Effects of molecular structures on the olfactory responses of phospholipid membranes to four alcohols

Article abstract of DOI:10.1016/S0009-3084(98)00054-1

In order to understand the relationship between phospholipid molecular structures and their olfactory responses to odorants, we designed and synthesized four phosphatidylcholine analogues with different long hydrocarbon (CH) chains and selected three natural phospholipids with different head-groups. By using interdigital electrodes (IEs) as olfactory sensors (OSs), we measured the responses of the IEs coated with these seven different lipid membranes to four alcohol vapors in a gas flow system. The IEs voltage changes were recorded and the voltage-relative saturate vapor pressure (V-P/P°) curves were also plotted. It was found that with a methyl (-CH₃) placed at the C-8 position in the 18-carbon chain, the olfactory responses could be improved about ten times and with conjugated double bonds (C=C) in the long chains, the sensitivity could be increased by 3~4 orders of magnitude. As to head-groups, choline is preferred over ethanolamine and serine in phospholipid structures in terms of high olfactory sensitivity. These results are expected to be useful in further designing and manufacturing lipid-mimicking OSs. Copyright (C) 1998 Elsevier Science Ireland Ltd.

Full text of DOI:10.1016/S0009-3084(98)00054-1

Chemistry and Physics of Lipids
95 (1998) 1–9
Effects of molecular structures on the olfactory responses of
phospholipid membranes to four alcohols
Qingzhong Zhou a,*, Yanan Yang a, Zhikuan Chen a, Anna Sun b
a College of Chemistry and Molecular Engineering, Peking Uni6ersity, Beijing 100871, People’s Republic of China
b Institute of Semiconductors, Chinese Academy of Sciences, People’s Republic of China
Received 6 March 1998; received in revised form 28 May 1998; accepted 28 May 1998
Abstract
In order to understand the relationship between phospholipid molecular structures and their olfactory responses to
odorants, we designed and synthesized four phosphatidylcholine analogues with different long hydrocarbon (CH)
chains and selected three natural phospholipids with different head-groups. By using interdigital electrodes (IEs) as
olfactory sensors (OSs), we measured the responses of the IEs coated with these seven different lipid membranes to
four alcohol vapors in a gas flow system. The IEs voltage changes were recorded and the voltage-relative saturate
vapor pressure (V-P/P°) curves were also plotted. It was found that with a methyl (–CH3) placed at the C-8 position
in the 18-carbon chain, the olfactory responses could be improved about ten times and with conjugated double bonds
(CꢀC) in the long chains, the sensitivity could be increased by 3ꢀ4 orders of magnitude. As to head-groups, choline
is preferred over ethanolamine and serine in phospholipid structures in terms of high olfactory sensitivity. These
results are expected to be useful in further designing and manufacturing lipid-mimicking OSs. © 1998 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Olfactory sensor; Interdigital electrode; Hydrocarbon chain; Head-group; Voltage response
Abbre6iations: IE, interdigital electrode; OS, olfactory sensor; CH, hydrocarbon; 2-(8-Me)DSPC, 1-stearoyl-2-(8-methyl)stearoyl-
sn-glycero-3-phosphatidylcholine; 1,2(8-Me)DSPC, 1,2-di-(8-methylstearoyl)-sn-glycero-3-phosphatidylcholine; DEPC, 1,2-
dielaeostearoyl-sn-glycero-3-phosphatidylcholine; LPC, lysophosphatidylcholine; PC, L-h phosphatidylcholine; PE, L-h
phosphatidylethanolamine; PS, L-h phosphatidylserine; MeOH, methanol; EtOH, ethanol; PrOH, propanol; iso-PrOH, iso-
propanol; Tc, temperature of phase transition; DSC, differential scanning calorimetry; V-P/P°, voltage-relative saturate vapor
pressure.
* Corresponding author. Tel.: +86 1 067291869; fax: +86 1 062574819; e-mail: postmaster@chemms.chem.pku.edu.cn
0009-3084/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(98)00054-1

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Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
1. Introduction
hydrocarbon (CH) chains, for example, there is
a methyl substituent at the C-8 position in the
18 carbon chain or there are conjugated double
bonds in the long chain. The three natural phos-
pholipids have different head-groups which are
choline, ethanolamine and serine. These seven
lipids were coated on the IEs by using the
method described previously (Cai et al., 1993;
Sun et al., 1995). In a gas flow system, the IEs
gave the responses to four alcohol (methanol,
ethanol, n-propanol and iso-propanol) vapors at
different relative saturate vapor pressure. The
voltage responses of IEs were then detected and
the voltage-relative saturate vapor pressure (V-P/
P°) curves were given. Our results show that the
olfactory responses to odorants vary greatly
with lipid molecular structures.
Olfaction is a specific chemical sense of ani-
mals to odorants. Its sensitivity and resolution
power are so remarkable that they can reach
molecular level. In 1987, Kurihara K. et al. put
forward the olfactory proposal of lipid-adsorp-
tion and the model of interface potential
changes (Nomura and Kurihara, 1987a,b). They
suggested that olfactory stimulation initially in-
volve odorant molecules being adsorbed to a
lipid layer of olfactory cell membranes. This
causes a potential change of the membrane and
then generates a nervous impulse. Olfactory dis-
crimination is due to the differences of lipid
components and structures in each olfactory cell
membrane that produce different membrane po-
tentials to different kinds of odorant molecules
(Nomura and Kurihara, 1987a; Entomoto et al.,
1991). As a result, it has been found that lipids
could be used as sensitive membranes for olfac-
tory and taste sensors (Okahata et al., 1989;
Tamiya et al., 1990; Nakamoto et al., 1991).
Based on the hydrophobic adsorption or solu-
tion in the hydrophobic area, odorant molecules
can affect the potential and other electronic
properties of olfactory cell membranes by
changing the conformation of membrane
molecules, the liquid crystallinity (ordering and
mobility) and the polar head-group charges or
dipolar ions’ strength and orientation (Nomura
and Kurihara, 1987b). Sun et al. had made ol-
factory sensors (OSs) by using interdigital elec-
trodes (IEs) coated with phosphatidylcholine
and similar chemicals as sensitive membranes.
The characteristics of these sensors are quite
similar to the human olfactory sense (Sun et al.,
1994, 1995). In this paper, in order to have a
better understanding about the relationship be-
tween phospholipid molecular structures and
their olfactory responses to odorants, we de-
signed and synthesized (Charp et al., 1988;
Menger et al., 1988a; Zhou et al., 1988; Zhou,
1992) four phosphatidylcholine analogues, 2-(8-
Me)DSPC, 1,2(8-Me)DSPC, DEPC and LPC.
We also selected three natural phospholipids,
PC, PE and PS as objects. The four phos-
phatidylcholine analogues have different long
2. Experiment
2.1. Lipids
Four phosphatidylcholine analogues (1)–(4)
and three natural phospholipids (5)–(7) are as
follows:
1. 1-stearoyl-2-(8-methyl)stearoyl-sn-glycero-3-
phosphatidylcholine
(2-(8-Me)DSPC
for
short), a methyl at C-8 of one long CH chain.
2. 1,2-di-(8-methylstearoyl)-sn-glycero-3-phos-
phatidylcholine (1,2(8-Me)DSPC), a methyl at
C-8 in both chains.
3. 1,2-dielaeostearoyl-sn-glycero-3-phosphatidyl-
choline (DEPC), conjugated double bonds in
both chains.
4. lysophosphatidylcholine (LPC), with only one
long chain.
5. L-h phosphatidylcholine (PC), from egg yolk,
purchased from Sigma Co., purity approx.,
99%, with choline as head-group
6. L-h phosphatidylethanolamine (PE), pur-
chased from Sigma Co., with ethanolamine as
head-group
7. L-h phosphatidylserine (PS), from bovine
brain, purchased from Sigma Co., with serine
as head-group
Fig. 1 shows the structures of the seven phos-
pholipids.

Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
3
2.2. The syntheses of the four analogues
5. PBr3/Pyridine/heating
6. H2/PtO2
2.2.1. The synthesis scheme of 8-methyl stearic
acid
7. KOH/H2O–EtOH
Fig. 2 demonstrates the synthesis scheme of
8-methyl stearic acid. The conditions of every
reaction step:
1. EtOH, EtO2C (CH2)6CO2Et/Conc. HCl,
dibutylether/heating
2.2.2. The syntheses scheme of the four analogues
Fig. 3 demonstrates the syntheses scheme of the
four analogues. The conditions of every reaction
step:
1. Et4N+OH−, ether/CdCl2, EtOH
2. SOCl2
3. [CH3(CH2)9]2Cd/benzene
4. CH3MgBr/diethyl ether
2. Snake venom, CaCl2/EtOH, Et2O, H2O
3. 8-methyl stearic acid, DCC, DMAP/CHCl3 or
elaeostearic acid, DCC, DMAP/CHCL3
Fig. 1. Molecular structures of 2-(8-Me)DSPC, 1,2(8-Me)DSPC, DEPC, LPC, PC, PE and PS.

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Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
Fig. 2. Synthesis scheme of 8-methyl stearic acid.
4. 8-methylstearic anhydride, DMAP/CHCl3
2.3. Measurements of membrane properties
The membrane structures were measured by
small angle X-ray diffraction and the temperature
of phase transition (Tc) was obtained by differen-
tial scanning calorimetry (DSC).
The phospholipid was dissolved in organic sol-
vent, commonly a mixture of chloroform and
ethanol (1:3 v/v). Then it was coated on an IE to
form the olfactory sensitive membrane by self-as-
sembly. The coating procedure was as follows. A
2 vl solution was added to the IE chip by mi-
crosyringe at room temperature. After evapora-
tion of the solvent and storage for 2 h, the IE was
ready for use. The IE was suspended in a gas flow
system. In the system, the carrier gas saturated by
an odorant vapor at constant temperature
(25.090.5°C) had been diluted to different rela-
tive vapor pressures with dry air. The voltage
changes of IE to odorants were measured. The
V-P/P° curves were plotted.
3. Results and discussion
According to the measurements described in
Section 2, we found that the IEs have no voltage
change to alcohol vapors without the phospho-
lipid membranes. After having been coated with
these phospholipid membranes, they can give sim-
ilar V-P/P° curves which all belong to adsorption
isotherm III. At a low concentration of the odor-
ant vapor, there is only adsorption, which means
that alcohol molecules only interact with lipid
molecules. At a high concentration, adsorption
co-exists with condensation, in other words, alco-

Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
5
hol molecules interact not only with lipid
molecules but also with themselves. The V-P/P°
curves shown in Fig. 4 indicate 1,2(8-Me)DSPC,
2-(8-Me)DSPC, LPC, PC and DEPC membranes’
responses to three alcohol vapors, ethanol
(EtOH), propanol (PrOH) and iso-propanol (iso-
PrOH).
In Fig. 4, these five lipid membranes have the
same olfactory response pattern. All obey Steven’s
Law. It indicates that 1,2(8-Me)DSPC, 2-(8-
Me)DSPC, PC and DEPC membranes have the
same order of the response strength to the three
these five kinds of membranes to methanol and
ethanol at 50% relative saturate vapor pressure
(25°C, P/P°).
From Fig. 4 and Table 1, the membrane re-
sponses increased greatly with a methyl sub-
stituent or conjugated double bonds in the CH
chains of phospholipids. As to 1,2(8-Me)DSPC
and 2-(8-Me)DSPC with methyl substituents in
the long CH chains, their voltage responses are
10–20 times higher than that of PC. As to DEPC
with three conjugated double bonds in the long
chains, the response has been improved four or-
ders of magnitude compared with that of PC.
These results are related to the effects of the
methyl and conjugated double bonds on the con-
formation and liquid crystallinity of lipid mem-
branes.
alcohols.
The
order
is:
DEPCꢁ1,2(8-
Me)DSPC\2-(8-Me)DSPC\PC. However, LPC
is more special, it has not a fixed position in the
order. With only one long CH chain in the
molecule, the LPC membrane is not stable and
this flexibility can produce a special response pat-
tern. In order to demonstrate the results more
clearly, Table 1 gives the voltage responses of
When all the C–C bonds of long CH chains of
fatty acid are at the all-trans position, the confor-
mation is more stable and the carbon chain is in
Fig. 3. Syntheses scheme of 2-(8-Me)DSPC, 1,2(8-Me)DSPC, DEPC and LPC.

6
Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
Fig. 4. Voltage-relative saturate vapor pressure (V-P/P°) curves to EtOH, PrOH and iso-PrOH vapors (a): 1,2(8-Me)DSPC; (b):
2-(8-Me)DSPC; (c): LPC; (d): PC; and (e): DEPC.
the shape of sawteeth in a plane. When there is a
methyl group in the chain, the conformation tends
to convert from trans to gauche (Menger et al.,
1988b). For instance, see Fig. 5A, with a methyl
at C-8 position of stearic acid, the linearity of the
latter half chain can be reestablished through a
second gauche to give a so-called 2gl kink, which
means that at the methyl substituted carbon, the
C–C bond rotates by 120° (g+), the next C–C
bond is trans (t) and the third C–C bond gives

Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
7
Table 1
Voltage responses (mv) of 2-(8-Me)DSPC, 1,2(8-Me)DSPC, DEPC, LPC and PC membranes to 50% relative saturate vapor pressure
of EtOH and MeOH
1,2(8-Me)DSPC
2-(8-Me)DSPC
LPC
DEPC
PC
50% EtOH
50% MeOH
20.2
130.0
13.4
102.3
1.4
3.2
11 300
\40 000
0.8
5.1
another rotation by −120° (g−). Consequently,
atoms are overlapped and easily polarized, which
can produce a stronger electric signal. So double
bonds can make a stable and rigid conformation
and impart a bow-shaped molecule, which enhances
the adsorption of odorants.
˚
the overall chain length decreases 1.3 A and the
3
˚
molecule volume increases 25–50 A (Menger et
al., 1988a). As a unit which can induce disorder in
the molecule, the methyl permits a degree of
conformational flexibility at the site of disruption.
The result of calculations reveals that the molecu-
lar across area of 8-methylstearic acid increases
50%. Another consequence of introducing methyl
at C-8 position is that it can stabilize the flexible
bend at the center of the alkyl chain. Accordingly
the segment of the long alkyl chain nearer to the
head group lies at a 30° angle to the membrane
surface and the terminal part is perpendicular to
the surface. This induces a bend at the center of
the chain (without any substituent at the center,
the bend is not stable). This central bend provides
the second half of the chain about 12% more
space in which to maneuver. More space can
benefit the adsorption of membranes to odorants
and then increase the olfactory sensitivity.
The introduction of substituents and double
bonds induces the change of membrane liquid
crystallinity, increases the mobility and decreases
the phase transition temperature. The membrane
tends to change from gel to liquid crystal phase. All
these results agree with the measurements of DSC,
Tc: PC: 54.8°C; 2-(8-Me)DSPC: 18.6°C; 1,2(8-
Me)DSPCB0°C. Therefore, increasing the mem-
brane mobility can enhance olfactory sensitivity.
As to head-groups, PC, PE and PS have differ-
ent olfactory responses to odorants. From Table
2, PE and PS only have a small response to
methanol and ethanol at high concentration (80%
more). It indicates that choline as the head-group
of PC is essential. It is a zwitter ion in which a
positive charge balances a negative charge. The
positive and negative charges in the head-group
benefit the change of membrane electronic proper-
ties. The three methyls in the choline provide a
more extensive conformation to the head of the
molecule which in turn provides more space for
the hydrophobic chains to maneuver. This struc-
ture is important for the entry of odorants into
membrane and for the inducement of the change
in electronic properties. Fig. 6 shows the head-
group structure of PC.
Elaeostearic acid is cis, trans, trans-9,11,13-oc-
tadectrienoic acid. Double bonds also make the
conformation of long CH chain change and stabilize
the center bend. Fig. 5B shows the conformation
of elaeostearic acid. Double bonds are rigid and the
conjugated system averages the relevant single and
double bonds. The y-electrons between carbon
The membrane structure was measured with
small angle X-ray diffraction. It shows that the
˚
adsorption peak lay at 40–60 A, which is lower
˚
than the thickness of 60–75 A of lipid bilayer.
Here we can conclude that the lipid bilayer is
formed through self-assembly in inclining or non-
perpendicular packing. The membrane structure is
a laminar accumulation in two dimensions.
Fig. 5. Conformations of two kinds of CH chains. A: 8-methyl
stearic acid, B: cis, trans, trans-9,11,13-octadectrienoic acid.

8
Q. Zhou et al. / Chemistry and Physics of Lipids 95 (1998) 1–9
Table 2
of the membrane molecules. Such a result is
useful in further designing and manufacturing
lipid-mimicking OSs.
Voltage responses (mv) of PC, PE and PS to 80% relative
saturate vapor pressure of EtOH and MeOH
PC
PS
PE
80% EtOH
80% MeOH
7.2
45.7
0
6.0
0.4
2.4
Acknowledgements
This work was supported by the National Nat-
ural Sciences Foundation of China.
4. Conclusion
1. The membranes of PC, 2-(8-Me)DSPC, 1,2(8-
Me)DSPC, DEPC and LPC by self-assembly
have a similar olfactory response pattern.
2. The structural differences of two long CH
chains in phospholipids, which include their
conformation and polarity, influence the mem-
brane order and mobility. This can result in a
different response sensibility of membranes to
odorants. Methyl and conjugated double
bonds introduced in the long chains can im-
prove the sensitivity substantially.
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