Stereochemical Analysis of Glycerophospholipids by Vibrational Circular Dichroism

Article abstract of DOI:10.1021/jacs.5b05832

The stereochemistry of glycerophospholipids (GPLs) has been of interest for its roles in the evolution of life and in their biological activity. However, because of their structural complexity, no convenient method to determine their configuration has been reported. In this work, through the first systematic application of vibrational circular dichroism (VCD) spectroscopy to various diacylated GPLs, we have revealed that their chirality can be assigned by the sign of a VCD exciton couplet generated by the interaction of two carbonyl groups. This paper also presents spectroscopic evidence for the stereochemistry of GPLs isolated from bacteria, eukaryotes, and mitochondria.

Full text of DOI:10.1021/jacs.5b05832

Communication
pubs.acs.org/JACS
Stereochemical Analysis of Glycerophospholipids by Vibrational
Circular Dichroism
Tohru Taniguchi,*,† Daisuke Manai,† Masataka Shibata,† Yutaka Itabashi,‡ and Kenji Monde*,†
†Faculty of Advanced Life Science, Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Kita
21 Nishi 11, Sapporo 001-0021, Japan
‡Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan
S
* Supporting Information
ABSTRACT: The stereochemistry of glycerophospholi-
pids (GPLs) has been of interest for its roles in the
evolution of life and in their biological activity. However,
because of their structural complexity, no convenient
method to determine their configuration has been
reported. In this work, through the first systematic
application of vibrational circular dichroism (VCD)
spectroscopy to various diacylated GPLs, we have revealed
that their chirality can be assigned by the sign of a VCD
exciton couplet generated by the interaction of two
carbonyl groups. This paper also presents spectroscopic
evidence for the stereochemistry of GPLs isolated from
bacteria, eukaryotes, and mitochondria.
lycerophospholipids (GPLs) are generally the most
Figure 1. (a) Stereochemistry of glycerophospholipids. (b)
Representative structures of the glycerophospholipids studied in this
work. PC = phosphatidylcholine; LPC = lysophosphatidylcholine; PE
1
Gabundant lipids in all mammalian membranes.
Not
only do these molecules provide suitable membranous
environments for various biomolecules to function, but also,
= phosphoethanolamine; PS = phosphatidylserine; BMP = bis-
some of them are known to act as signaling molecules called
lipid mediators to regulate cell behavior.2 GPLs consist of a
polar phosphoester headgroup, a prochiral glycerol, and acyl
chains that typically range from C14 to C20 with various levels of
unsaturation. Phosphoesterification to glycerol was believed to
occur solely at the sn-3 position in eukaryotic and bacterial
GPLs but at the sn-1 position in archaeal ones (Figure 1a),
which led to several hypotheses concerning the pivotal roles of
their chirality in the evolution of life.3 Interestingly, the
presence of an unusual sn-1-phosphorylated mammalian GPL,
bis(monoacylglycero)phosphate (BMP),4 was confirmed in a
recent study.5 This finding has highlighted the need for
exploration of other GPLs with unnatural configuration and
verification of the previously speculated stereochemistry to
obtain further insight into the evolution of life. Such
stereochemical studies may also lead to the development of
the biochemistry of GPLs with abnormal chirality, similar to the
blossoming field of D-amino acids.6 Nonetheless, because of the
structural diversity of GPLs, no universal method to determine
their chirality has been reported.
(monoacylglycero)phosphate; CL = cardiolipin.
sitates a variety of enzymes, which has limited its applicability to
the repertoire of GPLs. Furthermore, enzymatic results can be
ambiguous when their stereospecificity is not thoroughly
examined or when harsh reaction conditions are involved as a
pre- or post-treatment.4a,7 Other methods such as NMR
analysis of diastereomeric derivatives5 and chiral HPLC7
require an array of authentic compounds.
Seeking a direct analytical method that obviates the need for
authentic samples, we thought of applying vibrational circular
dichroism (VCD) spectroscopy. The VCD technique com-
bined with density functional theory (DFT) calculations has
become one of the most reliable methods to determine the
chirality of small molecules.8 However, GPLs may not be
amenable to normal computation because of their amphiphi-
licity, flexibility, and structural complexity. These properties
have also deterred a systematic VCD study of biological lipids,
in stark contrast to widely studied biomolecules such as
proteins, nucleic acids, and glycoconjugates.9 Recently, inspired
by the electronic CD (ECD) exciton chirality method by
Harada and Nakanishi,10 we found that two CO groups in
GPLs are composed of several classes of molecules that are
categorized according to the structure of the headgroup. The
major classes include phosphatidylcholines (PCs), phosphoe-
thanolamines (PEs), phosphatidylserines (PSs), and cardioli-
pins (CLs), and there are other classes such as BMPs (Figure
1b). Conventional enzymatic stereochemical analysis neces-
Received: June 12, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.5b05832
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Journal of the American Chemical Society
Communication
close vicinity exhibit an intense bisignate VCD signal whose
sign reflects the chirality of the molecule.11 This result led us to
hypothesize that VCD signals originating from the carbonyls in
diacylated GPLs could be indicative of their stereochemistry. In
the present work, through the first systematic VCD measure-
ments of GPLs, we demonstrate that the chirality of various
GPLs can be determined using a VCD exciton approach.
A fundamental challenge to our hypothesis is the possible
fluctuation of the orientation of the CO groups in their
acyclic structures, unlike the rigid cyclic molecules we studied
previously.11 Therefore, it was necessary to examine whether
two carbonyl groups exhibit a meaningful VCD signal and
whether the sign of the couplet is consistent for different classes
of GPLs. To this end, we first measured the VCD spectra of
phosphatidylcholines, a representative class of GPLs. Both sn-3
and sn-1 phosphatidylcholines were chemically synthesized (see
the Supporting Information, (SI)), and their VCD spectra were
measured in CDCl3. Figure 2a shows the VCD results for
weak intensities of their VCD signals compared with those
observed for cyclic molecules11 are probably due to the
conformational flexibility of GPLs. Importantly, the sign of the
exciton signals was not affected by biologically abundant longer
saturated and unsaturated acyl chains (PC-C18 and PC-
C18Δ9) (Table 1 and Figure S1). These results proposed that
the stereochemistry of phosphatidylcholines can be assigned
simply by observing their carbonyl stretching VCD, irrespective
of the types of their acyl chains.
According to the coupled oscillator model,13 the positive−
negative couplet in sn-3 phosphatidylcholines corresponds to a
clockwise carbonyl orientation. To better understand the
tolerance of the couplet to acyl chains, a preliminary
conformational calculation was performed for a phosphatidyl-
choline with C4 acyl chains (sn-3-PC-C4) to see the geometry
around the glycerol core (see the SI for details of the
calculation procedure and the choice of the model com-
pound).14,15 Molecular mechanics force field (MMFF) and
subsequent DFT calculations yielded multiple stable con-
formers, in accordance with its expected flexibility. Among
them, the conformers with a clockwise carbonyl twist are
predominant over those with a counterclockwise twist (Figures
2b and S2). The linear fatty acid chains point away from the
glycerol moiety and thus are less likely to interfere with the
orientation of the CO groups, which corroborates the
validity of the VCD analysis of the chirality of various
phosphatidylcholines.
The applicability to other types of headgroup was then
confirmed by a study of a phosphatidylserine and BMP. As
summarized in Table 1, the L-serine in PS-C8 did not alter the
sign of the VCD couplet. A similar positive−negative pattern
was also observed for a dimeric sn-3,3′ BMP derivative that has
four acyl chains (BMP-C8c). Meanwhile, BMP-C8 did not
show a couplet in spite of the presence of two carbonyl groups
(Figure 3). These results indicated that the CO groups at the
sn-1 and -2 positions do not cause any significant excitonic
interaction with those at the sn-1′ and -2′ positions. Such
independence of each glycerol moiety is advantageous in
applying this VCD method to GPLs with other headgroups.
Having verified the compatibility of the VCD exciton
approach with various GPL structures, we applied this method
to a synthetic BMP with exceptional sn-1 stereochemistry (sn-
1,1′-BMP-C8) and a lysophosphatidylcholine (LPC), a lipid
mediator, isolated from avian egg (LPC-C16). In the case of sn-
1,1′-BMP-C8, which did not show an exciton VCD in its intact
state, two more octanoyl groups were introduced using
Yamaguchi reagent in a manner similar to a reported
procedure.16 The resultant sn-1,1′-BMP-C8c yielded a
negative−positive couplet, consistent with its sn-1,1′ config-
uration (Figure 3a). Similarly, installation of a C16 fatty acid
into LPC-16 led to a positive−negative VCD couplet, which
unambiguously confirmed its eukaryotic sn-3 configuration
(Figure 3b).
Figure 2. (a) VCD and IR spectra of phosphatidylcholines. Each
spectrum was measured in CDCl3 (l = 100 μm) at a concentration of
0.1 M (sn-3-PC-C8a and sn-3-PC-C8b) or 0.08 M (sn-3-PC-C8 and
sn-1-PC-C8). (b) Schematic orientation of the two carbonyl groups of
sn-3-PC (left) and the relationship between the arrangement of the
electric transition moments (red arrows parallel to the CO bonds)
and the sign of the VCD couplet (right). The carbonyl orientation is
depicted on the basis of the predicted stable conformers of sn-3-PC-
C4, where the CO group at C1 prefers a syn orientation with regard
to C1−HS (see Figure S2).
The utility of this method for other complex lipids was
examined in the analysis of cardiolipins from Escherichia coli
(CL-EC) and bovine heart (CL-Mt, where Mt refers to
mitochondria, as the biosynthesis of cardiolipins in eukaryotic
cells occurs exclusively in mitochondria). CL-EC and CL-Mt
not only belong to one of the most structurally complex classes
of GPL but also are composed of mixtures of isomers differing
in acyl chains (see Figure S3), which poses difficulties in the
analysis of their chirality by other methods. Nevertheless, both
cardiolipins yielded positive−negative couplets that were clearly
phosphatidylcholines with C8 acyl chains (PC-C8). Despite our
concern, a characteristic bisignate signal was observed in the
carbonyl stretching region: positive−negative (from lower to
higher frequency) for the sn-3 enantiomer and negative−
positive for the sn-1 enantiomer. On the other hand, only a
broad positive band was seen for the monoacyl monoalkyl
derivatives PC-C8a and PC-C8b. This comparison suggested
that the observed VCD couplet of PC-C8 was produced by the
intramolecular interaction of the two carbonyl groups.12 The
B
DOI: 10.1021/jacs.5b05832
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Journal of the American Chemical Society
Communication
a
Table 1. VCD Couplets of Glycerophospholipids
b
b
c
c
GPL
R1
R2
Δε1 (ν [cm−1])
Δε2 (ν [cm−1])
d
d
sn-3-PC-C8a
sn-3-PC-C8b
sn-3-PC-C8
COC7H15
C8H17
C8H17
+0.014 (1724)
+0.014 (1728)
COC7H15
COC7H15
COC17H35
COC17H33
COC7H15
COC17H35
COC17H33
COC7H15
COC7H15
COC7H15
COC7H15
COC15H31
COC7H15
COC17H35
COC17H33
COC7H15
COC17H35
COC17H33
COC7H15
H
+0.020 (1724)
+0.010 (1724)
+0.012 (1720)
−0.026 (1724)
−0.014 (1724)
−0.019 (1720)
+0.027 (1717)
−0.027 (1740)
−0.013 (1740)
−0.021 (1740)
+0.017 (1744)
+0.011 (1740)
+0.011 (1736)
−0.011 (1736)
sn-3-PC-C18
sn-3-PC-C18Δ9
sn-1-PC-C8
sn-1-PC-C18
sn-1-PC-C18Δ9
e
sn-3-PS-C8
e
f
sn-1,1′-BMP-C8
ND
e
e
sn-1,1′-BMP-C8c
sn-3,3′-BMP-C8c
COC7H15
COC7H15
COC15H31
−0.029 (1713)
+0.028 (1720)
+0.010 (1724)
+0.018 (1724)
+0.038 (1740)
−0.027 (1740)
−0.013 (1740)
−0.038 (1736)
−0.025 (1740)
g
PC-C16
eh
i
,
CL-EC
mixtures ranging from COC13H27 to COC18H37
ej
,
i
CL-Mt
mostly COC17H31
+0.035 (1717)
a
b
c
Measured in CDCl3 (l = 100 μm, c = 0.03−0.1 M). See Figure 1 for the parent structure of each GPL. Intensities at the extrema of each VCD
d
e
f
g
band, in M−1 cm−1. Only a monosignate band was observed. Sodium salt. No significant band was detected. Derived from avian egg LPC-C16.
h
i
j
From E. coli. See Figure S3. From bovine heart.
recognized even with a relatively small amount of sample
(Figure 3c,d). Thus, simple observation of VCD exciton signals
allowed spectroscopic verification of their sn-3 stereochemistry.
Further development of more sensitive VCD spectrometers
should allow chiral analysis of even smaller amounts of GPLs in
the future.8c,17
This analytical method relies on the conformational
preference of GPLs in CDCl3.18 Therefore, GPLs not soluble
enough to CDCl3 may require chemical modification to
increase the solubility, as often needed in computation-based
VCD protocols.19 On the other hand, extensive VCD studies
on GPL conformations in liposomes or micelles may be of
interest, especially in relation to their functions in cell
membranes.
Lastly, this work has shown the effectiveness of the VCD
exciton approach for the stereochemical analysis of acyclic
molecules. Considering the success of the ECD exciton chirality
method for acyclic systems,10b,20 the VCD method also should
be of help in solving stereochemical problems of various acyclic
molecules that are abundant in nature.21
In summary, we have established a convenient method to
assign the stereochemistry of GPLs based on the sign of
bisignate VCD signals in the CO stretching region. Bacterial
and eukaryotic sn-3 GPLs exhibited a positive−negative
couplet, whereas the sn-1 configuration, which is characteristic
of archaeal GPLs and mammalian BMPs, showed a negative−
positive couplet. The applicability of this method to structurally
complex GPLs such as BMPs and isomeric mixtures of
cardiolipins was demonstrated. This work has also presented
spectroscopic evidence of the chirality of GPLs originating from
bacteria, eukaryotes, and mitochondria. Through the elucida-
tion of the chirality of GPLs, this VCD approach should
provide further insight into the evolution of life and advance
the biochemistry of GPLs with irregular stereochemistry.
Figure 3. VCD and IR spectra of (a) a synthetic BMP and its
derivative, (b) phosphatidylcholine derived from avian egg lysophos-
phatidylcholine, (c) cardiolipin from E. coli, and (d) cardiolipin from
bovine heart. Each spectrum was measured in CDCl3 (l = 100 μm) at a
concentration of 0.1 M (PC-C16) or 0.05 M (others). The
concentrations of CL-EC and CL-Mt were calculated on the basis of
their average molecular weights. Wavenumbers at the VCD extrema
are labeled in italics. The derivatization schemes are shown in (a) and
(b). TCBC = 2,4,6-trichlorobenzoyl chloride.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b05832.
■
S
Experimental details and additional VCD, DLS, and
structural data (PDF)
C
DOI: 10.1021/jacs.5b05832
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Journal of the American Chemical Society
Communication
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Mele, A. Chirality 2014, 26, 532.
(15) Dynamic light scattering (DLS) experiments excluded the
possibility of micelle formation of PC-C16 and PC-C18 in chloroform
(see Figure S4).
AUTHOR INFORMATION
■
Corresponding Authors
*ttaniguchi@sci.hokudai.ac.jp
*kmonde@sci.hokudai.ac.jp
Notes
The authors declare no competing financial interest.
(16) Acharya, H. P.; Kobayashi, Y. Synlett 2005, 2015.
(17) (a) Rhee, H.; June, Y.-G.; Lee, J.-S.; Lee, K.-K.; Ha, J.-H.; Kim,
ACKNOWLEDGMENTS
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Pfeifer, M.; Fischer, P. J. Am. Chem. Soc. 2011, 133, 5704.
(18) Performing measurements on PC-C8 in coordinating solvents
such as DMSO-d6 resulted in the disappearance of VCD couplet,
probably because of disruption of its conformation.
This paper is dedicated to Prof. Koji Nakanishi on the occasion
of his 90th birthday. The authors thank Mr. Katsuyuki Nambara
and other members of Prof. Kuniharu Ijiro’s group for their
assistance with DLS measurements and Ms. Haruka Satoh for
help with the TOC graphic. This work was partially supported
by the Hoansha Foundation, the Akiyama Life Science
Foundation, the Shimadzu Science Foundation, the Innovation
COE Project for Future Medicine and Medicinal Research, and
Grants in Aid for Scientific Research (26702034 to T.T. and
24310151 and 15H03111 to K.M.).
̈
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DOI: 10.1021/jacs.5b05832
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