FT-IR and NMR studies on the conformational and structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidyloligoglycerols

Article abstract of DOI:10.1021/jp026509t

The conformational behavior of membranes derived from a new class of phospholipids, 1,2-dipalmitoyl-sn-glycero-phosphatidyloligoglycerols DPPG_x, is studied for the first time by FT-IR spectroscopy in the liquid crystalline and gel state. The phospholipids examined here are characterized by oligoglycerol chains of variable length in the headgroup, ranging from one (x = 1, DPPG₁ = DPPG) to four glycerol units (x = 4, DPPG₄). The transition from the gellike to the liquid crystalline phase is monitored via CH₂ and CD₂ stretching bands as well as CH₂ wagging band progressions. CH₂ wagging bands are used to determine the relative amounts, i.e., integral values over the whole chains, of kink/gauche-trans-gauche, double gauche, and end gauche conformers in the acyl chain region. Information about the absolute amount of gauche conformers at a specific chain segment is obtained by a quantitative analysis of the CD₂ rocking band region for phospholipid samples with selectively deuterated acyl chains. These latter data are combined with the results from an independent ²H NMR study on the same compounds, which allows a distinction between the overall chain order and the local conformational order of the phospholipid chains. In addition, results from solid state ³¹P and ¹³C NMR studies are presented which provide additional support for the conclusions based on the FT-IR data. The present work clearly shows that the conformational and structural properties strongly depend on the headgroup structure and hydrophilicity, sample composition as well as sample temperature. The derived data are discussed by considering related phospholipid systems, such as DPPC, to demonstrate the particular impact of the headgroup length and hydrophilicity on the ordering characteristics of the acyl chains.

Full text of DOI:10.1021/jp026509t

J. Phys. Chem. B 2003, 107, 75-85
75
FT-IR and NMR Studies on the Conformational and Structural Properties of
1
,2-Dipalmitoyl-sn-glycero-3-phosphatidyloligoglycerols
†
Ren e´ Lehnert, Hans-J o1 rg Eibl, and Klaus M u1 ller*
Institut f u¨ r Physikalische Chemie, UniVersit a¨ t Stuttgart, Pfaffenwaldring 55, D- 70569 Stuttgart, Germany
ReceiVed: July 12, 2002; In Final Form: October 24, 2002
The conformational behavior of membranes derived from a new class of phospholipids, 1,2-dipalmitoyl-sn-
glycero-phosphatidyloligoglycerols DPPG , is studied for the first time by FT-IR spectroscopy in the liquid
crystalline and gel state. The phospholipids examined here are characterized by oligoglycerol chains of variable
length in the headgroup, ranging from one (x ) 1, DPPG ) DPPG) to four glycerol units (x ) 4, DPPG ).
The transition from the gellike to the liquid crystalline phase is monitored via CH and CD stretching bands
as well as CH wagging band progressions. CH wagging bands are used to determine the relative amounts,
x
1
4
2
2
2
2
i.e., integral values over the whole chains, of kink/gauche-trans-gauche, double gauche, and end gauche
conformers in the acyl chain region. Information about the absolute amount of gauche conformers at a specific
chain segment is obtained by a quantitative analysis of the CD rocking band region for phospholipid samples
2
with selectively deuterated acyl chains. These latter data are combined with the results from an independent
2
H NMR study on the same compounds, which allows a distinction between the overall chain order and the
3
1
13
local conformational order of the phospholipid chains. In addition, results from solid state P and C NMR
studies are presented which provide additional support for the conclusions based on the FT-IR data. The
present work clearly shows that the conformational and structural properties strongly depend on the headgroup
structure and hydrophilicity, sample composition as well as sample temperature. The derived data are discussed
by considering related phospholipid systems, such as DPPC, to demonstrate the particular impact of the
headgroup length and hydrophilicity on the ordering characteristics of the acyl chains.
Introduction
tional changes at a specific CH2 segment as a function of
1
4-17
In recent years spectroscopic techniques, such as NMR1-3
temperature can be obtained.
combined with those from supplementary H NMR experiments.
On this basis it is possible to distinguish between conformational
These data can be further
2
and IR spectroscopy,4-7 have been extensively used in the field
of biological membranes. In this connection it has been
demonstrated that IR spectroscopy is particularly suitable to
monitor the conformational properties in the acyl chain region
of the phospholipid molecules along with their changes as a
function of external parameters, such as temperature or sample
composition. Here, several conformation sensitive vibrational
bands are accessible, from which the desired information about
(or segmental) order and overall chain order of the phospholipid
molecules, as already demonstrated during an earlier work on
1
6
other phospholipids.
The variable temperature FT-IR and NMR investigations pre-
sented here were performed on a new class of model membranes
that differ from the biologically relevant and widely studied1
1,2-dipalmitoyl-sn-glycero-phosphatidylglycerol DPPG (in the
following: DPPG1) in the length of the glycerol unit of the
headgroup. Thus, we examine 1,2-dipalmitoyl-sn-glycero-phos-
phatidyl-oligoglycerols DPPGx (x ) 1-4) with one to four
8,19
4
-8
the conformational state can be obtained.
On the basis of these former IR investigations in the present
work data will be provided, that are derived from the analysis
of the following well-known conformation sensitive IR re-
gions: the symmetric CH2 stretching band at around 2850 cm
and antisymmetric CD2 stretching band at around 2172 cm ,
from which information about conformational order-disorder
transitions can be received; the CH2 wagging progressions
between 1100 and 1380 cm , which reflect changes in the
amount of all-trans chains upon temperature variation;
wagging band region between 1315 and 1400 cm , which yield
quantitative information about the integral amount of various
gauche conformers (kink/gtg (gauche-trans-gauche), double
gauche (dg), and end gauche (eg) conformers) along the aliphatic
chains in the liquid crystalline state;
in the region from 620 to 670 cm from which, using selec-
tively deuterated compounds, data about the local conforma-
2
0
-
1
glycerol units attached to the phosphate unit (Figure 1). Like
2
1
-
1
the “Stealth phospholipids”, liposomes on the basis of these
DPPGx show a prolonged in ViVo blood circulation time of up
2
0
9
to 24 h and are therefore of great interest as drug delivery
systems. It has been demonstrated that this ability is more
-
1
1
0,11
3 4
pronounced in DPPG and DPPG than that for membrane
the
-1
systems bearing two glycerol units (DPPG ) or one glycerol
2
unit (DPPG1), respectively. It is therefore of interest in which
manner the length and hydrophilicity of the headgroup affects
the conformation and structure of the lipid and how that, in
return, alters the stability of these systems under physiological
conditions.
1
2,13
the CD2 rocking bands
-
1
A first attempt to characterize this new class of phospholipid
systems on a molecular level is the combined IR and NMR
investigation presented in this work. The studies have been
performed on fully hydrated bilayers containing either pure lipids
or lipid/cholesterol mixtures, with a 40% molar fraction of
*
Author for correspondence. Phone: +49(711) 685 4470. FAX: +49-
(
711) 685 4467. E-mail: k.mueller@ipc.uni-stuttgart.de.
†
Max-Planck-Institut f u¨ r Biophysikalische Chemie, Am Fassberg 11,
D-37070 G o¨ ttingen, Germany.
1
0.1021/jp026509t CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/10/2002

76
J. Phys. Chem. B, Vol. 107, No. 1, 2003
Lehnert et al.
Synthesis of Oligoglycerols and 1,2-Dipalmitoyl-sn-glycero-
2
6-29
phosphatidyloligoglycerols.
The synthetic routes for the
preparation of the different phosphatityloligoglycerols have been
described earlier and follow the synthesis of phosphatidylglyc-
2
6
erol. To obtain the phosphatidyloligoglycerols, in the phos-
phorylation step isopropylidene glycerol was exchanged for
oligoglycerols with the respective protecting groups. For instance
2
-O-tetrahydropyranyl-glycero-1.3′-(1′.2′-isopropylidene)-glyc-
erol was used for the preparation of phosphatidyl-diglycerol,
2-tetrahydropyranyl)-glycero-1.3′-O-(2′-O-tetrahydropyranyl)-
′.3′′-O-(1′′.2′′-isopropylidene)-glycerol for the preparation of
(
1
phosphatidyl-triglycerol and (2-tetrahydropyranyl)-glycero-1.3′-
O-(2′-O-tetrahydropyranyl)-1′.3′′-O-(2′′-O-tetrahydropyranyl)-
1
′′.3′′′-O-(1′′′.2′′′-isopropylidene)-glycerol for the preparation
of phosphatidyl-tetraglycerol. The synthesis of these protected
oligoglycerols will be described in detail elsewhere.27
The purity of the described products is shown by thin layer
chromatography. 1.2-Dipalmitoyl-sn-glycero-3-phospho-glycerol
Figure 1. Chemical structures of the phospholipid systems used in
this work.
(
DPPG1) does not contain DPPG2, DPPG3 or DPPG4. Cor-
respondingly, 1.2-Dipalmitoyl-sn-glycero-3-phospho-diglycerol
DPPG2) is free from DPPG1, DPPG3, or DPPG4, etc. On the
(
cholesterol. In the case of DPPG2 a selectively deuterated sample
bases of HPTLC data, with Rf-values of 0.65 for DPPG1, 0.55
for DPPG2, 0.45 for DPPG3, and 0.35 for DPPG4 in the solvent
system CHCl3:CH3OH:CH3COOH:H2O 100:60:20:5 (per vol-
ume), the purity of the four phosphatidyloligoglycerols DPPG1,
DPPG2, DPPG3, and DPPG4 is better than 99%. This is much
better than for the products described earlier by Maryama et
(
deuteration at carbon C-6) was available which was examined
2
by both FT-IR and H NMR spectroscopy. Apart from the
analysis of the above-mentioned conformation sensitive IR bands
and the H NMR data, results from preliminary solid state
2
31
P
NMR and 13C MAS NMR studies will be presented. In the
discussion we will compare the influence of the length of the
glycerol unit as well as sample temperature and cholesterol
content on the conformational and ordering behavior of 1,2-
dipalmitoyl-sn-glycero-phosphatidyl-oligoglycerols. In addition,
the data of the present systems will be contrasted with those
from “classical” phospholipids, such as DPPC or DMPC.
2
8
al. which have been obtained by transesterification of phos-
phatitylcholines with phospholipase D in the presence of impure,
2
9
technical oligoglycerols.
(b) Sample Preparation. The multilamellar, cholesterol free
lipid dispersions were prepared by the addition of 15 mL
deuterium depleted water to 10 mg of lipid to ensure a complete
hydration of the glycerol headgroup. Homogenization was
obtained by repeated freeze-thawing, centrifuging, vortexing,
and incubation for about 3 h at temperatures well above the
main transition of the membrane. The cholesterol containing
dispersions were prepared by dissolving the appropriate amounts
of lipid and cholesterol (40% molar ratio) in a small quantity
of freshly distilled chloroform. The solvent was removed in a
dry nitrogen stream, followed by evaporation in a vacuum for
a few hours. The subsequent hydration and freeze-thawing steps
were carried out as described above.
Experimental Section
(a) Materials. The present spectroscopic investigations have
been performed on nondeuterated (DPPGx, DPPC) as well as
on selectively deuterated phospholipids (DPPG2, DPPC). Se-
lectively deuterated lipids were obtained by the attachment of
hexadecanoic acid-6,6-d2 that also was synthesized during the
present work. Likewise, for the synthesis of DPPGx the
preparation of several oligoglycerols was required. In the final
step the deuterated or nondeuterated hexadecanoic acid was
coupled with choline or an oligoglycerol headgroup to give
DPPC and DPPGx, respectively. In the following, the various
synthesis steps will be described briefly.
(c) Variable Temperature FT-IR Measurements. The
nondeuterated phospholipid dispersions were measured using
CaF2 windows and a 25 µm zinc spacer, whereas for the
Synthesis of 6,6-d2-Hexadecanoic Acid (Palmitic Acid).2
The initial step of this synthesis is the preparation of the
corresponding selectively deuterated 1-undecanol by reduction
of the appropriate ester or carboxylic acid with LiAlD4 in dry
THF. The next step includes the bromination of the alcohol.
After that, the Grignard agent of the resulting bromide was
coupled with the magnesium bromide of the 5-bromo-valeric
acid in the presence of stoichiometric amounts of CuCl2/LiCl
catalyst to give the desired selectively deuterated palmitic acid.
2-24
detection of CD rocking bands KRS5 windows and spacer with
2
6 µm thickness were used. All IR spectra were recorded on a
Nicolet Nexus 470 FT-IR spectrometer (Nicolet, Madison, WI)
using the OMNIC E. S. P. 5.1 software (Nicolet, Madison, WI)
equipped with a DTGS detector. The phospholipid samples were
thermostated in a variable temperature transmission cell (L. O.
T.-Oriel GmbH, Langenberg, Germany), equipped with NaCl
windows. The sample temperature was regulated with an
automatic temperature control unit. The temperature accuracy
was estimated to (0.5 °C. Typically, 1024 interferograms,
25
Synthesis of 1,2-Dipalmitoyl-sn-glycero-phosphatidylcholine.
-1
In a first step the palmitic acid is transferred to the corresponding
acid imidazolide using carbodiimidazolide (CDI; Fluka Chemie
AG, Buchs, Germany). In the next step sn-glycero-3-phosphati-
dylcholine (GPC, Lukas Meyer GmbH, Hamburg, Germany)
and 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU, Fluka Chemie
AG, Buchs, Germany) were added to the reaction mixture, which
was stirred for 24 h, and the solvent was removed. The resulting
lipid was purified via recrystallization in dry acetone.
covering a spectral range from 4000 to 400 cm at a resolution
-
1
of 2 cm , were collected in the temperature range between
223 and 353 K, apodized with a triangular function and Fourier
transformed after one zero filling. Correction for background
absorption was done by recording the background spectrum of
the empty cell (measured with twice the number of interfero-
grams as that used for the sample). The background spectrum
was automatically subtracted from the subsequent spectra of

Conformational and Structural Properties of DPPGx
J. Phys. Chem. B, Vol. 107, No. 1, 2003 77
2
the phospholipid samples. Data from three independent samples
were acquired at all temperatures for all samples studied. The
whole series of variable temperature FT-IR spectra was mea-
sured twice for each sample. The data analysis is thus based on
an average of six measurements.
(e) NMR Measurements. H NMR and 31P NMR measure-
ments were carried out on a CXP 300 NMR spectrometer
(Bruker, Karlsruhe, Germany) at resonance frequencies of 46.01
and 121.5 MHz, respectively. The spectrometer was controlled
by a MacSpect unit (Tecmag, Houston, TX) and was equipped
with a 1 kW linear amplifier (LPI-10H, ENI, Rochester, NY).
Measurements were done with a commercial double resonance
(d) IR Data Analysis. The processing of the CH2 and CD2
stretching vibrations has been carried out using the OMNIC E.
S. P. 5.1 software (Nicolet, Madison, WI). The analysis of the
CH2 wagging modes, CH2 wagging progressions, and CD2
rocking bands was performed on the Grams/32 software
3
1
probehead (Bruker, Karlsruhe, Germany). P NMR measure-
ments were performed with broadband proton decoupling during
data acquisition (decoupling power: 20 W). The B1 field
strength was adjusted to give a π/2-pulse length of 2 µs for the
(Galactic, Salem, NH).
2
31
The frequencies of the CH2 and CD2 stretching bands were
H and 3 µs for P.
13C MAS NMR measurements were carried out on a MSL
300 NMR spectrometer (Bruker, Karlsruhe, Germany), equipped
with a 1 kW linear amplifier stage (LPPA-13010, Dressler,
Stollberg/Germany), at a C resonance frequency of 75.47 MHz.
C MAS NMR measurements were done under cross-polariza-
determined from the interpolated zero crossing of the first
derivative spectra. For the analysis of the CH2 wagging band
1
0,11
progressions
it was necessary to subtract the underlying
-
-1
13
PO2 antisymmetric stretching band at around 1230 cm .
Therefore, an IR spectrum at the highest available temperature
13
(
here 353 K) was recorded and subtracted from the sample
tion conditions using a commercial 4 mm MAS probehead
(Bruker, Karlsruhe, Germany). The sample spinning frequency
was 2.5 kHz. The B1 field strengths were adjusted to give a
π/2-pulse of 5.5 µs.
spectrum with progressions. Afterward, a flattened baseline was
generated in the appropriate spectral region by selecting
appropriate spectral minima. All progression intensities, the sum
of the k ) 1 to k ) 6 components, were further normalized
with respect to the progression band intensity obtained for the
sample at 223 K.
(f) Differential Scanning Calorimetry (DSC). The phase
behavior of the various samples was examined by differential
scanning calorimetry, using a Netzsch DSC 204 calorimeter
(Selb, Germany). Typically, a temperature range between 240
For the analysis of the CH2 wagging band region (1315-
-
1
-1
1
400 cm ) a linear baseline correction was applied and it was
and 330 K at heating rates of 2 K min was covered.
made sure, that for each spectrum the same baseline points were
taken. Afterward, the spectra were iterated using five to six
vibration bands with Gaussian and Lorentzian contributions of
Results and Discussion
In the following we present variable temperature FT-IR and
NMR studies on model membranes based on the phospholipids
DPPG , DPPG , DPPG , and DPPG , which are distinguished
1 2 3 4
-
1
variable amount. Their initial positions were 1378 cm
-
1
(
symmetric methyl deformation mode), 1368 cm (kink and
-1
by the length of the glycerol moieties in the headgroup. It should
be noted that, unlike the situation for phosphatidylcholines,9
the number of FT-IR studies on phosphatidylglycerols is very
gtg sequences), 1354 cm (double gauche sequences), and 1342
cm (end gauche sequences). In addition, we had to account
for two other bands at 1328 cm (a δ(O-H) mode from the
glycerol moieties) and at 1385 cm (band not further specified)
in cholesterol rich samples. During the following least-squares
fit analysis the band intensities and widths were varied
independently, whereas the band positions were restricted to
3 cm of the values given above. The obtained integrated
intensities of the CH2 wagging bands were normalized with
-13
-
1
-
1
1
9,31
-1
limited.
In fact, a comprehensive FT-IR investigation of
phosphatidylglycerols, such as DPPG ()DPPG1), including
several conformation sensitive bands and CD2-rocking bands,
has not been reported so far. The same holds for the present
systems with oligoglycerol units in the headgroup DPPG to
DPPG4, whose structures are given in Figure 1. The present
work thus comprises experiments on pure DPPG /water disper-
sions (water content: 150 wt %) as well as DPPGx/cholesterol
mixtures (content of cholesterol: 40 mol %). For comparison,
the same IR and NMR experiments were also performed on
DPPC bilayers. In the case of DPPC and DPPG selectively
-
1
(
2
-
1
respect to the methyl deformation band at 1378 cm . The
x
amounts of the various gauche conformers were determined
according to the procedure by Senak et al.,13 which is based on
reference measurements on n-alkanes and a theoretical approach
30
using the rotational isomeric state (RIS) model. It is important
to point out that, unlike free alkanes, a phospholipid contains
just one methyl group per fatty acid chain. The estimated
uncertainty for the kink/gtg and double gauche sequence is 10-
2
deuterated samples (deuteration at carbon C-6) were available
which were examined by both FT-IR and H NMR spectroscopy.
2
(a) Calorimetry and Phase Behavior. Representative DSC
curves of the present phospholipids are shown in Figure 2. The
derived phase transitions are summarized in Table 1. It can be
seen that for pure DPPG1 (Figure 2a) a pretransition (Lâ f Pâ′)
1
5%. Due to the partly overlap by the δ(O-H) mode, the
uncertainty for the end gauche conformer is higher and estimated
to 25-35%, depending on the length of the oligoglycerol chain.
For the processing of the CD2 rocking bands we followed
the same procedure as described for the CH2 wagging bands.
Again, care was taken that for each spectrum the same baseline
correction has been applied. For the final iteration of the
processed FT-IR spectra three bands were initially positioned,
1
9,32
occurs at 309 K,
which also is known for example for DMPC
3
2
and DPPC bilayers. The main transition to the liquid crystalline
phase (LR phase) is registered at 315 K, which also shows up
in the case of the pure DPPG2/water dispersion (Figure 2b).
For pure membranes consisting of DPPG3 (Figure 2c) or DPPG4
(Figure 2d), apart from a “high temperature” transition at about
315 K, a second transition at 278 K is found (indicated by
arrows) that is pretty close to the melting point of water. As
before, the transition at higher temperature is attributed to the
main transition from the gellike to the liquid-crystalline phase.19
The observed slight decrease of the main transition by 4 K for
DPPG4 membranes as compared with DPPG1 bilayers might
be related to a decrease in packing density of the acyl chains in
the same direction.
-
1
-1
namely at 622 cm (tt segments), at 646 cm (ttgg or g′tgg)
-
1
and at 651 cm (g′tgt, ttgt). From the intensities of the two
latter bands the percentage of gauche bonds at a specific CD2
1
5
segment at a given temperature was calculated by using eq 1:
-
1
-1
I[651 cm ] + I[646 cm ]
%
gauche )
-1
-1
-1
2
I[622 cm ] + I[651 cm ] + I[646 cm ]
(
1)

78
J. Phys. Chem. B, Vol. 107, No. 1, 2003
Lehnert et al.
Figure 2. Experimental DSC curves between 240 and 330 K: (a)
DPPG , (b) DPPG , (c) DPPG , and (d) DPPG (top, pure lipid/water
1
2
3
4
dispersion; bottom, sample with 40 mol % cholesterol). Arrows indicate
additional phase transition (see text).
TABLE 1: Phase Transition Temperatures and Phase
Assignments from Calorimetric and IR Studies on DPPG
x
(x
)
1, 2, 3, and 4) Bilayersa
pure
315 K (313 K)
lipid
40 mol % cholesterol
DPPG
DPPG
DPPG
DPPG
1
2
3
4
309 K
L
â
to Pâ′
â
L to LC
315 K (314 K)
to LC
300-320 K (do.)
to LC
314K (314 K) 278 K 314 K (310 K)
to Q n. a. to LC
311 K (310 K) 280 K 311 K (310 K)
to LC n. a. to LC
Figure 3. Position of symmetric CH2 stretching bands of DPPGx
samples (x ) 1, 2, 3, and 4) as a function of temperature. Top: pure
lipid/water dispersion. Bottom: samples containing 40 mol % choles-
L
â
L
â
278 K
n. a.
b
L
â
R
L
â
1 2 3 4
terol. (O) DPPG , (4) DPPG , (9) DPPG , and ([) DPPG .
278 K
n. a.
L
â
L
â
-1
absorption wavenumbers fall in the range between 2850 cm
a
Data in brackets taken from the IR stretching data. b n.a.: not
-1
(gel phase) and 2854 cm (liquid crystalline phase), which
coincides with that from earlier work on related model
assigned.
1
9,34
membranes.
In the gel phase the acyl chains predominantly
For DPPG1 membranes the addition of 40 mol % cholesterol,
as in other phospholipids, such as DPPC or DMPC,33 results in
an almost complete suppression of the main transition, while
for membranes from DPPG2 a shift of the transition toward
lower temperatures along with a broadening of the DSC
transition can be observed. However, the addition of cholesterol
does not alter the main transition in DPPG3 and DPPG4
membranes. For these latter systems the “low temperature”
transition at 278 K is still visible, although being partially
obscured by the transition due to the melting of the water.
At present, the origin of the low-temperature transition in
the DPPG3 and DPPG4 samples is not understood. It might be
speculated whether it is related to the melting of the oligoglyc-
erol units or at least to some structural changes in the headgroup
region, although further information on this topic is lacking. In
this connection it is interesting to note that prolonged blood
circulation times can be observed for lipid mixtures containing
DPPG3 and DPPG4, while this effect is less pronounced for
mixtures with DPPG2 and completely absent for mixtures
containing DPPG1.20
exist in the all-trans conformation which is responsible for the
shift of the CH stretching band toward low wavenumbers. The
2
decrease of conformational order (i.e., increase of amount of
gauche conformers) at the transition to the liquid crystalline
phase is directly reflected by the pronounced shift of the CH2
stretching band in the opposite direction, as shown earlier during
investigations on pure hydrocarbons and other phospholipid
9
,36
membranes.
It should be emphasized that a similar shift is
also observed for the antisymmetric stretching band (data not
shown).
The absence of an additional shift or discontinuity at the
calorimetric “low temperature” transition at 278 K for the
DPPG and DPPG samples again supports the earlier assump-
3
4
tion of a structural change that mainly involves the glycerol
units in the headgroup. The hydrophobic part of the phospholipid
molecules, i.e., the acyl chain region, therefore should remain
unaffected, as indicated by the present FT-IR data. However, a
final proof for this assumption has still to be found.
The data from the symmetric CH stretching band analysis
2
for the membranes containing 40 mol % cholesterol support
(
b) CH2 Stretching Bands. The results from the analysis of
the results from the aforementioned DSC measurements. Thus,
for DPPG1/cholesterol a gradual shift of the absorption band
with increasing temperature can be found; a similar discontinu-
ous change in the vicinity of the main transition of the pure
phospholipid, as discussed above, now is completely missing.
In addition, the overall shift of the absorption band is limited
the symmetric CH2 stretching bands, referring to the pure lipid
bilayers of DPPGx (upper graph) and the mixtures with
cholesterol (lower graph), are summarized in Figure 3. It can
be seen that, without exception, all DPPGx/water dispersions
exhibit a sudden band shift of the symmetric stretching
absorption at the respective calorimetric main transitions. The
-1
to only 2 cm while in the pure DPPG1/water dispersion a shift

Conformational and Structural Properties of DPPGx
J. Phys. Chem. B, Vol. 107, No. 1, 2003 79
2
Figure 4. Experimental CH wagging band progressions of pure
-
1
DPPG
1
between 1360 and 1100 cm at 298 K.
of 4 cm- has been found. Very similar results have been
provided by former studies on DMPC or DPPC (own data from
reference measurements on DPPC bilayers not shown), where
after addition of cholesterol again a continuous shift for the CH2
stretching band has been reported.36,40
1
For the DPPG2/cholesterol sample the absorption frequency
again changes gradually with temperature, however, exhibiting
a larger overall shift than in the previous case. This is in line
with the results from the calorimetric studies where the main
transition is not completely suppressed. Rather, it is found to
be smeared out over a temperature range of about 30 K. The
graphs of the DPPG3 and the DPPG4/cholesterol mixtures are
almost identical to those from the corresponding pure model
membranes discussed above. Again, an abrupt band shift at the
main transition is observed. This is in line with the calorimetric
data where, unlike the situation for DPPG1 and DPPG2/
cholesterol mixtures, the main transition remains unaffected by
the addition of the steroid.
Figure 5. Experimental progression band intensities of DPPG
1, 2, 3, and 4) between 220 and 353 K. Top: pure lipid/water dispersion,
bottom: samples containing 40 mol % cholesterol. (O) DPPG , (4)
DPPG , (9) DPPG , and ([) DPPG
x
(x )
1
2
3
4
.
the intensity of the progression bands drops down by about 50%,
whereas the lipids containing one or two glycerol groups show
a decrease by only 20%. It should be recalled that a loss in
progression band intensity of 50% corresponds to 5% gauche
(c) CH2 Wagging Band Progressions. It has been shown
previously that a high conformational order in the hydrocarbon
chain region causes a coupling of the methylene wagging
1
0
1
0,11,35,37
probability or 0.6 gauche conformers per chain.
modes.
The resulting progression of bands between 1150
-
1
and 1350 cm can be described by the model of coupled
harmonic oscillators. The intensity of the progression bands
again can be used as a qualitative measure for the amount of
all-trans chains in the sample. In Figure 4 representative
progression bands for a pure DPPG1/water dispersion at 298 K
are shown. The existence of such progression bands clearly
demonstrates that highly ordered fatty acid chains, being in an
almost all-trans conformational state, prevail at temperatures
well below the main transition. In Figure 5 these progression
band intensities for the pure DPPGx membranes and the mixtures
with cholesterol are plotted as a function of temperature.
As for the CH2 stretching band data, the intensity of the
progression bands can be used to monitor the change of the
conformational properties in the vicinity of the main transition.
In the present case it can be seen that the progression bands
almost vanish at about 7 K above the main phase transition,
reflecting the sudden increase of conformational disorder, along
with a loss of the all-trans conformers, on going from gel to
liquid crystalline phase. Again, the experimental curves clearly
demonstrate that two kind of data sets can be distinguished,
namely those from the samples with DPPG1 and DPPG2 on the
one hand and from samples with DPPG3 and DPPG4 on the
other hand. In the case of the pure lipid/water dispersions from
DPPG3 and DPPG4, the conformational order starts to decrease
at about 20 K lower than for the samples with DPPG1 and
DPPG2. Thus, between 260 and 300 K for DPPG3 and DPPG4,
On a molecular level, the addition of cholesterol usually leads
to an enhanced conformational order and to the formation of
3
3
the so-called liquid ordered phase. This also holds for the
DPPG1 and DPPG2/cholesterol mixtures, as a finite progression
band intensity can be found at even higher temperatures than
for the corresponding lipid/cholesterol mixtures. In general, the
presence of the steroid is accompanied by a more gradual change
of the progression band intensities. Moreover, in the gel phase
for the DPPG3 and DPPG4/cholesterol mixtures the experimental
progression band intensities reflect a significantly lower amount
of all-trans chains as compared with the samples without
cholesterol. A reduction in intensity, although to a much smaller
extent, also can be found for the DPPG1 and DPPG2/cholesterol
mixtures, which again is very similar to the findings in DPPC
or DMPC/cholesterol mixtures. Obviously, in the gel phase the
impact on the amount of all-trans conformers by the addition
of the steroid is much stronger for the higher oligoglycerols
DPPGx (x ) 3 and 4) than for their shorter counterparts with x
)
1 and 2 or for other phospholipids bearing small headgroups.
(d) CH2 Wagging Bands. Figure 6 presents FT-IR spectra
for DPPG1 (a), DPPG2 (b), DPPG3 (c), and DPPG4 (d) above
the main transition temperature in the liquid crystalline phase.
For comparison a spectrum of DPPC (e) is also given. The
-1
spectral range from 1315 to 1395 cm covers the conformation
sensitive wagging mode region of the methylene groups. In the
12
well-known case of DPPC four absorption bands are observed.

80
J. Phys. Chem. B, Vol. 107, No. 1, 2003
Lehnert et al.
Figure 7. Number of gauche conformers per chain for DPPC bilayers
(
from CH
dispersion, open symbols: samples containing 40 mol % cholesterol.
O, b) gg conformers, (0, 9) kink/gtg conformers, and (3, 1) eg
conformers.
2
wagging band analysis). Filled symbols: pure lipid/water
(
Figure 6. Experimental FT-IR spectra covering the CH
region (pure lipid/water dispersion): (a) DPPG , (b) DPPG
d) DPPG , and (e) DPPC. Dotted lines: results from least-squares fit
analysis.
2
wagging band
1
2
, (c) DPPG
3
,
(
4
The intense band near 1378 cm-1 arises from the methyl
umbrella” deformation mode and is used as an internal
“
reference for the calibration of the other band intensities. At
lower wavenumbers three CH2 wagging bands, centered at 1368,
-
1
1
354, and 1342 cm , are visible that have been assigned to
kink/gauche-trans-gauche (gtg), double gauche (gg), and end
gauche (eg) sequences in the fatty acid chains. The relative
amounts of these conformers (i.e., their integral values over the
whole chain) are obtained via a curve fitting procedure, as
described in the Experimental Section.
The corresponding analysis of the wagging band region of
the oligoglycerols is complicated by an additional absorption
-
1
38
band near 1330 cm . It was assigned to the δ (O-H) mode,
which was verified by IR spectra of glycerol at different
concentrations of water (data not shown). Moreover, the δ(O-
H) band is shifted by the addition of 50 wt % water by five
-
1
Figure 8. Number of gauche conformers per chain for DPPG (x ) 1,
wavenumbers to 1335 cm . It becomes a critical factor as the
number of glycerol units, and therefore the amount of C-OH
groups in the headgroup, increases from DPPG1 to DPPG4. Due
x
2
, 3, and 4) bilayers (from CH
pure lipid/water dispersion. Right column: samples containing 40 mol
4
cholesterol. (O) DPPG , (4) DPPG , (9) DPPG , and ([) DPPG .
2
wagging band analysis). Left column:
%
1
2
3
-
1
to the overlap with the eg band at 1342 cm it becomes
increasingly difficult to separate these two bands and to derive
the amount of eg conformers. The uncertainty for the amount
of eg sequences in DPPG3 and DPPG4 therefore is estimated in
this case to be between 25 and 35%.
membrane, which is in good agreement with the data from other
12,31,39
investigations.
For DPPG2 the values are found to be about
20% higher than for DPPG1. For the two lipids containing larger
headgroups, the amount of gg conformers increases to values
between 0.3 and 0.36 that are 30-50% higher than those of
their shorter counterparts. In addition, the temperature depen-
dence of the amount of gg sequences is less pronounced than
for the low molecular weight oligoglycerols.
The data of the kink/gtg conformers exhibit quite similar
trends. Again, we can distinguish between the values of the low
molecular weight (DPPG1, DPPG2) and high molecular weight
lipids (DPPG3, DPPG4). For the former ones values in the range
from 0.3 to 0.4 are found, with DPPG2 showing about 30%
larger values. Again, the values for DPPG1 are identical with
those from DPPC, with values for kink/gtg conformers between
0.28 and 0.38. The higher amount of kink/gtg conformers in
DPPG2 can be understood by the presence of a bulkier
headgroup (due to the hydration of the diglycerol headgroup)
which creates free volume in the upper part of the fatty acid
To have a common basis for the comparison of the results
from the oligoglycerols and DPPC, a complete analysis of the
wagging band modes for the latter phospholipid was performed
as well (data are given in Figure 7). In Figure 8 the amounts of
eg, kink/gtg, and end gg conformers for DPPGx (left) and
DPPGx/cholesterol samples (right) are plotted as a function of
temperature. In close analogy to the previous discussions of the
calorimetric data, CH2 stretching band and CH2 wagging band
progression data, the results from the wagging band analysis
again imply a separation into two groups, namely, (i) DPPG1
and DPPG2 on one hand and (ii) DPPG3 and DPPG4 on the
other hand.
In the case of the pure lipid bilayers, we get a similar amount
of gg conformers for DPPG1 and DPPC, ranging from 0.21 to
0
.37 for the anionic lipid and 0.29 to 0.4 for the DPPC

Conformational and Structural Properties of DPPGx
J. Phys. Chem. B, Vol. 107, No. 1, 2003 81
chain region, where the kink sequences are expected to possess
the highest probability.34 A further support for this assumption
can be obtained from the data of the high molecular weight
systems, DPPG3 and DPPG4, which follow the trend toward an
higher amount of kink/gtg conformers.
The derived values for the eg conformers behave very similar.
Again, there is a clear tendency toward a higher amount of eg
conformers with increasing number of glycerol units in the
headgroup. However, it should be kept in mind that there is a
higher uncertainty of these values for DPPG3 and DPPG4
samples due the aforementioned overlap with the δ(O-H) band.
The higher amount of eg conformers points to a lower packing
density now even at the lower end of the fatty acid chains.
The addition of 40 mol % cholesterol significantly lowers
the amount of all gauche conformers in the systems studied here.
Especially, the amount of gg conformers is reduced up to 70%,
with values now ranging from 0.04 to 0.12. At the same time,
the amount of kink/gtg and eg conformers also drop down,
although the absolute reduction is less than for the gg conform-
ers. Now, the values for the kink/gtg conformers increase from
DPPG1 (0.24 to 0.3) via DPPG2 (0.32 to 0.34) and DPPG3 (0.29
to 0.35) to DPPG4 (0.4 to 0.41). Again, the values for the
DPPG1/cholesterol mixture are very close to those derived for
the DPPC/cholesterol sample (0.26 to 0.32). Likewise, reduced
values of the eg conformers are registered that again increase
from DPPG1 to DPPG4, with values from 0.09 to 0.15 and values
from 0.39 to 0.43, respectively. The above results and the minor
temperature dependence of the gauche conformers in the
cholesterol mixtures imply the presence of a higher molecular
packing of the membranes which mainly arises from the
incorporation of the cholesterol molecules in the acyl chain
regions.
Figure 9. Representative solid state 31P NMR line shapes pure lipid/
1 2 3
water dispersions at 325 K. (a) DPPG , (b) DPPG , (c) DPPG , (d)
DPPG , and (e) DPPC.
4
31
13
(
e) P and C NMR Spectroscopy. During the present study
31
also some orienting P NMR experiments have been performed
which can provide information about the mobility and ordering
41-43
effects in the headgroup region.
In Figure 9 representative
31
P NMR line shapes are given which refer to the pure
phospholipid/water dispersions from DPPGx and DPPC in the
liquid crystalline phase at 325 K. In general, the overall width
31
and shape of the P NMR spectra prove the presence of
motionally averaged systems. Moreover, the dependence of these
NMR line shapes on the length of the oligoglycerol chains is
quite obvious. Except for the DPPG2 bilayers, characteristic
From the results presented so far the influence of the
oligoglycerol chain length on both the conformational properties
and the calorimetric data is quite obvious. The comparison of
the pure lipid/water dispersions implies that the low mass
system, DPPG1, clearly can be distinguished from the higher
oligomeric systems, DPPG3 and DPPG4, which behave very
similar. The conformational behavior of DPPG2 lies between
both extremes, but nevertheless appears to be more close to
that of DPPG1. These differences can be explained by the
assumption that highly hydrated oligoglycerol chains prevail
in the DPPG3 and DPPG4/water dispersions. Owing to the rather
bulky headgroup, these phospholipid bilayers should therefore
possess a less dense packing in the acyl chain region along with
a larger free volume. In fact, this assumption is supported by
the distinct higher amount of gauche conformers in DPPG3 and
DPPG4 bilayers, as revealed during the present FT-IR studies.
31
axially symmetric P NMR spectra are observed. Again, the
spectra from DPPG3 and DPPG4 bilayers are different, as
expressed by a lower and reversed chemical shift anisotropy
(
DPPG1, 4.2 kHz; DPPG2, 3.9 kHz; DPPG3, -2.1 kHz; DPPG4,
-1.9 kHz; DPPC, 4.9 kHz).
The experimental 31P NMR spectra of the latter samples are
usually encountered for hexagonal and inverted hexagonal
41,44
phases.
On the basis of the chemical structure (i.e., very
bulky headgroup region) and with the known high amount of
gauche conformers from the IR investigations (i.e., low packing
density in the acyl chain region), the formation of both types
of hexagonal phases for DPPG3 or DPPG4/water dispersions is
very unlikely. In this connection, it should be recalled that the
31
overall P NMR line shapes also depend on the vesicle size
As for other phospholipid bilayers, the addition of 40 mol %
cholesterol is accompanied by an ordering effect in the acyl
chain region. In particular, the amount of gg sequences is
reduced, since it is the sterically most demanding conformation.
For DPPG3 and DPPG4 bilayers; however, the ordering effect
by cholesterol is much less pronounced than for the low mass
analogues DPPG1 and DPPG2 and other common lipid sys-
and the chemical shift (CSA) tensor orientation with respect to
45
the motional axis. Owing to the large size of the oligoglycerol
chains, it seems to be more appropriate to assume for DPPG3
and DPPG4 membranes a CSA tensor orientation that is different
from that of the low mass analogues. A final proof of this
assumption, however, is not possible on the basis of the present
data. In this connection, X-ray investigations as well as variable
34,40
31
tems.
The most likely explanation is the aforementioned high
temperature P NMR investigations, including experiments on
hydration of the oligoglycerol units, resulting in very bulky
headgroups along with a reduced packing of the acyl chain
region. Unlike the situation in DPPG1/cholesterol bilayers, for
DPPG3 and DPPG4/cholesterol mixtures the steroid molecules
can only partially compensate the free volume in the acyl chain
region. This also would explain the observation that for the latter
systems the main transition is not altered upon cholesterol
addition.
macroscopically oriented samples, would be very helpful. Again,
3
1
we note that the overall appearance of the P NMR spectrum
of the DPPG2/water dispersion appears to be between the
limiting cases DPPG1 on one hand and DPPG3 or DPPG4 on
the other hand: a situation which was encountered before during
the discussion of the FT-IR data.
As shown earlier by various investigations on n-alkanes and
related systems, an increase in conformational disorder gives

82
J. Phys. Chem. B, Vol. 107, No. 1, 2003
Lehnert et al.
TABLE 2: Temperature Dependent 13C Chemical Shift Values Referring to Carbons C-4 to C-13 in the Acyl Chain Region of
DPPG
x
and DPPC Bilayers
chemical shift [ppm]
DPPG
DPPC
DPPG
40 mol %
2
DPPG
3
DPPG
4
40 mol %
40 mol %
40 mol %
40 mol %
T [K]
pure
cholesterol
pure
cholesterol
pure
cholesterol
pure
cholesterol
pure
cholesterol
2
3
3
3
88
18
33
43
33.3
31.1
30.9
30.7
33.0
32.3
31.5
31.3
33.1
31.0
30.6
30.5
33.3
31.8
31.3
31.2
33.0
30.7
30.5
30.3
33.2
31.5
31.0
30.9
32.9
30.5
30.2
30.0
33.0
31.1
30.7
30.2
32.9
30.6
30.0
29.9
33.0
31.1
30.7
30.2
a result was expected, since the wagging band data already
pointed toward a higher gauche amount for DPPG2 bilayers.
Especially the kink/gtg sequences were found to be higher than
in the DPPC membrane, most likely due to the bulkier diglycerol
headgroup. In fact, the kink/gtg conformation generally is
expected to have the highest probability in the upper part of
3
4
the acyl chains. For this reason the results from the rocking
band analysis are consistent with the findings from the wagging
band data, although the latter can only provide integral values
over the whole acyl chain. Furthermore, the amount of gauche
conformers derived from the CD2 rocking bands also exhibit a
characteristic discontinuity at the main transition for both the
DPPG2 and DPPC bilayers. Again, in a qualitative way this
behavior has been seen earlier during the analysis of the variable
temperature CH2 stretching band and wagging band progres-
sions.
The incorporation of 40 mol % cholesterol decreases the
percentage of gauche conformers for the DPPG2 and DPPC
bilayer systems. The reduction, however, is more pronounced
in the liquid crystalline phases from pure phospholipid/water
dispersions which inherently contain a higher amount of gauche
conformers. Moreover, the above-mentioned discontinuity at the
main transition of the pure lipid/water dispersions does not show
up in the curves of the cholesterol mixtures. This again is in
agreement with the above results from the CH2 stretching bands
and wagging band progressions as well as calorimetric data from
which a similar suppression of the main transition after addition
of cholesterol has been deduced.
Figure 10. Representative FT-IR spectra (CD
lipid/water dispersions from DPPG (top) and DPPG
bottom) at 323 K, labeled at position C-6 in acyl chains. Dotted lines:
results from least-squares fit analysis.
2
rocking band region)
2
2
/cholesterol
(
rise to a high-field shift of the 13C resonances.46,47 Table 2
provides the experimental 13C chemical shift values for the inner
13
chain carbons, C-4 to C-13, obtained from C MAS NMR
investigations as a function of temperature and cholesterol
content. In general, these chemical shift data display the
expected high-field shift toward higher sample temperatures and
with increasing length of the oligoglycerol chains due to the
increase of the amount of gauche conformers which is in
agreement with the findings of the FT-IR studies.
In a qualitative way the conformational behavior at a
particular chain segment also can be followed by the position
6
of the CD2 stretching bands. In close analogy with the CH2
stretching bands (see above), a change of the conformational
state gives rise to a shift of the symmetric and antisymmetric
CD2 stretching bands. This is demonstrated in Figure 11c, where
for the DPPG2 bilayers the positions of the antisymmetric CD2
stretching band are plotted as a function of temperature. It can
be seen that the overall appearance of these curves is very close
to those given in Figure 11 for the percentage of gauche
conformers. This holds for both the temperature dependence
and the influence by the incorporation of cholesterol. The
correlation of the these two data sets is further demonstrated
by the straight lines given in Figure 11d, where the position of
the antisymmetric CD2 stretching band is plotted against the
percentage of gauche conformers. Similar plots have been shown
earlier for example during FT-IR investigations on hydrocarbon
(
f) CD2 Rocking and CD2 Stretching Bands. The CD2
-1
rocking bands between 620 and 670 cm , in combination with
selectively deuterated compounds, can be used to determine the
amount of gauche sequences at a specific methylene unit.
The CD2 rocking region contains three conformation sensitive
14-17,48
-
1
-1
bands, centered at around 622 cm (tt), 645 cm (ttgt, g′tgg),
-
1
and 651 cm (g′tgt, ttgt), reflecting the conformational states
given in brackets. In Figure 10 representative FT-IR spectra
covering the rocking band region are shown that were recorded
for the DPPG2/water dispersion and the DPPG2/cholesterol
mixture. In both cases the acyl chains were selectively deuterated
at position C-6.
The results in terms of percentage of gauche conformers at
position C-6, deduced from a band iteration process (see
Experimental Section), are summarized in Figure 11. The graphs
also contain data from the corresponding DPPC samples, that
also have been deuterated at the same position. The inspection
of these data reveals that the amount of gauche conformers at
position C-6 of the fatty acyl chain in the DPPG2/water
dispersion is higher than in the corresponding DPPC bilayer.
For the pure DPPC bilayer the gauche percentage at 323 K is
about 20%, whereas DPPG2 exhibits values of about 30%. Such
6
49
melts and on chemically modified silica gels.
2
(g) Combination of CD2 Rocking and H NMR Data. In
2
Figure 12 solid state H NMR spectra are depicted that refer to
the pure DPPG2/water dispersion and a DPPG2/cholesterol
mixture well above the main transition. As for other phospho-
5
1-53
2
lipid membranes,
these H NMR spectra are narrowed
considerably when compared to the spectra of a completely rigid
2
system. Detailed H NMR studies on other phospholipid

Conformational and Structural Properties of DPPGx
J. Phys. Chem. B, Vol. 107, No. 1, 2003 83
Figure 11. Results from analysis of the CD
the acyl chains. Open and filled symbols refer to the pure lipid/water dispersions sample containing 40 mol % cholesterol, respectively. (a) Amount
of gauche conformers in DPPC bilayers, (b) amount of gauche conformers in DPPG bilayers, (c) position of antisymmetric CD stretching band
stretching band vs amount of gauche conformers in DPPG bilayers.
2 2
rocking and antisysmmetric stretching band region for DPPC and DPPG , labeled at position C-6 in
2
2
in DPPG
2
bilayers, and (d) position of antisymmetric CD
2
2
molecules with respect to a local preferential axis, the di-
5
3
rector. Sγ represents the segmental order parameter at the
corresponding CD2 unit and thus reflects its conformational
state. With the assumption that only kink conformers are
contributing, the amount of gauche conformers, derived from
the CD2 rocking modes, can be related to the segmental order
1
6,53
parameter Sγ by
pt ) 1 - pg
1
S ) / p
(4)
γ
2
t
Here, pt and pg are the trans and gauche populations, respec-
tively. pg can be directly taken from the analysis of the CD2
rocking modes. With the known order parameters Sγ and SCD,
it is therefore possible to calculate the orientational order
parameter SR.
2
Figure 12. Representative H NMR spectra of a DPPG
3
4
1
membrane at
23 K. Top: pure lipd water dispersion. Bottom: sample containing
0 mol % cholesterol.
2
Figure 13 summarizes the order parameters SCD from the H
membranes5
1-53
have revealed the presence of various overall
and internal motions of the lipid molecule, such as long-axis
rotation, long-axis fluctuation, as well as trans-gauche isomer-
ization. If these motions are fast on the NMR time scale, then
the quadrupolar splitting is directly connected with the overall
order parameter SCD of the lipid molecule by the following
expression:
NMR splittings and the derived values for SR from the DPPC
and DPPG2 samples, comprising pure phospholipid bilayers and
mixtures with cholesterol. For the pure lipid bilayers only the
temperature range above the main transition is covered, since
here at lower temperatures the overall motions are slowed and
the above equations for the order parameters are not valid any
more. However, for the cholesterol mixtures the liquid crystalline
phase (with highly mobile lipid molecules) is stabilized and
extended toward lower temperatures; a phenomenon which is
5
3
2
e qQ
3
∆
νq )
SCD
(2)
52
well-known also from other lipid bilayers.
(
)
4
h
In general, the derived order parameters SCD and SR show an
almost linear decrease with increasing temperature which holds
for both the pure lipid bilayers and the mixtures with cholesterol.
2
Here, e qQ/h represents the static quadrupolar coupling constant
and ∆νq is the experimental quadrupolar splitting, i.e., the
2
1
Experimental SCD data are also available from other H NMR
distance between the perpendicular singularities. The order
parameter SCD can be further expressed by53
studies on pure DPPC bilayers, which are very close to the
values reported here. For example, at 323 K our value is given
5
4
16
SCD ) SRSγ
(3)
to SCD ) 0.26, whereas values of 0.29 , 0.22, (position C-4),
5
5
and 0.20 have been reported by other authors. Moreover, the
order parameters SCD of the samples with DPPC are found to
be somewhat higher than those from DPPG2. For the pure DPPC
SR is the orientational order parameter which expresses the
orientational order or degree of long-axis orientation of the lipid

84
J. Phys. Chem. B, Vol. 107, No. 1, 2003
Lehnert et al.
Figure 13. Experimental SCD (top) and derived S
R
(bottom) order parameters for DPPC (left column) and DPPG
2
bilayers (right column), labeled
at position 6 in the acyl chains. Open and filled symbols refer to pure lipid/water dispersions and samples containing 40 mol % cholesterol,
respectively.
bilayers values between 0.21 and 0.28 are obtained in the liquid
crystalline phase, whereas for the DPPG2/water dispersion values
between 0.12 and 0.15 are observed. The addition of cholesterol
is accompanied by an increase of the SCD values. Now in the
liquid crystalline phase the SCD values range from 0.29 to 0.34
and 0.21 to 0.32 for DPPC and DPPG2/cholesterol mixtures,
respectively.
The orientational order, expressed by the order parameter SR,
exhibits similar trends as discussed for SCD. That is, SR values
of the DPPG2 samples are lower as compared to those from
the DPPC systems. Likewise, the addition of cholesterol gives
rise to an increase of the orientational order. DPPG2 samples
exhibit SR values from 0.4 to 0.46 and from 0.5 to 0.82 for
the pure bilayer and the cholesterol mixture, respectively. For
the pure DPPC/water dispersion values between 0.52 and
Conclusions
In the present work new synthetic phospholipids DPPGx (x
)
1-4), bearing an oligoglycerol chain of variable length in
the headgroup, were examined by variable temperature FT-IR
and solid state NMR spectroscopy. Particular emphasis was
given on the influence of the headgroup size and hydrophilicity
on the conformational properties of the acyl chains, which was
examined as a function of temperature and sample composition
(i.e., presence or absence of cholesterol).
In the first step, the results from differential scanning
calorimetry were contrasted with the data derived from the
analysis of the symmetric CH2 stretching modes and CH2
wagging band progressions, both of which reflecting changes
of the conformational state in the acyl chains. Furthermore,
several conformational sensitive CH2 wagging bands were
analyzed from which the amount (i.e., integral value over the
whole chain) of various types of gauche conformers could be
obtained. It was found that the actual amounts of these
conformers critically depend on the headgroup size, sample
composition and sample temperature. The FT-IR investigations
were completed by solid state P and C MAS NMR
experiments. They could provide an independent support for
the conclusions that were drawn on the basis of the FT-IR data.
In addition, CD2 rocking modes of selectively deuterated DPPG2
have been analyzed in order to get information about the
conformational properties at a particular acyl chain segment (in
the present study at carbon C-6). These data were combined
0
.86 are derived, which are in good agreement with earlier
16
measurements on the same system, where a value of 0.54 in
the liquid crystalline phase at 324 K has been derived. Variable
temperature 31P NMR experiments also exist for DMPC
bilayers from which SR values between 0.43 and 0.53 were
reported.
56
3
1
13
These latter results clearly demonstrate that in general DPPG2
samples are characterized by a lower degree of overall chain
order than DPPC bilayers and most probably also DPPG1
bilayers, although for the latter system, due to the lack of a
deuterated sample, comparable data were not available. Again,
the lower degree of orientational order of the DPPG2 bilayers
is attributed to the higher hydration of the headgroup region.
The bulkier headgroup in DPPG2 prevents a close molecular
packing and therefore allows the system a greater conformational
and motional freedom, as expressed by the low values for the
order parameters SCD and SR. The rigid cholesterol increases
the conformational order and minimizes the fluctuation ampli-
tude of the lipid molecules, as reflected by the enhanced
conformational and orientational order parameters. Nevertheless,
due to the larger headgroup in DPPG2, the addition of cholesterol
is not sufficient in order to achieve a similar high packing
density as for DPPC bilayers.
2
with those from complementary H NMR investigations which
allowed a distinction between the conformational order and the
orientational order of the lipid molecules.
In general, the present membrane systems exhibit a similar
temperature dependence as known from other phospholipid
systems. That is, a higher sample temperature gives rise to a
decrease in conformational and orientational order. Likewise,
incorporation of cholesterol stabilizes the liquid crystalline
phase, as expressed on the molecular level again by higher
conformational and orientational order parameters. The actual
length of the oligoglycerol units in the phospholipid headgroup

Conformational and Structural Properties of DPPGx
J. Phys. Chem. B, Vol. 107, No. 1, 2003 85
also has a significant effect on these order parameters. Here,
the IR data showed a gradual decrease in conformational order
with increasing oligoglycerol length. A similar trend is also
discussed for the orientational order, although so far only data
for DPPG2 are available. These findings can be traced back to
the higher hydrophilicity of the systems with longer oligoglyc-
erol chains. Rather bulky lipid headgroups therefore prevail for
these membrane systems that are responsible for a lower packing
density in the acyl chain region which in turn can only partially
be compensated by the incorporation of cholesterol.
(17) Marconelli, M.; Strauss, H. L.; Snyder, R. G. J. Chem. Phys. 1985,
2, 2811.
8
(
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