2
04
J. Cui et al. / Bioorg. Med. Chem. Lett. 25 (2015) 203–206
signals of DOPC partitioned in L
known as the major phospholipid of human lens membranes,
and also reported to be involved in pathological events such as
o
even more difficult. DHSM is
outer doublet peak to the inner doublet peak is 28/72. As temper-
ature rose, the order of 9 in L phase decreased while that in L
1
4
o
d
phase increased, and the phase separation was poorly observed
at 40 °C. Then, at 45 °C, the spectra gave completely fused signals,
and the peak became sharp and less ordered at 50 °C, indicating the
1
5,16
viral infections.
As shown in Scheme 1, the improved synthesis began with the
preparation of phosphonium salt from brominated ester 1, fol-
lowed by Wittig olefination to give pure cis-olefin 2. Subsequent
2
membranes were uniformly mixed. The H NMR spectra also
shows isotropic components as shown by the central peak.
Although the sample contains a small amount of HDO in the water,
the isotropic signal is most likely due to the presence of non-ori-
ented structures (such as vesicles) tumbling rapidly in water. As
temperature rose, more amounts of isotropic non-oriented phases
were formed.
4
reduction of the ester with LiAlD afforded deuterated alcohol 3,
which was tosylated to give the deuterated intermediate 4. A
recent study suggested that the tosylated precursor is a more suit-
able coupling partner than the brominated one for Csp3–Csp3
17
KumadaÀCorriu coupling. Compound 4 was subsequently cou-
pled with the Grignard reagent 5 prepared from the commercial
benzyloxypentyl bromide to afford cross-coupled product 6 in high
Due to the superior performance of 9 with regard to the clear
L /L phase separation and high sensitivity for phase transitions,
o d
yield. Benzyl deprotection by boron trichloride followed by Jones’
we were convinced that this molecule could be further applied
to probe the membrane property alteration, which was thought
to be closely related to cellular functions. DHSM is the major
phospholipid of human lens membranes, whereas being a rather
2
oxidation led to the desired [C6- H
2
] oleic acid 8 in 24% overall
] DOPC 9 was prepared using
8:1-lyso PC under typical condensation conditions.
2
yield after 7 steps. Finally, [C6- H
2
1
2
13
With compound 9 in hand, we first measured the H NMR spec-
trum for its powder-type hydrated membrane and compared it
minor constituent in other tissues. However, an enrichment of
DHSM was found in the HIV-1 membrane, produced from the
2
20
with the previous probe [C11- H
2
] DOPC in ternary liposomes.
host cell through viral release. Recent studies indicated that
1
5,16
The standard wideline spectra were recorded as shown in Figure 1.
DHSM is important for HIV-1 gp41-mediated fusion.
The role
As expected, 9 generates much more clearly separated peaks than
of DHSM in regulating membrane properties can be speculated
by previous study that DHSM tends to form rigid domains due
to the lack of trans double bond in the ceramide backbone.14
To further examine the relevance of DHSM in domain formation,
2
[
C11- H
2
] DOPC does. The two doublet peaks in each spectra corre-
and L phase, respectively, the major fraction of
phase as shown by the larger integrated intensity
sponding to the L
DOPC is in the L
o
d
d
of the inner doublet. Notably, the peak distance between inner and
compound
system.
9 was applied to the DHSM-containing ternary
outer splittings in 9/SM/chol membranes increased by 1.6 fold
2
compared to [C11- H
2
] DOPC-containing mixtures. The above data
As compared with 9/SM/chol mixtures, less fraction of 9 should
confirms that 9 is the better probe appropriate for the L
o
d
/L -sepa-
be partition to the highly ordered DHSM-enriched phase. Fortu-
rated systems.
o
nately, small but clearly visible distribution of 9 in L phase was
The macroscopically aligned membranes, in which the bilayer
normal is perpendicular to the magnetic field direction, have
improved spectral resolution and sensitivity compared to powder
detected as shown by the outer doublet in the bottom spectra in
Figure 2b. At 30 °C, the integration area ratio of the outer doublet
peak to the inner doublet peak drops to 16/84, much lower than
that in the SM ternary mixtures, revealing that the greater amount
of DOPC is excluded from the DHSM-enriched phase. As tempera-
1
8,19
pattern samples.
Thus, we chose to study the thermodynamics
of 9 in such an oriented membrane system. In the bottom trace of
2
Figure 2a, the H NMR spectrum of oriented 9/SM/chol bilayers at
o
ture rose, the order of 9 in L phases decreased slower than that in
3
0 °C provided completely separated doublets with even higher
the SM mixture, and the apparent phase separation was clearly
observed even at 45 °C, at which only one doublet appeared in
the SM mixtures, implying the thermostability of the L domains
o
was obviously enhanced in the DHSM mixture. These results are
2
resolution than the powder-type H NMR spectra (Fig. 1a). It is
clear to see that small fraction of 9 is in the L phase, while major
fraction pertains to the L phase. The integration area ratio of the
o
d
i. PPh
3
, toluene,
o
LiAlD4
THF, 0 o
80%
120
C
O
O
7 15
C H
Br
C
ii. nonanal, KHMDS
o
O
O
THF, -78
C
1
2
50% (2 steps)
CuCl
2
, Ph
Me
BnO
MgBr
5
HO
D
C
7
H
15 TsCl, Et
3
N
TsO
D
7 15
C H
5
THF, 0 oC to rt
90%
D
CH
2
Cl
2
, rt
D
4
3
98%
BCl
3 2 2
, CH Cl
C
7
H
15
7 15
C H
BnO
HO
78 oC to rt
3
D
D
6
-
3
D
D
95%
7
1
8:1-lyso PC
O
MNBA, DMAP
Jones' reagent
acetone, rt
C
7
H
15
HO
CH Cl , rt
2
2
3
D
D
D
72%
8
60%
O
6
O
O
D
O
O
N
P
O
O
O
9
2
2
Scheme 1. Synthesis of [C6- H
2
] oleic acid 8 and [C6- H
2
] DOPC 9.
Cui, Jin
