New cardiolipin analogs synthesized by phospholipase D-catalyzed transphosphatidylation

Article abstract of DOI:10.1016/j.chemphyslip.2012.09.005

Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholipase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the synthesis of new CL analogs. The formation of this type of compounds from phosphatidylcholine was compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient production of the corresponding CL analogs. In contrast, the yields were comparably low in the reaction with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane, tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified diphosphatidyldiethanolamine and diphosphatidylserinol.

Full text of DOI:10.1016/j.chemphyslip.2012.09.005

Chemistry and Physics of Lipids 165 (2012) 787–793
Contents lists available at SciVerse ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
New cardiolipin analogs synthesized by phospholipase D-catalyzed
transphosphatidylation
a
a
b
a
a,∗
Anna O. Müller , Carmen Mrestani-Klaus , Jürgen Schmidt , Renate Ulbrich-Hofmann , Martin Dippe
a
Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Kurt-Mothes-Str. 3, D-06120 Halle, Germany
Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2012
Received in revised form
Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their
chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholi-
pase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the
synthesis of new CL analogs. The formation of this type of compounds from phosphatidylcholine was
compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols
such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine
derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient
production of the corresponding CL analogs. In contrast, the yields were comparably low in the reac-
tion with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane,
tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the
conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head
group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified
diphosphatidyldiethanolamine and diphosphatidylserinol.
1
8 September 2012
Accepted 21 September 2012
Available online 8 October 2012
Keywords:
Cardiolipin analogs
Diphosphatidyl lipids
Ethanolamine derivatives
Head group
Phospholipase D
Transphosphatidylation
©
2012 Elsevier Ireland Ltd. All rights reserved.
1
. Introduction
of the complexes of the respiratory chain (Arias-Cartin et al., 2012)
and of mitochondrial carriers (Klingenberg, 2009). Besides its struc-
tural function, CL serves as a reservoir for the electrochemical
gradient due to protonation of its head group at low pH (Haines and
Dencher, 2002) and thus contributes to proton conduction (Lange
et al., 2001). Additionally, it is involved in cell signaling related to
oxidative stress (Gohil and Greenberg, 2009) and apoptosis (Wright
et al., 2004; Joshi et al., 2009).
Because of its special structural features, the physicochemi-
cal properties of CL significantly differ from the corresponding
monophosphatidyl compounds (Lewis and McElhaney, 2009).
Since those properties are beneficial for the stabilization of lipid
vesicles, CLs and structurally similar anionic compounds are of
great interest for the preparation of liposomes used for DNA pack-
ing (Resina et al., 2009) and HIV prophylaxis (Malavia et al., 2011).
Positively charged CL analogs, which mediate cellular nucleic acid
uptake are attractive as components of liposome-based gene-
delivery systems (Zhang et al., 2006).
Unlike the common membrane phospholipids, cardiolipin (CL)
contains two phosphatidyl residues linked to the external hydroxyl
groups of glycerol. This dimeric phospholipid specifically occurs
in the membranes of bacteria and mitochondria harboring the
respiratory complexes. Therefore, the role of CL in membrane
organization and cellular energy metabolism has attracted much
attention (Schlame, 2008). For example, this unique phospholipid is
involved in the maintenance of the characteristic shape of the inner
membrane (Khalifat et al., 2008). CL is crucial for the stabilization
Abbreviations:
br, broad; dd, doubled doublet; dm, doubled multi-
plet; CL, cardiolipin; HPLC, high performance liquid chromatography; COSY,
homonuclear correlation spectroscopy; DSS, 4,4-dimethyl-4-silapentane sodium
sulfonate; FT-ICR, Fourier transform ion cyclotron resonance; HPTLC, high per-
formance thin layer chromatography; m, multiplet; PA, phosphatidic acid; PC,
1
-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PE, 1-palmitoyl-2-oleoyl-sn-
However, the chemical synthesis of CL or structurally related
compounds is complicated and involves various reaction steps
glycero-3-phosphoethanolamine; PG, phosphatidylglycerol; PLD, phospholipase D;
q, quartet; t, triplet; THA, tetrakis(2-hydroxyethyl)ammonium bromide; TOCSY,
total correlation spectroscopy; Tris, tris(hydroxymethyl)aminomethane.
∗ Corresponding author. Present address: Leibniz Institute of Plant Biochemistry,
Weinberg 3, 06120 Halle, Germany. Tel.: +49 34555 821412; fax: +49 34555 27303.
E-mail address: Martin.Dippe@ipb-halle.de (M. Dippe).
(Paltauf and Hermetter, 1994). Phospholipase D (PLD)-catalyzed
transesterification of the better accessible glycerophospholipids
seems to be a useful alternative strategy to produce CL com-
pounds. PLD is a well-known biocatalyst due to its capability to
0
009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemphyslip.2012.09.005

7
88
A.O. Müller et al. / Chemistry and Physics of Lipids 165 (2012) 787–793
Fig. 1. Ethanolamine derivatives assayed for the enzymatic production of CL analogs. The ethanolamine moiety of the N-substituted derivatives diethanolamine (a),
triethanolamine (b) and THA (c) and the C2-substituted derivatives serinol (d) and Tris (e) are drawn in gray.
exert a head group exchange reaction called transphosphatidy-
lation. In phospholipid synthesis, especially bacterial PLDs such
as the enzyme from Streptomyces sp. are used because they
strongly favor transphosphatidylation of the acceptor alcohol over
the undesired hydrolytic cleavage of the phospholipid substrate
2.2. Methods
2.2.1. Determination of PLD activity
PLD activity was assayed in adaption to D’Arrigo et al. (1995)
toward a micellar solution of the substrate phosphatidyl-4-
nitrophenol (0.42 mM) in 0.21 mM sodium dodecyl sulfate, 3.3 mM
Triton X-100, 120 mM CaCl2 and 300 mM acetate buffer, pH 5.6.
One unit (U) of enzyme activity represents the amount of enzyme
releasing 1 ␮mol of 4-nitrophenol per minute.
(
Ulbrich-Hofmann et al., 2005). In contrast to other PLDs, this
enzyme also mediates the conversion of phosphatidylglycerol (PG)
to CL (D’Arrigo et al., 1996; Piazza and Marmer, 2007).
Interestingly, a similar reaction was observed in the enzy-
matic synthesis of phosphatidylethanolamine derivatives with
multiple hydroxylated head groups (Dippe et al., 2008) lead-
ing to the formation of side products which were identified
as dimeric phospholipids with a positively charged hydrophilic
linker. In the present paper, we evaluate the applicability of
this reaction for the synthesis of dimeric phospholipids from
2
(
.2.2. Synthesis of tetrakis(2-hydroxyethyl)ammonium bromide
THA)
8 mmol of triethanolamine and 2-bromoethanol, respectively,
4
were dissolved in 15 ml of nitromethane and heated under reflux at
◦
9
5 C for 10 h. Upon cooling, white crystals of THA were obtained,
phosphatidylcholine and
a series of N- and C2-substituted
which were collected by filtration and washed with acetone. The
crude product was re-crystallized from methanol/acetone/water
derivatives of ethanolamine or non-nitrogenous acceptors (glyc-
erol and ethylene glycol). The reactions were compared with
respect to chemical structure and charge of the used acceptor
alcohols. Two of the CL analogs (diphosphatidylserinol and diphos-
phatidyldiethanolamine) are shown to be accessible in preparative
scale.
(15:10:1, v/v/v) and dried in vacuum in the presence of P O10,
4
1
yielding 1.20 g (4.4 mmol) of pure THA. H NMR (according to Sec-
tion 2.2.6, in D O-DSS-external): ı 3.41 ppm (t, 8H, CH2 NR3 ); ı
+
2
3
.89 ppm (t, 8H, CH2 OH).
2.2.3. Kinetic measurements
2
. Materials and methods
PLD-catalyzed reactions were performed in an aqueous-
organic two-phase system according to Hirche and Ulbrich-
Hofmann (2000). 620 ␮l of substrate solution (1.24 ␮mol PC, 1,2-
dioleoyl-sn-glycero-3-phosphoglycerol or 1,2-dioleoyl-sn-glycero-
3-phosphoethylene glycol in diethyl ether) were mixed with 80 ␮l
of enzyme solution (61.6 mU PLD in 300 mM sodium acetate buffer,
120 mM CaCl2, pH 5.6). In the reactions containing PC, 96 ␮mol of
acceptor alcohol was added to the aqueous phase. Before usage,
stock solutions of amino alcohols were adjusted to pH 5.6 by addi-
tion of HCl. Reaction mixtures were shaken (400 rpm) in HPTLC
2
.1. Materials
1
-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), 1-pal-
mitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE), 1-palm-
itoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1,2-dioleoyl-
sn-glycero-3-phospho-rac-glycerol, CL from bovine heart, PLD
from Streptomyces sp. (Type VII), and 2-bromoethanol were
products from Sigma (USA). The phosphatidic acids (PAs)
◦
1
-palmitoyl-2-oleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-
screw flasks (Roth, Germany) at 30 C. After a distinct reaction
sn-glycero-3-phosphate were from Avanti Polar (USA), and
phosphatidyl-4-nitrophenol was synthesized from egg yolk
phosphatidylcholine according to D’Arrigo et al. (1995). 1,2-
Dioleoyl-sn-glycero-3-phosphoethylene glycol was produced by
transphosphatidylation of the corresponding phosphatidylcholine
by recombinant PLD from white cabbage according to the pro-
cedure described by Dippe et al. (2008) Its molecular mass was
checked as described in Section 2.2.5. Ethylene glycol, water-free
glycerol, tris(hydroxymethyl)aminomethane (Tris) and serinol
were from Merck, Germany. All other reagents and solvents were
purchased from Roth, Germany.
time (0–17 h), samples (25 ␮l) were taken from the organic phase
which exclusively contained the phospholipids. The solvent was
removed by air-drying and the residuals were re-dissolved in
25 ␮l of toluene, applied to silica 60 plates (Merck, Germany) and
analyzed by HPTLC according to Dippe et al. (2008). After stain-
ing of the phospholipids with CuSO4/H3PO4 (Touchstone et al.,
1983), the resulting bands were evaluated by densitometry (den-
sitometer CD60, Desaga, Germany). The concentrations of the
individual phospholipids, which were identified according to their
Rf values (Dippe et al., 2008), were calculated using standards
of the corresponding PC, PA, PG, PE (for phospholipids having

A.O. Müller et al. / Chemistry and Physics of Lipids 165 (2012) 787–793
789
Fig. 2. Conversion of PC with ethanolamine derivatives by PLD-mediated transphosphatidylation. The reactions were performed in a two-phase system consisting of
diethylether and acetate buffer, pH 5.6, in the presence of (a) diethanolamine, (b) triethanolamine, (c) THA, (d) serinol, or (e) Tris as described in Section 2.2.3. Transphos-
phatidylation of PC (ꢀ) to mono- (ꢀ) and diphosphatidyl products (ꢀ) competes with the hydrolysis to PA (᭹).
ethanolamine-derived head groups), and CL. The presented data
are the means resulting from two independent experiments.
separation of phospholipids, 1 ml of the chloroform solution was
applied to a silica HPLC column (Lura Si 100, 100 ␮m, 4.3 × 250 mm)
(Phenomenex, USA). The stepwise elution of serinol-containing
transphosphatidylation products was achieved by a linear gradi-
2
.2.4. Preparative phospholipid transformation
Phospholipid conversions were performed similar to the kinetic
−
1
ent of CHCl /CH OH/H O (7:4:1, v/v/v) at a flow rate of 1 ml min
,
3
3
2
whereas for phospholipids having diethanolamine head groups
CHCl /CH OH/25% NH (7:4:0.05, v/v/v) was used. Phospholipid-
experiments (Section 2.2.3) but in a larger scale. The reaction mix-
tures contained 28.3 mg (37.2 ␮mol) of PC in 18.6 ml of diethyl ether
and 2.4 ml of enzyme solution (148 mU PLD in 300 mM sodium
acetate buffer, pH 5.6 containing 120 mM CaCl2 and 2.88 mmol
3
3
3
containing fractions were identified by a light scattering detector
Sedex 45 (Sedere, France).
◦
acceptor alcohol). After shaking (400 rpm) for 17 h at 30 C, the
organic phase was separated and washed with 5 ml of 1 mM
ethylendiaminetetraacetic acid (adjusted with NaOH to pH 8.0)
and water, respectively. The solvent was removed by rotary evap-
2.2.5. Mass spectrometry
The positive ion high resolution ESI mass spectra were obtained
from a Bruker Apex III Fourier transform ion cyclotron reso-
nance (FT-ICR) mass spectrometer (Bruker Daltonics, Billerica, USA)
oration and the residual was re-dissolved in 5 ml of CHCl . For
3

7
90
A.O. Müller et al. / Chemistry and Physics of Lipids 165 (2012) 787–793
Fig. 3. Scheme of the PLD-catalyzed reaction of PC with diethanolamine. In a first transphosphatidylation step (a), PC is converted to phosphatidyldiethanolamine, which
is subsequently transformed to its CL analog by reaction with a phosphatidyl donor (X = choline or diethanolamine moiety) (b). Hydrolytic degradation of PC (c) and both
transphosphatidylation products (d and e) yields PA.
equipped with an infinity cell, a 7.0 T superconducting magnet
Bruker), an RF-only hexapole ion guide and an external electron
spray ion source (Agilent). Nitrogen was used as drying gas at
to internal tetramethylsilane at 0 ppm. Standard methods were
used to perform 1D and 2D experiments, pulse programs being
taken from the Bruker Software Library. Resonance assignments
were made by combined analysis of H,H-COSY and TOCSY spectra.
(
◦
1
50 C. The sample solutions (in CH OH) were introduced contin-
3
−
1
uously via a syringe pump with a flow rate of 120 ␮l h . The data
were evaluated by the Bruker XMASS 7.0.8 software.
The following molecular masses were found (the theoretical
values are given in parentheses):
2.2.6.1. Di(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho)serinol.
3
ı
0.89 ppm (t,
m, 80H,
C3);
J = 6.7 Hz, 12H, CH3 );
1.61 ppm (br m,
2.03 ppm (br m, 8H,
ı
8
1.28 ppm (br
H, CH2 acyl
(CH )n );
ı
2
ı
CH CH CHCH2 );
ı
2
•
•
•
di(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho)serinol:
1.33 ppm (br, 8H,
CH CH CH CHCH CH2 );
ı
2.33 ppm
3.99 ppm (br, 4H,
4.18 ppm (ABX, m,
4.42 ppm (ABX, dd,
J = 12.1 Hz, J = 2.5 Hz, 2H, CH HBOR CHOR CH2 PO2 ); ı
PO2 ); ı 5.35 ppm (m,
CH2 ); ı 3.35 ppm
CH2 CH(NH3) ).
2
2
2
+
(q, 3J = 7.2 Hz, 8H,
m/z
1405.0277
[M+H] ,
(1405.0268,
calculated
for
CH2 acyl C2);
PO2 );
ı
+
[
C77H148NO16P ] );
CH OR CHOR CH2
O
ı
2
2
di(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho)diethanolamine:
2H,–CH H OR–CHOR–CH –O–PO –);
B
ı
A
2
2
+
3
2
m/z
1419.0429
[M+H] ,
(1419.0424,
calculated
for
O
A
+
[
C78H150NO16P ] ); and
5.16 ppm (br, 2H, CH OR CHOR CH2 O
2
2
1,2-dioleoyl-sn-glycero-3-phosphoethylene glycol m/z 743.5226
4H, CH CH ); ı 3.99 ppm (br, 4H, PO2
(buried by CD OD, 1H, PO2
O
−
−
[
M−H] , (743.5232, calculated for [C41H76O P] ).
O
9
3
2
.2.6. 1H NMR spectroscopy
2.2.6.2. Di(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho)
Spectra of the phospholipid solutions (2 mM in CD Cl/CD OD
diethanolamine. ı 0.86 ppm (br m, 12H, CH3 ); ı 1.27 ppm (br
3
3
(
7:4, v/v)) were recorded at 300 K on a Bruker Avance 400 spec-
m, 80H, (CH )n ); ı 1.61 ppm (br m, 8 H, CH2 acyl C3); ı
2
trometer (Rheinstetten, Germany). Chemical shifts were referred
2.03 ppm (br m, 8H, CH CH CHCH2 ); ı 1.32 ppm (br, 8H,
2

A.O. Müller et al. / Chemistry and Physics of Lipids 165 (2012) 787–793
791
CH CH CH CHCH CH ); ı 2.32 ppm (m, 8H, CH2 acyl C2);
2
2
2
2
3
ı 4.01 ppm (t, J = 6.3 Hz, 4H, CH OR CHOR CH2
O
PO2 ); ı
PO2 ); ı 4.39 ppm
CH HBOR CHOR CH2 PO2 );
PO2 ); ı 5.35 ppm
m, 4H, CH CH ); ı 4.20 and 4.15 ppm (dm, 2H, PO2 CH2 );
ı 3.23 ppm (br m, 2H, PO2 CH2 CH2 NH2 ); ı 3.81 ppm (m,
PO2 ); ı 3.13 and 3.01 ppm (dm, 2H,
PO2 ).
2
4
(
5
(
.20 ppm (m, 2H, CH HBOR CHOR CH2 O
A
buried by H O, 2H,
O
ı
2
A
.24 ppm (br m, 2H, CH OR CHOR CH2 O
2
O
O
2
H, NH2 CH2 CH2
NH2 CH2 CH2
O
O
3
. Results and discussion
3.1. PLD-mediated formation of CL analogs from ethanolamine
derivatives
To examine the production of CL-related phospholipids from
PC, transphosphatidylation reactions were performed with PLD
from Streptomyces sp. in the biphasic reaction system acetate
buffer–diethyl ether. As acceptor alcohols, compounds deriving
from ethanolamine by substitution of one, two or three amine
hydrogen atoms by hydroxyethyl groups (Fig. 1a–c) or of one or
two hydrogen atoms at the C2 position by hydroxymethyl groups
(
Fig. 1d and e) were used.
The time course of the reactions is shown in Fig. 2. In the
reaction with diethanolamine (Fig. 2a), a fast accumulation of phos-
phatidyldiethanolamine was observed in the initial period of the
reaction. As depicted in the reaction scheme (Fig. 3), this phospho-
lipid is produced by transphosphatidylation of PC. Due to a free
hydroxyl function in its head group, phosphatidyldiethanolamine
is able to participate in a subsequent step of transphosphatidylation
yielding the corresponding symmetric diphosphatidyl compound.
Thereby, PC or a second phosphatidyldiethanolamine molecule
was used as donor for the transferred phosphatidyl moiety. Thus,
the formation of the CL analog is limited by the concentration of
the monophosphatidyl compound, which is in accordance with
its slow increase (Fig. 2a). After 17 h reaction time, 50% diphos-
phatidyldiethanolamine were formed. In contrast to this strong
preference for transphosphatidylation, the hydrolytic production
of PA from PC or the transphoshatidylation products (Fig. 3) is
marginal (Fig. 2a).
Similar to diethanolamine, the more bulky triethanolamine is
rapidly transformed into its monophosphatidyl product (Fig. 2b).
However, the generation of the CL analog was comparably slow.
Since only 15% diphosphatidyl product was reached after 17 h, a
prolonged reaction time seems to be required to reach a maxi-
mum product yield. The differences in the reaction rates may be
related to the different molecular volume of both head group alco-
hols introduced into the phospholipid structure. This assumption
is supported by a similar trend observed for the C2-substituted
ethanolamines (Fig. 2d and e), where the small-sized serinol is pre-
ferred compared to the highly branched homologue Tris. In the
reaction with serinol, the yield of the diphosphatidyl product was
remarkably high, whereas the concentration of the correspond-
ing CL analog of Tris reached only 6% in the same reaction time.
That means that PLD from Streptomyces sp. efficiently catalyzes
the transfer of a single phosphatidyl moiety to all of the accep-
tor alcohols mentioned above (Fig. 2; Dippe et al., 2008), but the
modification of the introduced head group by a second phosphati-
date seems to be strongly size-controlled. Obviously, this difference
results from increased steric requirements of the phospholipid
acceptor limiting the access to the active site.
Fig. 4. Conversion of PC with glycerol (a) and ethylene glycol (b) by PLD-mediated
transphosphatidylation. The reactions were performed in a two-phase system con-
sisting of diethylether and acetate buffer, pH 5.6, as described in Section 2.2.3.
Transphosphatidylation of PC (ꢀ) to mono- (ꢀ) and diphosphatidyl products (ꢀ)
competes with the hydrolysis to PA (᭹).
the initial generation of phosphatidyl-THA. As indicated by the
strongly enhanced formation of PA (Fig. 2c), PC is mainly converted
by hydrolysis.
3.2. PLD-mediated formation of CL and its analog from ethylene
glycol
Beside the ethanolamine derivatives described in Section 3.1,
glycerol and ethylene glycol were used for the transphosphatidy-
lation of PC. Glycerol is the central part of the natural CL and
both alcohols are – in contrast to the ethanolamine derivatives –
uncharged. Surprisingly, in the reaction with glycerol only small
amounts of CL were formed (Fig. 4a), although the molecular vol-
ume of glycerol (87.1 A˚ 3, http://www.molinspiration.com) is very
similar to that of serinol (91.2 A˚ 3) which strongly promotes the pro-
duction of its CL analog (Fig. 2d). Similar results were obtained
for the even less voluminous ethylene glycol (Fig. 4b). Therefore,
the lack of a positive charge in the head group of PG or phos-
phatidylethylene glycol seems to be restrictive for the subsequent
transphosphatidylation step leading to CL. Obviously, beside its
size, also the charge of the head group which serves as nucleophile
is a significant factor in the transformation of phospholipids to CL
analogs, whereas this charge is not essential in the formation of the
monophosphatidyl derivative where 91% of products are reached
(Fig. 4a and b).
In contrast to the acceptor alcohols of the reactions mentioned
above (Fig. 2a, b, d, and e), THA (Fig. 1c) does not allow any signif-
icant transphosphatidylation (Fig. 2c). The high molecular volume
of THA (195.3 A˚ 3, http://www.molinspiration.com) even impairs

7
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A.O. Müller et al. / Chemistry and Physics of Lipids 165 (2012) 787–793
determined by high-resolution mass spectrometry were in accor-
1
dance with the theoretical masses. The experimental data of the H
NMR spectroscopy confirmed the identity of the compounds.
4. Conclusions
In addition to its ability to exchange the head group alcohol,
bacterial PLD is able to catalyze the conversion of phospholipids
with polyhydric head groups into diphosphatidyl compounds. This
enzymatic two-step reaction allows the synthesis of new CL analogs
bearing head groups with positive charge and different volume
which are scarcely accessible by chemical synthesis. However,
product yields are strongly dependent on both the molecular
volume and the charge of the used acceptor alcohol. Diphos-
phatidyldiethanolamine and -serinol are two newly described
compounds that can be easily produced in significant yields. In
contrast to the initial transphosphatidylation of PC yielding the
monophosphatidyl compound, the subsequent transfer of a second
phosphatidyl moiety is hindered if the first attached head group
alcohol is sterically challenging or uncharged.
Acknowledgements
We thank the LIPOID GmbH (Ludwigshafen, Germany) for
the gift of phospholipids and Christa Kuplens and Katja Fröhlich
for their excellent technical assistance. The financial support by
the State of Sachsen-Anhalt and by the Graduiertenkolleg 1026
(Deutsche Forschungsgemeinschaft, Bonn, Germany) is gratefully
acknowledged.
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3
(

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