Stereoselective synthesis of the head group of archaeal phospholipid PGP-Me to investigate bacteriorhodopsin-lipid interactions

Article abstract of DOI:10.1039/c5ob01252j

Phosphatidylglycerophosphate methyl ester (PGP-Me), a major constituent of the archaeal purple membrane, is essential for the proper proton-pump activity of bacteriorhodopsin (bR). We carried out the first synthesis of the bisphosphate head group of PGP-M

Full text of DOI:10.1039/c5ob01252j

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Stereoselective synthesis of the head group of
archaeal phospholipid PGP-Me to investigate
bacteriorhodopsin–lipid interactions†
Cite this: Org. Biomol. Chem., 2015,
13, 10279
Received 18th June 2015,
Jin Cui,‡a,b,c Satoshi Kawatake,a,b,c Yuichi Umegawa,a,b,c Sébastien Lethu,a,b,c
Masaki Yamagami,a Shigeru Matsuoka,a,b,c Fuminori Sato,b,c Nobuaki Matsumori§a
and Michio Murata*a,b,c
Accepted 14th September 2015
DOI: 10.1039/c5ob01252j
www.rsc.org/obc
Phosphatidylglycerophosphate methyl ester (PGP-Me), a major
Bacteriorhodopsin (bR) is a light-driven proton pump
constituent of the archaeal purple membrane, is essential for the of Halobacterium halobium that is localized in the purple
proper proton-pump activity of bacteriorhodopsin (bR). We carried membrane (PM) in a trimeric form.13–15 Because of the thermal
out the first synthesis of the bisphosphate head group of PGP-Me stability and facile preparation, reconstituted bR in artificial
using H-phosphonate chemistry that led to the production of a membranes has been regarded as a simple and well-character-
simplified PGP-Me analogue with straight alkyl chains. To investi- ised model to investigate LPI at the atomistic level.16 Membrane
gate the role of this head group in the structural and functional models composed of phospholipids (and sterols) have been
integrity of bR, the analogue was used to reconstitute bR into lipo- used to reproduce the original properties of biomembranes to a
somes, in which bR retained the original trimeric structure and certain extent. Among these, neutral phosphatidylcholine (PC)
light-induced photocycle activity. Enhanced ordering of an alkyl is often used for the reconstitution of bR.5–9 However, large
chain of the 2H-labelled analogue was observed in 2H NMR spectra differences in chemical structures between PCs and PM lipids,
upon interaction with bR. These results together suggest that the particularly in their head groups, lead to an altered membrane
bisphosphate moiety plays a role in the proper functioning of bR environment that disrupts the assembly and functionality of
through the lipid–protein interaction.
bR. It has been frequently reported that the two-dimensional
crystalline structure of bR molecules is disassembled in di-
myristoyl-PC (DMPC) vesicles above the phase transition temp-
erature,5,8,17 while bR in natural PM is stable even at high
temperatures;18 among the other artificial lipids, PCs with
fluorine-substituted acyl chains have been found to provide a
more suitable environment for bR.9 For a better understanding
of the atomistic mechanism underlying LPI, structurally stable
membrane proteins such as bR should be investigated in depth
by using model lipids with various head groups.
It is scientifically important to investigate the lipid–protein
interaction (LPI), which is a key factor for the proper function-
ing of membrane-integral proteins. However, the interpret-
ation of experimental results on LPI is usually difficult due to
the highly complex and dynamic nature of biological mem-
branes. Thus, as shown in the pioneering studies by Racker
and coworkers,1–3 artificial liposomes reconstituted with puri-
fied membrane proteins provide a useful model system4–9 to
investigate the interplay between membrane proteins and their
surrounding lipids.10–12
Phosphatidylglycerophosphate methyl ester (PGP-Me,
1
in Fig. 1), which is the major constituent of PM lipids,19,20
aDepartment of Chemistry, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka
560-0043, Japan. E-mail: murata@chem.sci.osaka-u.ac.jp; Fax: +81 66850 5774;
Tel: +81 66850 5774
bJST ERATO, Lipid Active Structure Project, Osaka University, 1-1 Machikaneyama,
Toyonaka, Osaka 560-0043, Japan
cProject Research Centre for Fundamental Science, Osaka University, 1-1
Machikaneyama, Toyonaka, Osaka 560-0043, Japan
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c5ob01252j
‡Current address: Institute of Microbial Chemistry (BIKAKEN) Tokyo, 3-14-
23 Kamiosaki, Shinagawa-ku, Tokyo, 141-0021, Japan.
§Current address: Department of Chemistry, Graduate School of Science, Kyusyu
University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan.
Fig. 1 Chemical structures of compounds 1–3 with two phosphate
esters a and b.
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possesses an acidic bisphosphate headgroup. In addition, 1
contains methyl-branched alkyl chains that are bound to gly-
cerol via ether linkages as opposed to straight acyl chains in
PCs. Previous studies have suggested that the double-charged
head group of 1 is important for the array structure and proton
pump activity of bR.21–24 Thus, a molecular probe, in which
the polar head of PGP-Me is preserved, may exhibit superior
performance of LPI in comparison with PCs with neutral head
groups. In addition, higher affinity of the negatively charged
bisphosphate moiety to the surface of the protein may
somehow facilitate the observation of the transient LPI; lipid
molecules usually undergo very rapid exchange between the
annular shell and the bulk phase in membranes.10,11 However,
to our knowledge, there is no previous report on a stereo-
selective synthetic method for the head group of PGP-Me.
In this study we established the first stereoselective route to
furnish the polar head of PGP-Me based on H-phosphonate
chemistry, and synthesised a PGP-Me analogue 2 bearing two
simple non-branched side chains that was used for the re-
constitution of bR. The analogue 2 was subjected to circular
dichroism (CD) and laser flash photolysis for evaluating its
ability to stabilise the structure and functions of membrane-
Scheme 1 Synthesis of compound 6.
ammonium cerium(IV) nitrate (CAN), acetone/H2O (9/1 v/v)).
However, in each case, the benzyl methylene protons of iso-
lated 6 showed two pairs of doublets in a ca. 1 : 1 ratio, which
was distinctly different from that of the substrate, indicating
that an isomerisation reaction occurred during deprotection
and/or purification. Presumably, the hydroxyl group of 6
underwent intramolecular nucleophilic attack at the phospho-
triester to give a phosphorane intermediate,29 resulting in the
racemisation of the sn-2 asymmetric centre in 6 (Scheme 1).
We turned our attention to an alternative approach based
on Route B (Fig. 2) via an H-phosphonate intermediate, which
could prevent the racemisation. As shown in Scheme 2, com-
pound 12, which was synthesised from (R)-solketal,28 was
2
2
bound bR. Based on H NMR experiments for the H-labelled
analogue 3 of PGP-Me, a moderate increase in the acyl chain
order was observed upon interaction with bR.
Retrosynthetically, we envisioned that 2 would be con-
structed by H-phosphonate coupling25–27 between 4 and 5.
Compound 4 could be obtained via phosphorylation either
from phosphate 6 (Route A), or from an H-phosphonate deriva-
tive 7 and a known phosphoramidite 8 (Route B) (Fig. 2). As
shown in Scheme 1, a protected glycerol 10 was synthesised
from the commercially available (S)-solketal 9 using a pro-
cedure that was partially modified from that in previous
reports.28 Compound 10 was phosphorylated with 8/1H-tetra-
zole followed by oxidation with tert-butyl hydroperoxide
(t-BuOOH) to produce phosphotriester 11. 1H NMR spectra
revealed that 11 was a mixture of diastereoisomers with
respect to the sn-2 carbon and the phosphate centre at a
diastereomeric ratio of 2 : 1 based on the methylene proton
signals of the benzyl phosphonate ester. To introduce
the second phosphorus, the p-methoxybenzyl (PMB) group
was removed by oxidative cleavage using typical conditions
(2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), wet CH2Cl2;
phosphorylated
using
2-chloro-4H-1,3,2-benzodioxaphos-
phinin-4-one 13 in pyridine followed by hydrolysis to afford
H-phosphonate 14. The PMB group in 14 was removed by tri-
Scheme 2 Synthesis of compound 2.
Fig. 2 Retrosynthetic analysis of compound 2.
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fluoroacetic acid (TFA) to give H-phosphonate 7 with a high
1
enantiomeric excess (>95%) as determined by H NMR using
Chirabite-AR30 as a chemical shift reagent.31 Phosphorylation
of 7 with 8 followed by selective oxidation of the phosphite
triester generated in situ, resulted in a high yield of bisphos-
phate 4. Using pivaloyl chloride (PvCl) as a condensing agent,
in the presence of 5,32 4 was completely converted into a phos-
phite, which was then oxidised with iodine followed by hydro-
lysis with water to give 15. Deprotection of benzyl ethers in the
presence of Adams’s catalyst followed by ion exchange using
NaClO4 produced 2 with a modest yield (50%) in two steps.
2
To examine LPI by using 2H NMR, a H-labelled analogue 3
was prepared from 4 with an sn-3-6,6-dideuterated alkyl chain
by employing the same method as used for 2 to detect the
mobility of the analogue molecule in the membrane.28,33–35
With the synthetic analogues 2 and 3 in hand, we first eval-
uated the functions of the PGP-Me head group in restoring the
protein structure and biological activity of bR. In PM, bR is
aggregated into protein trimers to form a two-dimensional
hexagonal lattice, and the assembly of bR is known to demon-
strate the split Cotton effect with a negative peak at ca. 600 nm
in visible CD spectroscopy.18 As shown in Fig. 3a and b, the
CD spectra of partially delipidated bR (dbR), where ca. 76% of
the original PM lipids were removed, showed a small and an
invisible negative peak at 600 nm at 25 °C and 50 °C, respect-
ively, indicating that the trimer structure was largely dis-
assembled. On the other hand, reconstitution of dbR into
dipalmitoyl-PC (DPPC) and 2 liposomes restored an asym-
metric split-Cotton effect that was similar to that with PM at
25 °C, indicating the presence of the trimer structure. When
the temperature was raised to 50 °C, the biphasic pattern was
retained in PM, which supported the notion that bR has high
thermodynamic stability at high temperature.18 However, the
CD spectrum of DPPC/dbR was markedly changed from that at
50 °C, exhibiting a monophasic curve with a maximum peak at
ca. 560 nm. This result indicates that bR trimers were largely
dispersed as monomers in the DPPC membrane. A similar
phenomenon has been reported for DMPC liposomes, in
Fig. 3 CD spectra of PM, 2/dbR (100/1 mol), dbR, and DPPC/dbR
(100/1 mol) at temperatures of 25 °C (a) and 50 °C (b).
which trimeric bR disaggregated into monomers.5,8,17 More and decay of the M-intermediate, O-intermediate, and ground
interestingly, 2/dbR vesicles showed a similar CD spectrum as state, respectively.9 The proton pump activity of bR was highly
that of PM at 50 °C, implying that bR could retain its lattice perturbed by the partial delipidation of PM, implying a signifi-
structure in 2/dbR complexes in the fluid phase at higher cant change in the conformation of bR, as inferred from the
temperatures. Since the phase transition temperature of 2 was slow-down of photocycle kinetics (Fig. 4, green line). Coupled
determined by differential scanning calorimetry to be 41 °C,28 with the CD results, this finding indicates the importance of
which is the same as that of DPPC, the difference between 2 the surrounding lipids in maintaining the structural and func-
and DPPC is possibly not due to differences in their phase tional integrity of bR. As seen in Fig. 4, 2/dbR liposomes
status, but rather due to the stabilising effect of the head showed similar kinetic profiles as those of PM in both magni-
group in 2, which could partly reproduce that of the original tude and lifetime although the photocycle was slightly
PGP-Me.
increased, again implying that the original activity of bR was
Similar to mammalian rhodopsin, the biological functions restored in the artificial membranes containing 2. Similar tran-
of bR in PM are based on the protein structural change trig- sient absorption changes at 410 nm, 640 nm, and 560 nm of 2/
gered by light-induced isomerisation of retinal.36–39 The photo- dbR and PM were obtained in the liquid crystalline phase at
cycle kinetics of the reconstituted bR were examined by laser 50 °C (Fig. S2†).
flash photolysis to evaluate the influence of membrane lipids
Based on visible CD and laser flash photolysis, it is likely
on the protein function. Fig. 4 shows the absorbance changes that the dynamic formation of structural units by the inter-
at 410 nm, 640 nm, and 560 nm at 25 °C due to the increase action between bR and 2 is important to retain the original
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ture while the sharp isotropic peak at 0 ppm indicated the
presence of rapidly moving lipid aggregates. Thus, analogue 2
tends to form micelles, probably due to its smaller side chains
than those of the natural PGP-Me. For further NMR measure-
ments, we used plain-water dispersions of 2 due to the propen-
sity that the lipid forms micelles more easily in buffer than in
pure water, particularly without bR, which prevented us from
using a buffer for suspending 2 in aqueous media. The effects
of a buffer on 31P NMR spectra were examined as shown in
Fig. S4;† no noticeable difference in lipid mobility and
packing was observed between the presence and absence of a
buffer.
In Fig. 6a, the 2H NMR spectra of 3 without bR provided
two patterns, comprising a centre peak and an anisotropic
Pake doublet with a splitting of 23.1 kHz. The strong central
peak was assignable to isotropic components such as micelles
as observed in the 31P NMR spectrum. The addition of dbR sig-
nificantly altered the spectra (Fig. 6b). The reconstituted 3/dbR
(100/1 mol) showed an obviously enhanced magnitude with a
quadrupolar splitting of 25.5 kHz. The difference in splitting
magnitude implies that the interaction between 3 and bR
reduced its mobility (or wobbling) and resulted in the for-
mation of higher order alkyl chains. In addition, the formation
of micelles was greatly reduced as indicated by the decreased
isotropic peak intensity. Since the rate of exchange of annular
lipids with bulk lipids is much faster than the NMR time
scale,41 only one pair of splitting was observed in the 3/bR
membrane. After delipidation to obtain dbR, a few PM lipids,
mainly composed of PGP-Me and other lipids, remained on
the protein surface as determined by mass chromatography.
Fig. 4 Flash-induced absorbance changes at 410 nm (M intermediate),
560 nm (ground state), and 640 nm (O intermediate) for dbR (green),
2/dbR (100/1 mol) (red) and PM (blue) at 25 °C.
structure and functions of bR in the membranes. In order to
obtain detailed information on the molecular dynamics of this
process, bR was incorporated into the liposomes composed of
analogues 2 and 3 for solid-state 31P and 2H NMR measure-
ments, respectively.
First, we examined the formation of the lamellar bilayered
structure of 2 using solid-state 31P NMR spectroscopy. There
are two phosphate ester groups a and b in 2, which give rise to
two different 31P signals; the one in the terminal position (b)
is known to show a narrow peak near 0 ppm while the other
between two glycerol moieties (a) shows a typical pattern for
the axially symmetric system.40 As shown in Fig. 5, the 31P
NMR spectrum of 2 gave rise to three sets of signals, two of
which fitted well with those of PGP-Me in the bilayered struc-
Fig. 5 31P NMR spectrum of aqueous multilamellar dispersion of 2 (a),
and simulated spectra by Simpson (b) showing isotropic signals such as
that due to micelles and anisotropic signals. The spectrum was
measured with 60% hydrated membrane dispersions at 50 °C. Simulated
31P static spectrum (b) composed of two uniaxially symmetric powder
patterns due to two phosphate esters (red, 79 mol%) and one isotropic
pattern (blue, 21 mol%) corresponding to the lamellar structure and
micelle, respectively.
Fig. 6 2H NMR spectra of aqueous multilamellar dispersions of (a) 3, (b)
3/dbR (100/1 mol), and (c) 3/PM lipids (100/4 w/w). All the spectra were
measured with 60% hydrated membrane dispersions at 50 °C.
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Thus, the membrane preparation of 3/PM lipids (100/4 w/w) the original trimeric structure and light-activated photocycling.
was subjected to NMR measurements to confirm that the The unique double-charged head group of the analogue
ordering effect of the proteins observed in Fig. 6b was due to should play an important role in forming the lipid/protein
bR. As shown in Fig. 6c, the addition of PM lipids slightly functional complex. Using a 2H-labelled PGP-Me analogue 3,
decreased the splitting magnitude of 3, probably due to the the interactions between lipids and proteins were detected by
disordering nature of the methyl-branched alkyl chains in PM solid-state 2H NMR as indicated by the higher order of the
lipids. These observations imply that the enhancement of probe in the bR-containing membranes. These results indicate
quadrupolar splitting in 3/dbR was largely due to LPI; quadru- the usefulness of the synthetic analogue as a tool for investi-
polar splitting is known to be markedly influenced by subtle gating the LPIs occurring in PM.
changes in the membrane conditions, and thus, further NMR
experiments under different lipid compositions, buffers and
pH values may be necessary to confirm the present results.
These results again reveal that the head group of PGP-Me is
Acknowledgements
crucial for providing an appropriate environment for bR to We thank Profs S. Mitaku and Y. Yokoyama (Nagoya University,
form its intrinsic structure and proper function. The role of Japan) for the generous gift of H. salinarum strain R1M1,
the phytanyl chains of PM lipids including PGP-Me remains Prof. M. Sonoyama (Gunma University, Japan) for supporting
unknown in the present study. Chemical synthesis of this the measurements of laser flash photolysis, Drs H. Tsuchikawa
tetramethyl-hexadecanol with three stereogenic centres poses and S. Hanashima for discussions, and Ms K. Oshimo for
another synthetic challenge, and we are currently attempting assistance with purifying bR. This work was supported by the
to prepare a 2H-labelled probe to examine the effects of Japanese Society of Technology ERATO, and JSPS KAKENHI
the hydrophobic portions of PM lipids on bR in a further study Grant Number 25242073.
on LPI.
Previous 2H NMR studies on LPI have revealed that lipids
are important to reproduce the original structure and function
Notes and references
of embedded proteins.42,43 Standard phospholipids, such as
DPPC in this study, sometimes cause dissociation of protein–
protein interactions, resulting in unnatural contacts of model
lipids with the intrinsic protein–protein interfaces. Annular
lipids usually stay on the protein interface for less than a
microsecond,11 which hampers the selective observation of
their associated form. The order parameter of 3 upon interact-
ing with bR is increased by 10% (Fig. 6a and b), which is rela-
tively high as compared with the ordering effect of rhodopsin
on a saturated chain of PC.42 Thus, the firm association of 3
with bR may be one of the reasons for stabilisation of the tri-
meric assemblage; hydrophobic matching is also known to
play an important role in oligomerisation and functions of
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