FTIR-spectroscopic characterization of phosphocholine-headgroup model compounds

Article abstract of DOI:10.1016/S0022-2860(00)00830-9

The polar headgroups are an important molecular subregion of phospholipids since they mediate a substantial part of the interactions emerging with other constituents of biological membranes, be it inherent macromolecules (proteins) or water as forming the natural environment. FTIR spectroscopy is well proven for characterizing aspects of weak interactions, above all hydrogen bonding. We have used this method to study solid deposits of a number of selected lipid models, such as choline, acetylcholine and methylphosphocholine (MePC), and compared them with the common phospholipid dimyristoylglycero-phosphocholine (DMPC), at different degrees of hydration, which were varied via relative humidity (RH). At low RHs, only MePC and DMPC take up considerable amounts of water, thus elucidating the essential role of phosphate groups in the first stages of lipid hydration. The progress of PC-headgroup hydration can be sensitively monitored by the wavenumber decrease of the band owing to antisymmetric PO₂^- stretching vibration. Concomitant variations of the spectral parameters of vCH bands of MePC reveal that conformational changes may simultaneously occur in the headgroup. Surprisingly to us, the model compounds display qualitative differences in the appearance of their vCH bands, which are most probably a result of substituent effects.

Full text of DOI:10.1016/S0022-2860(00)00830-9

Journal of Molecular Structure 563±564 /2001) 463±467
www.elsevier.nl/locate/molstruc
FTIR-spectroscopic characterization of phosphocholine-headgroup
model compounds
a,
a
W. Pohle , D.R. Gauger , H. Fritzsche , B. Rattay , C. Selle , H. Binder , H. B ohlig
a
b
c
c
d
*
È
aInstitute of Molecular Biology, Friedrich-Schiller University Jena, Winzerlaer str. 10, D-07745 Jena, Germany
bInstitute of Pharmaceutical Chemistry, Martin-Luther University, Halle, Germany
cInstitute of Experimental Physics I, University of Leipzig, Leipzig, Germany
dInstitute of Physical Chemistry, University of Leipzig, Leipzig, Germany
Received 31 August 2000; accepted 29 September 2000
Abstract
The polar headgroups are an important molecular subregion of phospholipids since they mediate a substantial part of the
interactions emerging with other constituents of biological membranes, be it inherent macromolecules /proteins) or water as
forming the natural environment. FTIR spectroscopy is well proven for characterizing aspects of weak interactions, above all
hydrogen bonding. We have used this method to study solid deposits of a number of selected lipid models, such as choline,
acetylcholine and methylphosphocholine /MePC), and compared them with the common phospholipid dimyristoylglycero-
phosphocholine /DMPC), at different degrees of hydration, which were varied via relative humidity /RH).
At low RHs, only MePC and DMPC take up considerable amounts of water, thus elucidating the essential role of phosphate
groups in the ®rst stages of lipid hydration. The progress of PC-headgroup hydration can be sensitively monitored by the
wavenumber decrease of the band owing to antisymmetric PO22 stretching vibration. Concomitant variations of the spectral
parameters of nCH bands of MePC reveal that conformational changes may simultaneously occur in the headgroup. Surpris-
ingly to us, the model compounds display qualitative differences in the appearance of their nCH bands, which are most probably
a result of substituent effects. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Phospholipids; Choline; FTIR spectroscopy; Hydration; Conformation
1. Introduction
phospholipids can be assumed to play a major role
in the interplay of the weak forces relevant in mole-
cular cell biology since it both bears charges which
may exert Coulombic forces against the environment
and provides sites for the speci®c binding from water
molecules as the main constituents of the natural
medium or, alternatively, other membrane compo-
nents /proteins, sterols) and membrane-active
compounds /peptides, drugs). Among phospholipids
as the class of lipids most abundant in eucaryotic
organisms, phosphatidylcholines /PCs) are especially
widespread and generally enriched in the outer lea¯et
Lipids represent an important class of biologically
essential molecules. Their most meaningful property
can be seen in amphiphilicity, i.e. the coexistence of
polar and apolar regions in one and the same mole-
cule, enabling lipids to form supramolecular assem-
blies and aggregations. The polar domain of
* Corresponding author. Tel.: 149-3641-657566; fax: 149-3641-
657520.
E-mail address: wapo@molebio.uni-jena.de /W. Pohle).
0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S0022-2860/00)00830-9

464
W. Pohle et al. / Journal of Molecular Structure 563±564 82001) 463±467
of the bilayers constituting the biological membranes
[1] presumably in order to improve the barrier func-
tion against hydrophobic matter.
DMPC was from Bachem /Heidelberg, Germany).
All these substances were used without further puri-
®cation. MePC, being unavailable commercially,
had to be synthesized. Among the different protocols
given in the literature, the procedure according to
Geilen et al. [18] was selected as the most suitable
one. The details of MePC synthesis will be given
elsewhere /Rattay et al., in preparation). Brie¯y,
tetrabutylammoniumphosphorylcholine was reacted
with methyliodide under appropriate conditions, and
the resulting MePC was re®ned in several steps and
analyzed by different methods /elemental analysis,
mass spectrometry, NMR spectroscopy).
FTIR spectroscopy is one of the most powerful and
frequently used methods to characterize lipid systems
on a submolecular level [2±4]. However, previous IR-
spectroscopic studies were devoted mostly to the apolar
region [2,3]. In a series of recent papers, efforts have
beenmadetofocusthe potentialofIRspectroscopyonto
the headgroup region of phospholipids mainly by
considering the spectral implications owing to hydra-
tion-driven isothermal /lyotropic) phase transitions
[5±8]. Conformational changes have been suggested
to occur in PC-headgroups on the basis of spectroscopic
data obtained by NMR [9,10] as well as IR spectroscopy
[5±8,11]. Relevant interpretations of previous IR-spec-
troscopic results referred mostly to features of the phos-
phate moiety [4±7] or to the ammonium residue
[5,8,11]. Bands due to the two methylene groups of
the PC headgroups which might be also in¯uenced by
relevantconformationalchangescanhardlybeobserved
in the IR-spectra region of usual PCs since they are
overlapped by the spectroscopic features arising from
the large majority of chain methylene groups. To avoid
this disadvantageous situation, we have recorded IR
spectra of the basic PC model compound methylpho-
sphocholine /MePC). Moreover, choline and acetylcho-
line as close relatives with the same ±/CH2)2±N1/CH3)3
moiety were investigated correspondingly. It should be
mentionedthat all thesethree compounds are not onlyof
academic interest as suitable PC models, but each of
them also has some biological meaning. MePC naturally
occurs in the eggs of sea urchins [12], acetylcholine is a
neurotransmitter, and choline isanimportant metabolite
in biochemical pathways. The latter two compounds are
also interesting from the pharmacological point of view.
To model the phosphate moiety in biomolecules /DNA,
phospholipids), mainly dimethylphosphate salts have
been studied by IR spectroscopy [13,14] and in relevant
theoretical calculations [14±17] up to now.
2.2. FTIR-spectroscopic measurements
Samples of the substrates were obtained as solid
deposits by placing the material on IR-transparent
ZnSe windows either from aqueous /choline, acetyl-
choline, MePC) or chloroformic solutions /DMPC)
and measured in situ. Infrared spectra were recorded
by means of an IFS-66 FTIR spectrometer from
Bruker /Karlsruhe, Germany) in the DTGS-detection
mode using different accumulation rates between 2
and 32 scans at a resolution of 2 cm21 and a zero-
®lling factor of 2. Samples were investigated at
different values of the surrounding relative humidity
/RH) between 0 and 100% either isopiestically or in
non-stationary hydration or dehydration scans. Data
processing was done by the Opus software package
/Bruker) with a wavenumber accuracy of 0.1 cm21
.
All other experimental details were described
previously [7]. Deviating from the algorithm used
there [7], AOH for MePC was determined in the
following way: the total absorbance /AOHCH)0 obtained
by integration between 2500 and 3750 cm21 of the dry
sample was subtracted from the AOHCH value resulting
at either degree of hydration, and the difference was
then divided by /AOHCH)0.
3. Results and discussion
2. Experimental
The overall IR spectra of choline, acetylcholine,
MePC and DMPC are shown in Fig. 1. Some of the
due results are discussed in terms of different spectral
ranges, without regarding nCO and nCC bands.
2.1. Materials
Choline bromide and acetylcholine iodide were
purchased from Sigma Co. /Munich, Germany),

W. Pohle et al. / Journal of Molecular Structure 563±564 82001) 463±467
1500 1000
(a)
465
3600
3000
called residual water, which is usually retained in PCs
under conditions of 0% RH [7]. Upon progressive
hydration, choline and its derivatives display signi®-
cant differences in their capacity to imbibe water. First
of all, any of the studied substances can be considered
hygroscopic; this is simply revealed by the fact that
they take up water to an extent that the deposits even-
tually slip down the support. At low RHs /#23%),
only MePC and DMPC are able to bind substantial
amounts of water in contrast to choline and acetyl-
choline. This as well as the dif®culty to completely
dry MePC and DMPC illuminate the particular role
played by phosphate groups especially in the ®rst
steps of phospholipid hydration /cf. Refs. [7,19],
and papers cited therein). Besides, a strong water
uptake starts for choline much earlier /at 44% RH)
than for acetylcholine /at 86% RH). The ªhygrosco-
picityº of DMPC refers largely to the low-hydration
range and is manifested by the existence of the resi-
dual water /see above).
(b)
(c)
3.2. Region of nCH bands
The wavenumbers of the peak maxima of IR
absorption bands arising in the 3050±2800 cm21
region show clear differences for the PC-model
compounds. The only consistently occurring band is
that situated around 3030 cm21, which can be attrib-
uted to the nCH mode of the nitrogen-bound methyl
groups /this band will be discussed for MePC in the
next section, see Fig. 2b). Further signi®cant nCH
bands emerge at about 2952, 2904 and 2845 cm21
for choline, at ,3003, 2976 and 2956 in acetyl-
choline, around 2950, 2910 and 2840 cm21 for
MePC and at 2920 and 2850 cm21 for DMPC /as
for other long-chain PCs, too [2±8]). While the wave-
numbers of nCH bands, n~CH, are similar for choline
and MePC, those of acetylcholine deviate dramati-
cally, and we note here especially the absence of
any signi®cant absorption in the 2950±2800 cm21
region. Altogether, these data indicate that n~CH of
methylene groups can be very strongly in¯uenced
by their relevant substituents. We are currently trying
to explore these phenomena in more detail by the use
of theoretical methods. The hydration-dependent
behaviour of the MePC band at 2840 cm21 is explored
in Section 3.3.
(d)
3600
3000
1500
1000
Fig. 1. FTIR spectra of: /a) choline, /b) acetylcholine, /c) methyl-
phosphocholine, and /d) dimyristoylglycerophosphocholine
measured as water-depleted deposits in an environment of 0% RH.
3.1. Region of nOH bands
In the nOH range of dry compounds, only one
striking absorption is observed; this is a band near
3270 cm21 arising in the case of choline, which can
be assigned to OH stretching vibration. Whereas
acetylcholine does not exhibit remarkable features in
this region, the small broad bands with maxima
around 3370 and 3380 cm21 observed in MePC and
DMPC, respectively, belong most probably to the so-

466
W. Pohle et al. / Journal of Molecular Structure 563±564 82001) 463±467
(b)
(a)
(c)
b)
c)
1256
2850
3040
1248
1240
1232
3035
3030
3025
2845
2840
a)
0
1
2
3
4
5
A
(MePC)
OH
Fig. 2. Wavenumbers of the bands owing to antisymmetric PO22 stretch /a) and the C±H stretches near 3035 /b) and 2840 cm21 /c) of MePC
depicted versus AOH, the degree of hydration, which was evaluated as explained in Section 2.2. The vertical line marks discontinuities occurring
in the courses of all three graphs.
3.3. PC-headgroup bands
declines exponentially in the former case [7,20] and
decreases relatively weakly in the initial hydration
steps in the latter case. Supposedly, this is related to
the fact that MePC forms a stable hemihydrate in the
®rst stage of hydration as reported recently [22], and
the binding of more water is somewhat retarded.
Signi®cant discontinuities at the same water content
con®rming this interpretation appear also in the AOH
dependences of other spectral parameters of MePC
/cf. curves b and c in Fig. 2, where the locus of discon-
tinuities in graphs /a±c) is marked by a broken
vertical line; further corresponding data are not
shown).
The other two graphs in Fig. 2 display how the
wavenumbers of two C±H stretching-vibration
bands around 3035 /b) and 2845 cm21 /c) depend on
hydration. While the former band can be assigned
easily to the C±H stretch of N-bound methyl groups
/see above), the assignment of the latter band is dispu-
table. Interestingly, both these parameters behave like
reversed images referred to n~aPO22 ; thus suggesting
that all these magnitudes are correlated with each
other. Since direct water binding to C±H unities is
rather unlikely to occur, the course of the n~CH graphs
in Fig. 2b and c can be Ð in contrast with the graph
according to Fig. 2a Ð unambiguously interpreted as
re¯ecting the occurrence of conformational change/s)
in MePC accompanying the process of water binding
to phosphate /we consider highly improbable that the
From the several IR modes representing the phos-
phate moiety, the band arising from the antisymmetric
stretching vibration, naPO22, is particularly sensitive
against structural changes concerning relevant biomo-
lecules, as phospholipids or DNA /see Refs.
[4,7,8,19], and papers cited therein). Hydrating PCs
from 0 to 100% RH, the wavenumber of this band,
n~aPO22 ; decreases usually from ,1260 to
,1230 cm21, i.e. by about 30 cm21, as demonstrated
in several cases [7], and especially also for DMPC
[20]. Phosphate groups are accepted generally to act
as the primary binding sites for water in biomolecules.
Supported by the results of corresponding quantum-
chemical calculations, the strong hydration-induced
decline of n~aPO22 is in accord with the assumption
that water molecules are hydrogen-bonded to the
free oxygens of the phosphate group [21]. However,
this does not exclude the fact that n~aPO22 is addition-
ally affected by changes in the PC conformation,
which may occur simultaneously.
MePC shows a n~aPO22 shift nearly as large as that
of PCs /cf. Fig. 2a) suggesting strong binding of water
to the phosphate also in this case, in contrast with the
conclusions drawn in a previous IR-spectroscopic
study of MePC [22]. One striking difference in the
characteristics of the graphs of common PCs /cf.
e.g. Fig. 7 in Ref. [7]) and MePC is that n~aPO22

W. Pohle et al. / Journal of Molecular Structure 563±564 82001) 463±467
467
decrease of n~aPO22 re¯ects only headgroup-confor-
mation changes of MePC). Similar conformational
transitions triggered by hydration might proceed in
the headgroups of genuine PCs, too, but to detect
them would demand the use of isotopically labeled
compounds.
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Acknowledgements
The authors are indebted to Deutsche Forschungs-
gemeinschaft for granting this research in the frame of
SFB197 under the subprojects B10 /W.P., D.R.G.,
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