. Oxygen consumption was recorded on a computer and
analyzed with the Oxygraph plus software. Oxygen consumption rate (ORC) is defined as
consumed O2 (nmol)/min/total protein (mg).
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Synthesis of Cardiolipins.
General Experimental Conditions. All air and moisture sensitive reactions were carried out
under a nitrogen or argon atmosphere. All reactions carried out above room temperature were
performed using an oil bath set to the specified temperature and monitored with an external
thermometer. THF and diethyl ether were distilled from sodium/benzophenone ketyl.
Dichloromethane was distilled from CaH2. Diisopropylethylamine (DIPEA) and diisopropylamine
were distilled from and stored over KOH. Pyridine was filtered through activated basic alumina
and stored over freshly activated 4 Å molecular sieves for at least 24 h prior to use. Amberliteꢀ
IR120H resin was activated by stirring with 6 N HCl for 30 min, filtering, and rinsing once with
equal volumes of water and methanol. All other reagents were used as received. Concentrating
under reduced pressure refers to removing solvents by the use of a rotary evaporator connected
to a PIAB Lab Vac H40. Reactions were monitored by thin layer chromatography analysis (preꢀ
coated silica gel 60 F254 plates, 250 ꢁm layer thickness) and visualized with a 254 nm UV light
or by staining with a KMnO4 solution. Flash chromatography on SiO2 (Silicycle, 40ꢀ63 ꢁm) was
used for purification where indicated. All products were placed under high vacuum to remove
trace solvents. Melting points were determined using a Laboratory Devices MelꢀTemp II and are
uncorrected. Infrared spectra were obtained from neat solids or oils on a Perkin Elmer ATR IR
or Smiths Detection IdentifyIR FTꢀIR spectrometer. Highꢀresolution mass spectra were obtained
on a Thermo Scientific Exactive Orbitrap mass spectrometer using electrospray ionization. Lowꢀ
resolution mass spectra were obtained on an Advion Expression L compact mass spectrometer
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1
using electrospray ionization. H NMR spectra were obtained on a Bruker Avance at 300 MHz,
400 MHz, or 500 MHz in CDCl3, CD2Cl2, or CD3OD. Chemical shifts (δ) were reported in parts
per million with the residual solvent peak used as an internal standard δ 1H / 13C (solvent): 7.26 /
1
77.00 (CDCl3); 5.32/53.84 (CD2Cl2). H NMR spectra were obtained and are tabulated as
follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of
doublets, m = multiplet, bs = broad singlet), number of protons, and coupling constant(s). 13C
NMR spectra were recorded using a protonꢀdecoupled pulse sequence run at 100 MHz or 125
31
MHz and are tabulated by observed peaks. P NMR spectra were recorded using a complete
decoupled pulse sequence run at 162 MHz.
OH
C15H31
O
OH
O
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1-O-Palmitoylglycerol (1a) 28. General Procedure A. A solution of palmitic acid (5.5 g, 21.4
mmol, 1.0 eq.), 50% glycidol in CH2Cl2 (3.0 mL, 22.8 mmol, 1.06 eq.) and tributylamine (0.1 mL,
0.4 mmol, 0.02 eq.) was heated to 85 °C and stirred for 5 h. The crude product was
recrystallized from iꢀPr2O:Et2O (1:1) to yield 1ꢀOꢀpalmitoylglycerol (1a, 4.88 g, 14.8 mmol, 69%)
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9
1
as a colorless solid: Mp 71.9ꢀ72.5 °C; IR 3293, 2913, 2848, 1728 cmꢀ1; H NMR (CDCl3, 300
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MHz) δ 4.22 (dd, 1 H, J= 4.8, 11.7 Hz), 4.15 (dd, 1 H, J= 6.0, 11.4 Hz), 3.97ꢀ3.90 (m, 1 H), 3.70
(dd, 1 H, J= 4.2, 11.7 Hz), 3.60 (dd, 1 H, J = 5.7, 11.4 Hz), 2.35 (t, 2 H, J = 7.7 Hz), 1.66ꢀ1.58
(m, 2 H), 1.28ꢀ1.25 (m, 25 H), 0.85 (t, 3 H, J = 6.8 Hz); 13C NMR (CDCl3, 100 MHz) δ 174.4,
70.3, 65.2, 63.3, 34.1, 31.9, 29.7ꢀ29.6 (multiple peaks), 29.4, 29.3, 29.1, 24.9, 22.7, 14.1.
OH
C17H35
O
OH
O
1-O-Stearoylglycerol (1b) 28. According to General Procedure A but using stearic acid, 1ꢀOꢀ
1
stearoylglycerol (1b, 4.36 g, 12.2 mmol, 71%) was obtained as a colorless solid: H NMR
(CDCl3, 300 MHz) δ 4.21 (dd, 1 H, J = 4.8, 11.7 Hz ), 4.14 (dd, 1 H, J= 5.7, 11.4 Hz), 3.96ꢀ3.90
(m, 1 H), 3.70 (dd, 1 H, J = 3.9, 11.4 Hz), 3.60 (dd, 1 H, J = 6.0, 11.4 Hz), 2.71 (bs, 2 H), 2.35 (t,
2 H, J = 7.7 Hz), 1.65ꢀ1.60 (m, 2 H), 1.28ꢀ1.25 (m, 29 H), 0.88 (t, 3 H, J = 6.6 Hz).
C15H31
O
O
O
C15H31
ODMT
O
1,2-O-Dipalmitoyl-3-O-dimethoxyltriylglycerol (2a). General Procedure B. To a solution of
1a (4.57 g, 13.8 mmol, 1.0 eq.) in pyridine:CH2Cl2 (1:2, 35 mL) at 0 °C was added
dimethoxyltrityl chloride (DMTCl, 4.82 g, 14.1 mmol, 1.0 eq.) and dimethylaminopyridine
(DMAP, 0.177 g, 1.43 mmol, 0.1 eq.) The orange solution was stirred at this temperature for 15
min., then warmed to room temperature and stirred for 13 h. The orange reaction mixture was
quenched with water (30 mL) and extracted with CH2Cl2 (2x50 mL). The organic portion was
rinsed with water (40 mL), brine (40 mL), dried (MgSO4), and concentrated. The residue was
then concentrated from toluene (2x20 mL) and the resulting orange oil used without further
purification. A solution of 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl)carbodiimide (EDCI, 2.93 g, 15.3
mmol, 1.1 eq.), palmitic acid (3.91 g, 15.3 mmol, 1.1 eq.) and DMAP (0.851 g, 6.90 mmol, 0.5
eq.) in CH2Cl2 (60 mL) was allowed to stir at room temperature under nitrogen for 15 min. Then,
a solution of the previously obtained oil from the dimethoxyltrityl protection (8.77 g, 13.9 mmol
1.0 eq) in CH2Cl2 (30 mL) was added and stirring was continued for 4 h. The reaction mixture
was diluted with water (50 mL) and the organic layer was rinsed with water (50 mL), brine (50
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mL), dried (MgSO4), and concentrated. Purification by filtration through SiO2 (9:1
CH2Cl2:hexanes) gave 1,2ꢀOꢀdipalmitoylꢀ3ꢀOꢀdimethoxyltriylglycerol (2a, 7.94 g, 9.11 mmol,
66%) as a colorless solid. Pure 2a can also be obtained by repeated trituration with MeOH. Mp
1
64.5ꢀ66.0 °C; IR ν 2915, 2846, 1729 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 7.42 (d, 2.0 H, J = 2
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Hz), 7.40ꢀ7.20 (m, 7 H), 6.82 (d, 4 H, J = 8.7 Hz), 5.28ꢀ5.22 (m, 1 H), 4.34 (dd, 1 H, J = 3.6, 11.7
Hz), 4.25 (dd, 1 H, J = 6.6, 11.7 Hz), 3.79 (s, 6 H), 3.22 (d, 2 H, J = 5.1 Hz), 2.33 (t, 2 H, J = 7.5
Hz), 2.23 (t, 2 H, J = 7.5 Hz), 1.65ꢀ1.53 (m, 4 H), 1.28ꢀ1.25 (m, 48 H), 0.91 (t, 3 H, J = 6.6 Hz);
13C NMR (CDCl3, 125 MHz) δ 173.4, 173.0, 158.5, 144.5, 135.7, 130.0, 128.1, 127.8, 126.8,
113.1, 86.0, 70.5, 62.9, 62.0, 55.1 34.4, 34.1, 31.9, 29.7ꢀ29.1 (multiple peaks), 25.0, 24.8, 22.7,
14.1.
C17H35
O
O
O
C17H35
ODMT
O
1,2-O-Distearoyl-3-O-dimethoxyltriylglycerol (2b). According to General Procedure B but
using 1b and purification by trituration with MeOH gave 1,2ꢀOꢀdistearoylꢀ3ꢀOꢀ
dimethoxyltriylglycerol (2b, 6.68 g, 6.48 mmol, 53%) as a colorless solid: Mp 67ꢀ68.4 °C; IR ν
2915, 2848, 1730 cmꢀ1; 1H NMR (CDCl3, 300 MHz) δ 7.45 (d, 2 H, J = 7.2 Hz), 7.35ꢀ7.24 (m, 7
H), 6.85 (d, 4 H, J = 9.0 Hz), 5.29ꢀ5.28 (m, 1 H), 4.35 (dd, 1 H, J = 8.1, 11.7 Hz), 4.25 (dd, 1 H,
J = 6.6, 11.7 Hz), 3.82 (s, 6 H), 3.25 (d, 2 H, J = 3.3 Hz), 2.36 (t, 2 H, J = 7.5 Hz), 2.27 (t, 2 H, J
13
= 7.5 Hz), 1.69ꢀ1.56 (m, 4 H), 1.29 (m, 58 H), 0.91 (t, 3 H, J = 6.6 Hz); C NMR (CDCl3, 125
MHz) δ 173.4, 173.0, 158.5, 144.6, 135.8, 130.1, 128.1, 127.8, 126.8, 113.1, 113.0, 86.1, 70.5,
62.9, 62.1, 55.2, 34.4, 34.1, 31.9, 29.7ꢀ29.1 (multiple peaks), 25.0, 24.8, 22.7, 14.1.
C15H29
O
O
O
C15H31
ODMT
O
1-O-Palmitoyl-2-O-palmitoleoyl-3-O-dimethoxyltriylglycerol (2c). According to General
Procedure B but using 1a and purification by chromatography on SiO2 (95:5, hexanes:ethyl
acetate) gave 1ꢀOꢀpalmitoylꢀ2ꢀOꢀpalmitoleoylꢀ3ꢀOꢀdimethoxyltriylglycerol (2c, 0.619 g, 0.712
mmol, 65%) as a clear colorless oil: IR ν 2923, 2853, 2093, 1741, 1250 cmꢀ1; 1H NMR (CDCl3,
300 MHz) δ 7.43 (d, 2 H, J = 8.4 Hz), 7.31ꢀ7.25 (m, 7 H), 6.82 (d, 4 H, J = 8.7 Hz), 5.40ꢀ5.30 (m,
2 H), 5.29ꢀ5.22 (m, 1 H), 4.34 (dd, 1 H, J = 3.6, 11.7 Hz), 4.23 (dd, 1 H, J = 6.6, 12.0 Hz), 3.79
(s, 6 H), 3.23ꢀ3.21 (m, 2 H), 2.33 (t, 2 H, J = 7.5 Hz), 2.23 (t, 2 H, J = 7.7 Hz), 2.02ꢀ2.00 (m, 4
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H), 1.67ꢀ1.53 (m, 4 H), 1.29 (m, 40 H), 0.88 (t, 6 H, J = 6.8 Hz); 13C NMR (CDCl3, 125 MHz) δ
173.4, 173.0, 158.5, 144.6, 135.8, 135.7, 130.0, 129.7 128.1, 127.8, 126.8, 113.1, 86.1, 70.5,
62.9, 62.0, 55.2, 34.4, 34.1, 31.9, 31.8, 29.8ꢀ29.0 (multiple peaks), 27.2, 25.0, 24.9, 22.7, 22.6,
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14.1.
C15H31
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O
O
O
C15H31
OH
O
1,2-O-Dipalmitoylglycerol (3a) 29. General Procedure C. To a solution of 2a (7.94 g, 9.11
mmol, 1.0 eq.) in CHCl3:MeOH (1:1, 90 mL) was added Amberlite IRꢀ120H (4.0 g). The reaction
mixture was stirred for 2.5 h, then filtered through basic alumina (EtOAc). The resulting yellow
oil was precipitated from Et2O:hexanes (3:1) and filtered to give 1,2ꢀOꢀdipalmitoylglycerol (3a,
3.75 g, 5.50 mmol, 60%, purity by H NMR: 90%) as a colorless solid: 1H NMR (CDCl3, 400
1
MHz) δ 5.10ꢀ5.06 (m, 1 H), 4.32 (dd, 1 H, J = 4.8, 12.0 Hz), 4.24 (dd, 1 H, J = 6.0, 12.0 Hz),
3.74ꢀ3.73 (m, 2 H), 2.33 (apparent q, 4 H, J = 8 Hz), 1.65ꢀ1.58 (m, 4 H), 1.28ꢀ1.25 (m, 48 H),
0.88 (t, 3 H, J = 6.6 Hz).
C17H35
O
O
O
C17H35
OH
O
1,2-O-Distearoylglycerol (3b) 30. According to General Procedure C but using 2b, and
purification by trituration with Et2O:hexanes (3:1) gave 1,2ꢀOꢀdistearoylglycerol (3b, 2.08 g, 3.39
mmol, 72%) as a colorless solid: 1H NMR (CDCl3, 500 MHz) δ 5.10ꢀ5.06 (m, 1 H), 4.32 (dd, 1 H,
J1 = 4.5 Hz, J2 = 12 Hz), 4.24 (dd, 1 H, J1 = 5.5 Hz, J2 = 12 Hz), 3.79ꢀ3.72 (m, 2 H), 2.34 (m, 4
H), 1.99 (t, 1 H, J = 4.0 Hz), 1.64ꢀ1.54 (m, 4 H), 1.29ꢀ1.25 (m, 56 H), 0.88 (t, 3 H, J = 7.0 Hz).
C15H29
O
O
O
C15H31
OH
O
1-O-Palmitoyl-2-O-palmitoleoylglycerol (3c). According to General Procedure C but using 2c
and purification by chromatography on SiO2 (1:0 to 5:1, CH2Cl2:ethyl acetate) gave 1ꢀOꢀ
palmitoylꢀ2ꢀOꢀpalmitoleoylglycerol (3c, 64 mg, 0.113 mmol, 98%) as a light yellow oil: IR ν 3502,
2919, 2850, 2093, 1737, 1163 cmꢀ1; 1H NMR (CDCl3, 300 MHz) δ 5.40ꢀ5.30 (m, 2 H), 5.10ꢀ5.05
(m, 1 H), 4.32 (dd, 1 H, J = 4.8, 12.3 Hz), 4.24 (dd, 1 H, J = 5.4, 11.7 Hz), 3.73 (m, 2 H), 2.33
(apparent q, 4 H, J = 7.3 Hz), 2.02ꢀ2.00, (m, 4 H), 1.64ꢀ1.54 (m, 4 H), 1.30ꢀ1.27 (m, 40 H), 0.88
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(t, 6 H, J = 6.7 Hz); 13C NMR (CDCl3, 125 MHz) δ 173.8, 173.4, 130.0, 129.7 128.1, 72.1, 62.0,
61.6, 34.3, 34.1, 31.9, 29.7ꢀ29.0 (multiple peaks), 27.2, 25.0, 24.9, 22.7, 22.6, 14.1; HRMS
(ESI+) m/z calculated for C35H67O5 [M+H] 567.4983, found 567.4960.
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N,N-Diisopropylamidomethylchlorophosphite (4) . To a solution of PCl3 (3.2 mL, 36 mmol,
1.04 eq.) and pyridine (2.9 mL, 36 mmol, 1.04 eq.) in Et2O (120 mL) at ꢀ78 °C under argon was
added dry MeOH (1.4 mL, 35 mmol, 1.0 eq.) in Et2O (4 mL) over 30 min. The reaction mixture
was stirred for 3 h at room temperature. diluted with Et2O (40 mL) and cooled to 0 °C.
Diisopropylamine (9.6 mL, 67.9 mmol, 2.0 eq.) was added over 0.5 h and stirring was continued
for 22 h at room temperature. The salts were filtered off under argon, rinsed with Et2O (50 mL)
and the solvent was distilled off. The resulting yellow oil was purified by vacuum distillation to
give N,Nꢀdiisopropylamidomethylchlorophosphite (4, 3.73 g, 18.9 mmol, 56%) as a clear
colorless oil: Bp 88 °C (12 Torr); 1H NMR (CD2Cl2, 400 MHz) δ 3.74ꢀ3.64 (m, 2 H), 3.51 (d, 3 H,
J = 14.4 Hz), 1.20ꢀ1.16 (m, 12 H); 31P NMR (CD2Cl2, 162 MHz) δ 183.8.
O
O
BnO
30
4-((Benzyloxy)methyl)-2,2-dimethyl-1,3-dioxolane (5).
A slurry of 60% NaH in mineral oil
(360 mg, 9.00 mmol, 1.2 eq.) in THF (10 mL) at 0 °C under nitrogen was added to a solution of
2,2ꢀdimethylꢀ1,3ꢀdioxolaneꢀ4ꢀmethanol (1.0 mL, 7.8 mmol, 1.0 eq.) in THF (5 mL). The reaction
mixture was stirred for 30 min, then BnBr (0.98 mL, 1.4 mmol, 1.05 eq.) was added in a drop
wise fashion. The reaction mixture was warmed to room temperature and stirred for 2.5 h. The
reaction was quenched with water (8 mL), diluted with Et2O (20 mL), and the layers were
separated. The aqueous layer was extracted with Et2O (25 mL) and the combined organic
layers were rinsed with brine (10 mL), dried (MgSO4), and concentrated. Purification by
chromatography on SiO2 (95:5 to 9:1 to 85:15, hexanes:ethyl acetate) gave 4ꢀ
((benzyloxy)methyl)ꢀ2,2ꢀdimethylꢀ1,3ꢀdioxolane (5, 1.15 g, 5.17 mmol, 67%) as a clear colorless
oil: 1H NMR (CDCl3, 400 MHz) δ 7.35ꢀ7.29 (m, 5 H), 4.59, 4.56 (AB, 2 H, J = 12.0 Hz), 4.34ꢀ4.28
(m, 1 H), 4.06 (dd, 1 H, J = 6.4, 8.0 Hz), 3.75 (dd, 1 H, J = 6.4, 8.4 Hz), 3.56, 3.45 (ABd, 2 H, J
= 5.6, 9.6 Hz), 3.46 (dd, 1 H, J = 5.6, 9.6 Hz), 1.42 (s, 3 H), 1.37 (s, 3 H).
OTBS
BnO
OTBS
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1-O-Benzyl-2,3-O-bis(tert-butyldimethylsilyl)glycerol (6). A solution of 5 (1.15 g, 5.17 mmol,
1.0 eq.) in THF:H2O (2:1, 10 mL) was treated with 12 M HCl (2.1, 25 mmol, 4.9 eq.), and the
reaction mixture was stirred for 1 h at room temperature, poured into sat. NaHCO3 (30 mL) and
extracted with Et2O (3x 25 mL). The combined organic layers were rinsed brine (25 mL), dried
(MgSO4), and concentrated. The resulting yellow oil was dissolved in CH2Cl2 (8.7 mL) at 0 °C
under nitrogen and treated with Et3N (1.4 mL, 9.9 mmol, 2.3 eq.) and TBSOTf (2.2 mL, 9.5
mmol, 2.2 eq.). The cloudy white reaction mixture was then stirred for 3 h at room temperature,
quenched with H2O (8 mL) and diluted with CH2Cl2 (10 mL). The organic layer was rinsed with
brine (8 mL), dried (MgSO4), and concentrated. Purification by chromatography on SiO2 (99:1 to
95:5, hexanes:ethyl acetate) gave 1ꢀOꢀbenzylꢀ2,3ꢀOꢀbis(tertꢀbutyldimethylsilyl)glycerol (6, 1.72,
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4.31 mmol, 83% over 2 steps) as a clear colorless oil: H NMR (CDCl3, 500 MHz) δ 7.35ꢀ7.33
(m, 4 H), 7.28ꢀ7.26 (m, 1 H), 4.54 (s, 2 H), 3.89ꢀ3.84 (m, 1 H), 3.61 (dd, 1 H, J = 6.0, 10.0 Hz),
3.57ꢀ3.52 (m, 2 H), 3.42 (dd, 1 H, J = 5.5, 10.0 Hz), 0.87 (s, 18 H), 0.070 (d, 6 H, J = 1.5 Hz),
0.042 (s, 6 H).
OTBS
HO
OTBS
32
2,3-O-Bis(tert-butyldimethylsilyl)glycerol (7).
To a solution of 6 (1.62 g, 4.06 mmol, 1.0
eq.) in ethyl acetate (20 mL) was added 10% Pd/C (0.161 g, 0.152 mmol, 0.037 eq.). The
reaction mixture was submitted to hydrogenation on a Parr hydrogenator under H2 (5 atm) for 1
h, filtered through Celite (ethyl acetate) and concentrated to give 2,3ꢀOꢀbis(tertꢀ
butyldimethylsilyl)glycerol (7, 1.15 g, 3.59 mmol, 88%) as a clear colorless oil that was used
without further purification. An analytically pure sample was obtained by chromatography on
SiO2 (95:5 to 9:1, hexanes:ethyl acetate): 1H NMR (CDCl3, 300 MHz) δ 3.79ꢀ3.74 (m, 1 H), 3.69ꢀ
3.53 (m, 4 H), 2.10 (bs, 1 H), 0.90 (s, 18 H), 0.1ꢀ0.06 (m, 12 H).
C15H31
O
O
O
OTBS
OTBS
O
P
C15H31
O
O
O
OMe
3-(((2,3-Bis((tert-butyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propane-1,2-
diyl dipalmitate (8a).7 General Procedure D. To a solution of 3a (1.73 g, 2.89 mmol, 1.0 eq.)
and diisopropylethylamine (DIPEA, 0.53 mL, 3.2 mmol, 1.1 eq.) in CH2Cl2 (30 mL) under
nitrogen was added 7 (6.0 mL, 3.0 mmol, 1.1 eq.). The reaction mixture was stirred for 1 h, and
treated dropwise with a solution of 6 (0.921 g, 2.87 mmol, 0.99 eq.) and 4,5ꢀdicyanoimidazole
(0.700 g, 5.80 mmol, 2 eq.) in THF (4.9 mL). The solution was stirred for 1 h, cooled to 0 °C,
treated with Bu4NIO4 (1.46 g, 3.36 mmol, 1.2 eq.), and stirring was continued for 30 min. The
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mixture was diluted with water (15 mL) and the combined organic layers were rinsed with sat.
Na2S2O3 (15 mL), sat. NaHCO3 (15 mL), and brine (15 mL), dried (MgSO4), and concentrated.
The crude residue was filtered through a plug of SiO2 (1:1, hexanes:ethyl acetate) and
concentrated. Purification by chromatography on SiO2 (5:1 to 3:1, hexanes:ethyl acetate) gave
3ꢀ(((2,3ꢀbis((tertꢀbutyldimethylsilyl)oxy)propoxy)(methoxy)
phosphoryl)oxy)propaneꢀ1,2ꢀdiyl
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dipalmitate (8a, 1.42 g, 1.47 mmol, 51%) as a clear colorless oil: IR ν 2922, 2854, 1741, 1252,
1
835 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 5.25ꢀ5.19 (m, 1 H), 4.36ꢀ4.30 (m, 1 H), 4.19ꢀ4.09 (m, 4
H), 3.97ꢀ3.83 (m, 2 H), 3.76 (d, 3 H, J =11.1 Hz; diasteromers), 3.56ꢀ3.54 (m, 2 H), 2.34ꢀ2.27
(m, 4 H), 2.23 (t, 2 H, J = 7.5 Hz), 1.61ꢀ1.56 (m, 4 H), 1.28ꢀ1.25 (m, 48 H), 0.88ꢀ0.85 (m, 24 H),
0.08 (s, 3 H), 0.07 (s, 3 H), 0.05 (s, 6 H); 13C NMR (CDCl3, 125 MHz) δ 173.2, 172.7, 72.0 (d, J
= 7.5 Hz), 69.4 (d, J = 7.5 Hz), 69.1 (d, J = 6.5 Hz), 65.4ꢀ65.3 (multiple peaks), 64.0, 61.6, 55.4
(d, J = 6.3 Hz) 34.1, 34.0, 31.9, 31.6, 29.7ꢀ29.1 (multiple peaks), 25.8, 25.7, 24.8, 22.6, 18.3,
18.1,14.1, ꢀ4.7, ꢀ4.8, ꢀ5.5; HRMS (ESI+) m/z calculated for C51H106O10PSi2 [M+H] 965.7057,
found 965.7086.
C17H35
O
O
O
OTBS
OTBS
O
P
C17H35
O
O
O
OMe
3-(((2,3-Bis((tert-butyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propane-1,2-
32
diyl distearate (8b).
chromatography on
According to General Procedure D but using 3b and purification by
SiO2 (5:1, hexanes:ethyl acetate) gave 3ꢀ(((2,3ꢀbis((tertꢀ
butyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propaneꢀ1,2ꢀdiyl distearate (8b, 0.605
g, 0.592 mmol, 67%) as a waxy colorless solid: Mp 31.0ꢀ31.9 °C; IR ν 2923, 2852, 1743, 1252,
1
835 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 5.24ꢀ5.20 (m, 1 H), 4.37ꢀ4.31 (m, 1 H), 4.22ꢀ4.09 (m, 4
H), 3.97ꢀ3.84 (m, 2 H), 3.76 (d, 3 H, J =11.1 Hz; diasteromers), 3.57ꢀ3.54 (m, 2 H), 2.31
(apparent q, 4 H, J = 7.0 Hz), 1.61ꢀ1.57 (m, 4 H), 1.28ꢀ1.25 (m, 56 H), 0.88ꢀ0.86 (m, 24 H), 0.09
(s, 3 H), 0.08 (s, 3 H), 0.05 (s, 6 H); 13C NMR (CDCl3, 125 MHz) δ 173.2, 172.2, 72.0 (d, J = 8.8
Hz), 69.4 (d, J = 7.5 Hz), 69.2ꢀ69.1 (m), 65.4ꢀ65.3 (m), 64.0, 61.6, 54.4 (d, J = 5.0 Hz), 34.2,
34.0, 31.9, 29.7ꢀ29.1 (multiple peaks), 25.9, 25.7, 24.8, 22.7, 22.6, 18.3, 18.1,14.1, ꢀ4.7, ꢀ4.8, ꢀ
5.4, ꢀ5.5; HRMS (ESI+) m/z calculated for C55H114O10PSi2 [M+H] 1021.7688, found 1021.7766.
C15H31
O
O
O
OTBS
OH
O
P
C15H31
O
O
O
OMe
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3-(((2-((Tert-butyldimethylsilyl)oxy)-3-hydroxypropoxy)(methoxy)phosphoryl)oxy)
propane-1,2-diyl dipalmitate (9a). General Procedure E. To a solution of 8a (800 mg, 0.829
mmol, 1.0 eq.) in CH2Cl2:MeOH (1:1, 8.3 mL) at 0 °C was added camphorsulfonic acid (195 mg,
0.823 mmol, 1.0 eq.). The reaction mixture was maintained between ꢀ5 and 5 °C and stirred for
5.5 h. The solution was filtered through a small plug of basic alumina (ethyl acetate) and
concentrated. Purification by chromatography on SiO2 (3:1 to 1:1, hexanes:ethyl acetate) gave
3ꢀ(((2ꢀ((tertꢀbutyldimethylsilyl)oxy)ꢀ3ꢀhydroxypropoxy)(methoxy)phosphoryl)oxy)propaneꢀ1,2ꢀdiyl
dipalmitate (9a, 435 mg, 0.511 mmol, 62%) as a colorless solid: Mp 27.8ꢀ28.0 °C; IR ν 2922,
2854, 1741, 1252, 835 cmꢀ1; 1H NMR (CDCl3, 400 MHz) δ 5.29ꢀ5.20 (m, 1 H), 4.33 (dd, 1 H, J =
4.4, 12.0 Hz), 4.21ꢀ4.14 (m, 3 H), 4.06ꢀ4.01 (m, 2 H), 3.93ꢀ3.89 (m, 1 H), 3.80ꢀ3.76 (m, 3 H),
3.67ꢀ3.50 (m, 2 H), 2.35ꢀ2.31 (m, 5 H), 1.62ꢀ1.60 (m, 4 H), 1.28ꢀ1.25 (m, 48 H), 0.90ꢀ0.86 (m,
15 H), 0.11 (s, 6 H); 13C NMR (CDCl3, 100 MHz) δ 173.2, 172.8, 71.1 (d, J = 7.0 Hz), 69.4ꢀ69.3
(multiple peaks), 67.7 (m), 65.6ꢀ65.5 (multiple peaks), 63.0, 61.6, 54.6 (multiple peaks), 34.1,
34.0, 31.9, 29.7ꢀ29.1 (multiple peaks), 25.7, 24.8, 22.7, 18.0, 14.1, ꢀ4.8, ꢀ4.9; HRMS (ESI+) m/z
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calculated for C45H92O10PSi [M+H] 851.6192, found 851.6180.
C17H35
O
O
O
OTBS
OH
O
P
C17H35
O
O
O
OMe
3-(((2-((Tert-butyldimethylsilyl)oxy)-3-hydroxypropoxy)(methoxy)phosphoryl)oxy)
propane-1,2-diyl distearate (9b). According to General Procedure E but using 8b and
purification by chromatography on SiO2 (4:1 to 1:1, hexanes:ethyl acetate) gave 3ꢀ(((2ꢀ((tertꢀ
butyldimethylsilyl)oxy)ꢀ3ꢀhydroxypropoxy)(methoxy)phosphoryl)oxy)propaneꢀ1,2ꢀdiyl distearate
(9b, 470 mg, 0.518 mmol, 55%) as a waxy colorless solid: Mp = 41.2ꢀ41.7 °C; IR ν 3535, 2921,
1
2850, 1739, 733 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 5.25ꢀ5.22 (m, 1 H), 4.34 (dd, 1 H, J = 4.2,
12.0 Hz), 4.19ꢀ4.11 (m, 3 H), 4.05ꢀ4.02 (m, 2 H), 3.95ꢀ3.90 (m, 1 H), 3.78 (d, 3 H, J = 11.4 Hz;
diasteromers), 3.67ꢀ3.60 (m, 2 H), 2.36ꢀ2.29 (m, 5 H), 1.62ꢀ1.58 (m, 4 H), 1.28ꢀ1.25 (m, 56 H),
13
0.90ꢀ0.86 (m, 15 H), 0.11 (s, 6 H); C NMR (CDCl3, 125 MHz) δ 173.3, 172.8, 71.1 (d, J = 7.1
Hz), 69.4 (multiple peaks), 67.7 (multiple peaks), 65.6 (multiple peaks), 63.0, 61.6, 54.6
(multiple peaks), 34.2, 34.0, 31.9, 29.7ꢀ29.1 (multiple peaks), 25.7, 24.8, 22.7, 18.0, 14.1, ꢀ4.7, ꢀ
4.9; HRMS (ESI+) m/z calculated for C49H100O10PSi [M+H] 907.6818, found 907.6785.
C15H31
C15H31
O
O
O
O
O
OTBS
O
O
P
O
P
C15H31
O
O
O
O
C15H31
O
OMe
OMe
O
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3-(((3-(((2,3-(Palmitoyloxy)propoxy)(methoxy)phosphoryl)oxy)-2-((tert-
5
6
7
butyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propane-1,2-diyl
dipalmitate
32
(
10a).
To a solution of 9a (322 mg, 0.378 mmol, 1.0 eq.) in CH2Cl2 (3.7 mL) at 0 °C under
8
9
nitrogen was added DIPEA (0.069 mL, 0.42 mmol, 1.1 eq.) and a 0.5 M solution of 7 in CH2Cl2
(0.83 mL, 0.42 mmol, 1.1 eq.). The reaction mixture was warmed to room temperature, stirred
for 1.5 h, and treated with a solution of 3a (364 mg, 0.416 mmol, 1.1 eq.) and 4,5ꢀ
dicyanoimidazole (90 mg, 0.76 mmol, 2.0 eq.) in THF (1.5 mL) in a drowpwise fashion. The
reaction mixture was stirred for 2 h, cooled to 0 °C, treated with Bu4NIO4 (186 mg, 0.429 mmol,
1.1 eq.) and stirred for 40 min. The mixture was diluted with water (3 mL) and CH2Cl2 (5 mL)
and and the organic layer was rinsed with sat. Na2S2O3 (3 mL) and brine (3 mL), dried (MgSO4),
and concentrated. The crude product was filtered through a plug of SiO2 (1:1 hexanes:ethyl
acetate) and concentrated. Purification by chromatography on SiO2 (3:1 to 2:1 to 1:1,
hexanes:ethyl acetate) gave 3ꢀ(((3ꢀ(((2,3ꢀ(palmitoyloxy)propoxy)(methoxy) phosphoryl)oxy)ꢀ2ꢀ
((tertꢀbutyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propaneꢀ1,2ꢀdiyl dipalmitate (10a,
381 mg, 0.255 mmol, 67%) as a colorless solid: Mp 36.5ꢀ37.5 °C; IR ν 2913, 2848, 1737, 1465,
913, 744 cmꢀ1; 1H NMR (CDCl3, 400 MHz) δ 5.25ꢀ5.22 (m, 2 H), 4.36ꢀ4.31 (m, 2 H), 4.22ꢀ4.12
(m, 6 H), 4.05ꢀ3.97 (m, 5 H), 3.78 (d, 6 H, J = 11.2 Hz; diasteromers), 2.32 (apparent q, 8 H, J =
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7.9 Hz), 1.61ꢀ1.57 (m, 8 H), 1.28ꢀ1.25 (m, 96 H), 0.89ꢀ0.86 (m, 21 H), 0.11 (s, 6 H); C NMR
(CDCl3, 125 MHz) δ 173.1, 172.7, 69.8 (multiple peaks), 69.4 (d, J = 6.3 Hz), 67.8 (2, J = 6.3
Hz), 65.7 (multiple peaks), 61.6, 54.6 (d, J = 6.3 Hz), 34.2, 34.0, 31.9, 29.7ꢀ29.1 (multiple
peaks), 25.6, 24.9, 22.7, 18.0, 14.1, ꢀ4.9; HRMS (ESI+) m/z calculated for C81H161O17P2Si [M+H]
1496.0973, found 1496.0903.
C17H35
C17H35
O
O
O
OTBS
O
O
O
O
P
O
P
C17H35
O
O
O
O
C17H35
O
OMe
OMe
O
3-(((3-(((2,3-(Stearoyloxy)propoxy)(methoxy)phosphoryl)oxy)-2-((tert-
butyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propane-1,2-diyl
distearate
33
(10b). General Procedure F . To a solution of 3b (356 mg, 0.570 mmol, 1.0 eq.) and DIPEA
(0.10 mL, 0.63 mmol, 1.1 eq.) under nitrogen was added a 0.5 M solution of 7 (1.2 mL, 0.63
mmol, 1.1 eq.) in CH2Cl2. The reaction was mixture stirred for 1 h, and treated drowpwise with a
solution of 9b (470 mg, 0.518 mmol, 1.0 eq.) and 4,5ꢀdicyanoimidazole (125 mg, 1.04 mmol, 2.0
eq.) in THF (1.5 mL). The mixture was stirred for 3 h, cooled to 0 °C, treated with Bu4NIO4 (173
mg, 0.399 mmol, 1.2 eq.), stirred for 45 min and diluted with water (2 mL) and CH2Cl2 (5 mL).
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The organic layer was rinsed with sat. Na2S2O3 (2x3 mL), sat. NaHCO3 (3 mL), and brine (3
mL), dried (MgSO4), and concentrated. The crude residue was filtered through a plug of SiO2
(1:1, hexanes:ethyl acetate), concentrated, dissolved in CH2Cl2 (4 mL) and treated with TBSCl
(43 mg, 0.29 mmol 0.5 eq.), imidazole (21 mg, 0.31 0.6 eq.) and DMAP (3 mg, 0.024 mmol 0.04
eq.). The reaction mixture was stirred for 4.5 h, quenched with water (4 mL) and diluted with
CH2Cl2 (4 mL). The organic layer was rinsed with brine (4 mL), dried (MgSO4), and
concentrated. Purification by chromatography on SiO2 (5:1 to 3:1 to 1:1, hexanes:ethyl acetate)
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gave
3ꢀ(((3ꢀ(((2,3ꢀ(stearoyloxy)propoxy)(methoxy)phosphoryl)oxy)ꢀ2ꢀ((tertꢀbutyldimethylsilyl)
oxy)propoxy) (methoxy)phosphoryl)oxy)propaneꢀ1,2ꢀdiyl distearate (10b, 335 mg, 0.208 mmol,
1
40%) as a waxy colorless solid: Mp 48.1ꢀ48.2 °C; IR ν 2918, 2851, 1741, 1043 cmꢀ1; H NMR
(CDCl3, 300 MHz) δ 5.25ꢀ5.22 (m, 2 H), 4.35ꢀ4.30 (m, 2 H), 4.29ꢀ4.04 (m, 11 H), 3.78 (d, 6 H, J
= 11.4 Hz; diasteromers), 2.32 (apparent q, 8 H, J = 7.2 Hz), 1.61ꢀ1.57 (m, 8 H), 1.25 (m, 112
H), 0.89ꢀ0.86 (m, 21 H), 0.11 (s, 6 H); 13C NMR (CDCl3, 125 MHz) δ 173.2, 172.8, 69.7 (multiple
peaks), 69.4 (multiple peaks), 67.9 (multiple peaks), 65.6 (multiple peaks), 61.6, 54.6 (d, J = 5.9
Hz), 34.1, 34.0, 31.9, 29.7ꢀ29.1 (multiple peaks), 25.6, 24.8, 22.7, 18.0, 14.1, ꢀ4.9; HRMS (ESI+)
m/z calculated for C89H177O17P2Si [M+H] 1608.2225, found 1608.2190.
C15H31
C15H29
O
O
O
OTBS
O
O
O
O
P
O
P
C15H31
O
O
O
O
C15H31
O
OMe
OMe
O
3-(((3-(((2-Palmitoleoyloxy-3-(palmitoyloxy)propoxy)(methoxy)phosphoryl)oxy)-2-((tert-
butyldimethylsilyl)oxy)propoxy)(methoxy)phosphoryl)oxy)propane-1,2-diyl
dipalmitate
(10c). According to General Procedure F using 3c and 9c and a purification by chromatography
on SiO2 (5:1 to 3:1 to 1:1, hexanes:ethyl acetate) gave 3ꢀ(((3ꢀ(((2ꢀpalmitoleoyloxyꢀ3ꢀ
(palmitoyloxy)propoxy)(methoxy)phosphoryl)oxy)ꢀ2ꢀ((tertꢀbutyldimethylsilyl)oxy)propoxy)
(methoxy)phosphoryl)oxy)propaneꢀ1,2ꢀdiyl dipalmitate (10c,145 mg, 0.0970 mmol, 39%) as a
1
clear colorless oil: IR ν 2919, 2850, 1741, 1465, 1034 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 5.35ꢀ
5.33 (m, 2 H), 5.25ꢀ5.22 (m, 2 H), 4.36ꢀ4.32 (m, 2 H), 4.23ꢀ4.12 (m, 6 H), 4.05ꢀ4.00 (m, 5 H),
3.78 (d, 6 H, J = 11.4 Hz; diasteromers visible), 2.32 (apparent q, 8 H, J = 7.2 Hz), 2.02ꢀ2.00 (m,
4 H), 1.61ꢀ1.54 (m, 8 H), 1.30ꢀ1.26 (m, 88 H), 0.89ꢀ0.86 (m, 21 H), 0.11 (s, 6 H); 13C NMR
(CDCl3, 125 MHz) δ 173.2, 172.8, 130.0, 129.7, 69.7 (m), 69.4 (d, J = 6.5 Hz), 67.8 (d, J = 5.5
Hz), 65.6 (m), 61.6, 54.6 (d, J = 6.0 Hz), 34.1, 34.0, 31.8, 29.7ꢀ29.0 (multiple peaks), 25.6, 24.8,
22.7, 22.6 18.0, 14.1, ꢀ4.9; HRMS (ESI+) m/z calculated for C81H159O17P2Si [M+H] 1494.0816,
found 1494.0825.
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Tetrapalmitoylcardiolipin diammonium salt (11a). General Procedure G. A solution of 10a
and NaI (24 mg, 0.16 mmol, 4 eq.) in 2ꢀbutanone (0.8 mL) was heated at reflux for 13.5 h and
concentrated. The crude waxy yellow solid was dissolved in 1 M HCl:THF:H2O (0.1:2:1, 1 mL)
and the resulting yellow solution was stirred for 12 h, and quenched with 30% NH4OH (2 mL).
The resulting white slurry was extracted with CHCl3 (3x5 mL), concentrated, and triturated with
Et2O to give tetrapalmitoylcardiolipin diammonium salt (11a, 40 mg, 0.029 mmol, 73%) as a
colorless solid containing 5ꢀ7% (ESIꢀMS) monolyso (tripalmitoyl) cardiolipin as an impurity that
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was removed by thin layer chromatography: H NMR (CDCl3, 400 MHz) δ 7.46 (bs, 8 H),
5.21(bs, 2 H), 4.37 (m, 2 H), 4.17ꢀ4.13 (m, 2 H), 3.91 (bs, 9 H), 2.29 (apparent q, 8 H, J = 8.5
Hz), 1.58 (m, 8 H), 1.28ꢀ1.25 (m, 98 H), 0.87 (t, 12 H, J = 6.8 Hz); HRMS (ESIꢀ) m/z calculated
for C73H141O17P2 [MꢀH] 1351.9639, found 1351.9648.
C17H35
O
C17H35
O
O
OH
O
O
O
O
P
O
P
C17H35
O
O
O
O
C17H35
O
Oꢀ +NH4
Oꢀ +NH4
O
Tetrastearoylcardiolipin diammonium salt (11b) 33, According to General Procedure G using
10b and purification by precipitation from hot THF gave tetrastearoylcardiolipin diammonium salt
(11b, 53 mg, 0.035 mmol, 38%) as a colorless powder containing 5ꢀ7% (ESIꢀMS) monolyso
(tristearoylcardiolipin) cardiolipin as an impurity that was removed by thin layer chromatography:
1H NMR (CDCl3, 400 MHz) δ 7.44 (bs, 8 H), 5.21 (bs, 2 H), 4.37ꢀ4.35 (m, 2 H), 4.17ꢀ4.13 (m, 2
H), 3.92 (bs, 9 H), 2.30 (apparent q, 8 H, J = 8.3 Hz), 1.59ꢀ1.58 (m, 8 H), 1.28ꢀ1.25 (m, 112 H),
0.88 (t, 12 H, J = 6.8 Hz); MS (ESIꢀ) m/z calculated for C81H156O17P2 [Mꢀ2H]2ꢀ 731.5, found 731.8.
C15H31
C15H29
O
O
O
OH
O
O
O
O
P
O
P
C15H31
O
O
O
O
C15H31
O
Oꢀ +NH4
Oꢀ +NH4
O
Tripalmitoyl-monopalmitoleoylcardiolipin diammonium salt (11c). According to General
Procedure G using 10c and purification by trituration with Et2O gave tripalmitoylꢀ
monopalmitoleoylcardiolipin diammonium salt (11c, 83 mg, 0.060 mmol, 70%) as a colorless
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powder containing 5ꢀ7% (ESIꢀMS) monolysoꢀ11c as an impurity that was removed by thin layer
chromatography: 1H NMR (CDCl3, 400 MHz) δ 7.44 (bs, 8 H), 5.35ꢀ5.32 (m, 2 H), 5.21 (bs, 2 H),
4.39ꢀ4.35 (m, 2 H), 4.17ꢀ4.13 (m, 2 H), 3.84 (bs, 9 H), 2.30 (apparent q, 8 H, J = 8.1 Hz), 2.01ꢀ
1.99 (m, 4 H), 1.66ꢀ1.58 (m, 8 H), 1.28ꢀ1.25 (m, 106 H), 0.88 (t, 12 H, J = 6.4 Hz); MS (ESIꢀ) m/z
calculated for C73H138O17P2 [Mꢀ2H]2ꢀ 674.5, found 674.7.
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Assessment of Substrate Specificity of Purified Cld1. Activity of Cld1 was detected in liposomes
by accumulation of MLCL after CL hydrolysis. Liposomes containing DOPC and CL (10 µM, 1:1)
were prepared by sonication (Ultrasonic Homogenizer 4710 series, ColeꢀParmer Instrument
Co., Chicago, IL) and treated with Cld1 (0.1−0.5 ꢁg of protein per 250µl sample) in 50 mM
HEPES pH 7.45, containing 100 ꢁM DTPA for 20 min at 37°C. After incubation with Cld1, lipids
were extracted by Folch procedure and analysis of MLCL was performed using LC−ESIꢀMS.
Internal standards of MLCLs were prepared from respective CLs in PLA2 (porcine pancreatic,
Sigma) driven reaction as described by Kim and Hoppel (2011) 26 with light modifications.
Molecular modeling
The homology model of Cld1 was built using IꢀTASSER 34 webserver. Hsad, a steroidꢀdegrading
hydrolase from Mycobacterium Tuberculosis (PDB ID: 2VF213), was utilized as a template. Cꢀ
score for the constructed model was equal to 1.78. The Cꢀscore is a confidence score to
estimate the quality of predicted models by IꢀTASSER, in the range of [ꢀ5,2], where a Cꢀscore of
higher value defines a model with a high confidence 34. The model showed a TMꢀscore equal to
0.5. TMꢀscore is a scale for measuring the structural similarity of two protein models, with lower
sensitivity to the local error compared to RMSD (a TMꢀscore >0.5 indicates a model of correct
topology independent from protein length) 35. The overlay of the cavities and cartoon
representations was employed to explore the active site and a possible channel facilitating
access of CL acyl chain to the hydrolase domain of Cld1. Three homoꢀacylatedꢀCL species,
including (C14:0)4ꢀCL, (C16:0)4ꢀCL and (C18:2)4ꢀCL were docked to the predicted structure of
Cld1 using default settings by AutoDock Vina (http://vina.scripps.edu) 36. The pdb into pdbqt
format conversion of lipids and protein structures was performed using MGL Tools. A grid box
was centered at coordinates 39.861, 40.057, 74.975 with 100Å, 80Å and 90Å units in x, y and z
directions, respectively, to cover the entire protein structure. AutoDock Vina reports the 9 lowest
energy conformations, which were inspected using PyMOL software (www.pymol.org).
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Statistical Analysis. The results are presented as mean ± S.D. values from at least three
experiments, and statistical analyses were performed by paired/unpaired Student's tꢀtest. The
statistical significance of differences was set at p< 0.05.
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Acknowledgements: This work was supported by the National Institutes of Health (HL114453,
ES020693, U19AIO68021, NS076511, NS061817, NS052315, CA 165065, HL117880), Human
Frontier Science Program (HFSPꢀRGP0013/2014), and the Barth Syndrome Foundation, Inc.
and the Barth Syndrome Foundation of Canada.
Competing financial interests: The authors declare that they have no competing financial
interests.
References:
1.
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