General synthesis and physicochemical characterisation of a series of peptide-mimic lysine-based amino-functionalised lipids

Article abstract of DOI:10.1002/chem.201204529

A series of novel malonic acid diamides (second generation) with two long hydrophobic alkyl chains and an alkaline polar head group was synthesised and characterised as a new class of amino-functionalised lipids. These peptide-mimic lipids are suitable for polynucleotide transfer. The lipids bear a novel backbone consisting of a lysine unit and a malonic acid unit. Six different head-group structures, which vary in size and number of amino groups that can be protonated, were attached to the backbone structure. Furthermore, different alkyl chains were used to build the lipophilic part (namely tetradecyl, hexadecyl, and oleyl). Phase transitions of the new compounds in aqueous dispersions at pHa 10 were analysed and discussed in terms of head group and alkyl chain variations. The shape and size of the formed aggregates of selected lipid dispersions were investigated by dynamic light scattering and transmission electron microscopy. Copycats: Malonic acid diamides, a new class of amino-functionalised lipids, have been synthesised and characterised. These peptide-mimic lipids are suitable for polynucleotide transfer. Six head-group structures, which vary in size and number of amino groups, are used. The modularly composed synthesis of the backbone allows easy alkyl-chain variation (see figure). Phase transitions are discussed in terms of head-group and alkyl-chain variations. Copyright

Full text of DOI:10.1002/chem.201204529

DOI: 10.1002/chem.201204529
General Synthesis and Physicochemical Characterisation of a Series of
Peptide-Mimic Lysine-Based Amino-Functionalised Lipids
[
a]
[a]
[b]
[b]
Christian Wçlk, Simon Drescher, Annette Meister, Alfred Blume,
[
a]
[a]
Andreas Langner, and Bodo Dobner*
Abstract: A series of novel malonic
acid diamides (second generation) with
two long hydrophobic alkyl chains and
an alkaline polar head group was syn-
thesised and characterised as a new
class of amino-functionalised lipids.
These peptide-mimic lipids are suitable
for polynucleotide transfer. The lipids
bear a novel backbone consisting of a
lysine unit and a malonic acid unit. Six
different head-group structures, which
vary in size and number of amino
groups that can be protonated, were at-
tached to the backbone structure. Fur-
thermore, different alkyl chains were
used to build the lipophilic part
(namely tetradecyl, hexadecyl, and
oleyl). Phase transitions of the new
compounds in aqueous dispersions at
pH 10 were analysed and discussed in
terms of head group and alkyl chain
variations. The shape and size of the
formed aggregates of selected lipid dis-
persions were investigated by dynamic
light scattering and transmission elec-
tron microscopy.
Keywords: amides · lipids · phase
transitions
·
self-assembly
·
synthetic methods
Introduction
namely toxicity, immunogenicity and limitations with respect
[8]
to scale-up procedures. Due to these disadvantages syn-
thetic chemical vectors became a promising alternative, and
the lipid-based vectors are the most-used representatives of
Amino-functionalised and cationic lipids offer various appli-
cations in the field of medicine. For example, they are used
[
1]
[7,8b,9]
as non-viral nucleic acid delivery systems, drug-delivery
this group.
[2]
systems for neoplastic diseases and, more recently, as vac-
Recently, we reported on a new class of amino-functional-
[3]
cine carriers or adjuvants. Furthermore, their use as anti-
ised lipids with a malonic acid diamide structure as back-
[4]
[10]
microbial agents is described. During the last two decades
the application of alkaline or cationic lipids as nucleic acid
delivery systems (nucleic acid delivery with lipids is called
bone and two hydrophobic alkyl chains.
Therein, we
varied the hydrophobic molecule part and the head-group
region of the lipids with the aim to gain information about
structure-function relationships. Within that previous work,
some very effective amino-functionalised lipids have been
prepared exceeding commercially available synthetic vec-
tors, such as Lipofectamine, in their transfection efficacy.
Furthermore, selected lipid-based vectors of these novel cy-
tofectines have been investigated by physicochemical meth-
ods, such as X-ray scattering, differential scanning calorime-
try (DSC) and Langmuir film balance measurements, which
“
lipofection”) gained great importance. Within classical
gene therapy, which means the treatment of diseases
through the use of DNA-based drugs allowing the delivery
of DNA to cells followed by expression of a therapeutic
gene, cationic lipids are common and widely used delivery
[
1a,5]
systems besides cationic polymers and viral vectors.
Ad-
ditionally, the methods of gene silencing using antisense oli-
[6]
gonucleotides and siRNA, respectively, are applications
for amino-functionalised lipids. At present, viral vectors are
[11]
explained the observed structure–activity relationships.
[
7]
the most-used nucleic acid delivery systems. However, fun-
damental problems are associated with viral vector systems,
Herein, we describe the synthesis of a series of malonic
acid diamides with a modified backbone (called second gen-
eration) based on a malonic acid structure combined with a
lysine unit (Figure 1). The hydrophobic part contains two
long alkyl chains of variable length and saturation (tetradec-
yl, hexadecyl, and oleyl chains); the first alkyl chain is
bound through an amide bond to the lysine unit and the
second one is bound to the a-carbon of the malonic acid
unit, thus resulting in a larger distance of the alkyl chains in
comparison to the alkyl chains in glycerol-based phospholi-
pids. Due to the complexity of the transfection pathway, no
general schemes have emerged correlating the structure of
amino-functionalised/cationic lipids with their transfection
efficacy and, hence, the approaches for optimising the struc-
[
a] Dipl.-Pharm. C. Wçlk, Dr. S. Drescher, Prof. Dr. A. Langner,
Prof. Dr. B. Dobner
Institute of Pharmacy, MLU Halle-Wittenberg
Wolfgang-Langenbeck-Strasse 4, 06120 Halle/Saale (Germany)
Fax : (+49)345-55-27018
E-mail: bodo.dobner@pharmazie.uni-halle.de
[
b] Dr. A. Meister, Prof. Dr. A. Blume
Institute of Chemistry, MLU Halle-Wittenberg
Von-Danckelmann-Platz 4, 06120 Halle/Saale (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201204529. It contains further
experimental details and the detailed characterisation of the lipid pre-
cursors 9a–c, 11a–c, 12a–c, 13a–c, 15a–c, and 16a–c.
12824
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FULL PAPER
Figure 1. Chemical structures and abbreviations of the 18 malonic acid di-
amides synthesised in this work.
ture of these lipids are still largely empirical. To close this
gap, we varied the head-group volume of our lipids by using
different spacers (ethylenediamine type, tris(2-aminoethyl)-
amine type), and different numbers of coupled lysines (zero
to three). We chose the amide bond as the main connecting
bond between the different building blocks of the lipid be-
cause of its biodegradability (which is necessary for low tox-
Scheme 1. Synthesis of the lipid backbone: a) tetradecylamine/oleyl-
AHCTUNGTRNNEGaU mine, PyBOP, DIPEA, CH Cl , RT; b) piperidine, DMF; c) tetradecyl
2 2
bromide/hexadecyl bromide, NaH, toluene; d) 1) KOH, ethanol, RT;
2) HCl, H O; e) PyBOP, DIPEA, CH Cl , RT; f) 1) KOH, THF, H O;
2
2
2
2
[12]
icity), and its higher stability to hydrolysis compared with
the ester bond. Furthermore, we used the proteinogenic
amino acid lysine as a main unit for the head group and the
backbone, because of its biocompatibility and known ability
to form complexes with DNA as a main element of histones
2) H
2
SO
4
2
, H O. See text for details.
starting material was the orthogonally amine-protected l-
lysine derivative 1 with an amine labile 9-fluorenylmethoxy-
carbonyl (Fmoc) protective group in the a position and an
acid labile tert-butoxycarbonyl (Boc)-protected amino group
in the e position. Compound 1 was first coupled with tetra-
decylamine and oleylamine with (benzotriazol-1-yloxy)tri-
pyrrolidinophosphonium hexafluorophosphate (PyBOP) as
coupling agent and diisopropylethylamine (DIPEA) as ac-
cessory base, yielding the orthogonally protected lysine
amides 2 (yield: 72–82%). The property that DIPEA
cleaves the Fmoc group very slowly in comparison with
other tertiary amines (50% DIPEA in dimethylformamide
[13]
and poly-l-lysine. The structural modifications result in 18
novel amino-functionalised lipids with peptide-like structure
elements (peptide-mimic lipids, Figure 1).
The main focus of this work is the detailed description of
the lipid synthesis. Furthermore, the report summarises the
chemical and physicochemical properties of the novel lipids.
We investigated the pure aqueous lipid dispersions at pH 5
and 10 by means of DSC to obtain information about the
thermal behaviour of the pure lipids and to explore the in-
fluence of structural variations in the lipophilic part and the
head-group region of the molecules on the thermal behav-
iour. In addition, we investigated the formed aggregate
structures of selected aqueous lipid dispersions at pH 10 by
means of dynamic light scattering (DLS) and transmission
electron microscopy (TEM).
[14]
(DMF): t =10.1 h ) ensures its application as proton
1
=
2
catching agent for this coupling reaction. PyBOP is an effec-
tive coupling agent for the synthesis of amides successfully
[15]
used in peptide synthesis. Afterwards, the Fmoc deprotec-
tion of compounds 2 with piperidine yields the e-Boc-pro-
tected lysine derivatives 3 with a free a-amino group needed
for a further amide coupling reaction. Piperidine cleaves the
Results and Discussion
Fmoc group very fast (20% in DMF: t =3 s; 5% in DMF:
1
=
2
[14]
t =20 s ). In addition, we also used DMF as solvent, ac-
cording to the Merrifield peptide synthesis technique.
The starting material for the synthesis of the second part
of the backbone was the malonic acid diethyl ester 4, which
was transformed into the monoalkylated derivatives 5 by
1
=
2
[16]
Synthetic methods: The synthetic strategy developed in this
work allows easy variation of the alkyl chains by different
combinations of separately prepared molecule parts.
Scheme 1 describes the synthesis of the lipid backbone. The
Chem. Eur. J. 2013, 19, 12824 – 12838
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12825

B. Dobner et al.
treatment with sodium hydride
and tetradecyl and hexadecyl
bromide. This reaction is descri-
[17]
bed in various procedures,
but our experience showed that
the use of toluene as solvent
and sodium hydride as deproto-
nating agent gave the best re-
sults. The following monosapo-
nification step by using potassi-
um hydroxide yields the malon-
[18]
ic acid monoethyl esters 6.
The two backbone parts 3 and
can readily be prepared in
higher quantities (up to 10 g).
different combination of
6
A
both parts in the following
amide coupling step allows an
easy alkyl chain variation,
which opens the way for a wide
spectrum of lipids for further
structure–function relationship
studies. The carboxylic acids 6
and the amines 3 were coupled
with PyBOP/DIPEA, resulting
Scheme 2. Synthesis of the lipids with an ethylenediamine moiety: a) ethylenediamine, PyBOP, TEA, CH
b) 1) TFA, CH Cl ; 2) H O, NH ; c) Boc-Lys(Boc)-OSu, TEA, CH Cl
2 2
Cl ;
2
2
2
3
A
H
N
T
E
N
N
2
2
.
in the formation of the amides 7 (yield: 93–97%). We used
diamine due to the large excess (about 20 equiv) of ethyle-
nediamine used in this coupling reaction. After chromato-
graphic purification, the resulting compounds 9 were treated
with trifluoroacetic acid (TFA) to cleave the Boc groups
and to yield compounds 10 (lipids 1a–c) that contain the
smallest head group, with two amino moieties that can be
protonated. For a further enlargement of the head-group
region we used the Boc-protected N-hydroxysuccinimide
ester of the proteinogenic amino acid l-lysine (Boc-Lys-
DIPEA in this coupling reaction because of its increasing
[19]
effect on the yields in sterically hindered reactions. In a
further saponification step the ethyl ester function of com-
pounds 7 was converted into the free carboxylic acid of
compounds 8. This reaction was carried out under mild con-
ditions by using only an excess of potassium hydroxide of
0.5 equivalents with regard to compounds 7.
The synthesis of the head-group region was designed in
such a way that the number of free amino groups that can
be protonated and, hence, the resulting volume of the head
group could be easily varied for further structure-activity re-
lationship studies. For this purpose, the lipid backbone pre-
pared so far offers two connecting positions for the amino-
functionalised head group: on the one hand, the e-amino
group of the lysine, which can be coupled with various car-
boxylic acids, and on the other hand, the free carboxylic
acid moiety of the malonic acid unit that can be connected
with different amines. Thus, the lipid backbone consists, in
principle, of two different head groups, which can be syn-
thesised and connected separately allowing a huge variety of
structures.
ACHTUNGTRENNU(GN Boc)-OSu)—an activated amide bond forming ester suc-
cessfully used in peptide synthesis. Compounds 9, as well
as 10, were coupled with one or two molecules of Boc-Lys-
[20]
AHCTUNGTRENNUNG( Boc)-OSu, resulting in the formation of the Boc-protected
compounds 11 and 12, respectively. The subsequent cleavage
of the Boc groups with TFA yields lipids 2a–c, with three
amino groups, and lipids 3a–c, bearing four amino groups.
The final purification of lipids 1–3a–c was carried out with
column chromatography on silica gel and a gradient techni-
que by using chloroform, methanol, and ammonia as elu-
ents.
For the synthesis of lipids 4–6a–c, we used tris(2-aminoe-
thyl)amine instead of ethylenediamine as spacer to finally
increase the number of amino groups that can be protonated
and, hence, to enlarge the head-group region. The synthetic
procedures are comparable to the aforementioned syntheses
(Scheme 3). Starting from compounds 8, the amidation of
the carboxylic acid moieties with tris(2-aminoethyl)amine
by using PyBOP/TEA led to the formation of compounds
13. After the subsequently performed cleavage of the Boc
group, compounds 14 (lipids 4a–c) were obtained. Both
compounds 13 and 14 were used for the coupling with differ-
ent numbers of lysine residues, leading to compounds 15
The free carboxylic acid moiety of compounds 8 can be
coupled with either ethylenediamine, resulting in the lipids
1
6
–3a–c, or with tris(2-aminoethyl)amine, yielding lipids 4–
a–c. Both amine spacers further increase the structural di-
versity of the lipids prepared in this work.
The synthesis of lipids 1–3a–c, which contain ethylenedia-
mine as spacer, is described in Scheme 2. The carboxylic
acids 8 were coupled with ethylenediamine through amide
bond formation by using PyBOP and triethylamine (TEA).
This reaction step needs no monoprotection of the ethylene-
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Peptide-Mimic Amino-Functionalised Lipids
FULL PAPER
This module-like synthesis
strategy allows
a straightfor-
ward combination of different
alkyl chains with diverse head
groups, which are variable in
size and number of amino
groups, thereby resulting in a
series of amino-functionalised
lipids applicable for further
structure influence investiga-
tions.
Stereochemical investigations:
To achieve biocompatibility of
the lipids, we used the proteino-
genic amino acid l-lysine within
the lipid backbone and head
group. Hence, we had to pay at-
tention to the stereochemistry
of the amino acid units. We ap-
plied the commercially availa-
ble Boc-Lys ACHUTNGRENNUG( Boc)-OSu exhibit-
ing an S configuration for the
lysine coupling reaction. The
coupling methods with N-hy-
droxysuccinimide esters are de-
scribed to be free of racemisa-
Scheme 3. Synthesis of the lipids with a tris(2-aminoethyl)amine moiety: a) tris(2-aminoethyl)amine, PyBOP,
TEA, CH
2
Cl
2
2 2 2 3 2 2 2
; b) 1) TFA, CH Cl ; 2) H O, NH ; c) Boc-Lys ACUNTHGRNEUN(G Boc)-OSu, TEA, CH Cl ; d) 1) HCl, H O, ethyl
acetate; 2) DMAP, CHCl
3
, methanol.
[24]
tion. In addition, the coupling
reaction by using PyBOP is
[25]
and 16, respectively. For the final cleavage of the Boc moiet-
ies of 15 and 16, we used a modified method because the
TFA salts, which were achieved with the generally applied
method, were difficult to work up. Instead, we used hydro-
chloric acid as the cleaving agent. Normally, HCl-saturated
organic solvents, for example, ethyl acetate, are appropriate
nearly free of racemisation. Furthermore, we chose every
reaction step so that we used only reaction conditions that
do not cause amino acid racemisation, that is, the protective
group cleaving agents were used under conditions that are
common in peptide synthesis, and the saponification reac-
tions in the presence of amino acid moieties were performed
under mild conditions. Therefore, we can assume that all of
the lysine moieties in the lipids are S configured.
[21]
for this purpose, but due to the easier handling we utilised
[22]
aqueous HCl, as described previously.
Additionally, we
worked with a two-phase system containing water and ethyl
acetate as solvents because of the insolubility of the Boc-
protected compounds in water. A comparable procedure
Surprisingly, the cleavage of Boc groups resulted in the
formation of two different substances if the product mixture
was investigated by thin-layer chromatography (TLC). How-
ever, ESI-MS measurements of these two substances, ob-
tained by preparative TLC, showed the same mass spectra
of the desired product. With regard to the synthetic path-
way, we conclude the formation of diastereomers: the alkyl
amide of the Boc-protected derivative of l-lysine (3) and
the racemate of the alkylated malonic acid monoethyl ester
(6) were connected by the formation of a rigid amide bond
resulting in two diastereomers with SR and SS configura-
tion. At first we tried to separate both diastereomers by
column chromatography, but purification could not be real-
was previously described for
a
toluene–water–HCl
[23]
system. Furthermore, we used a novel method of workup:
after the deprotection of the amino moieties of lipids 5a–c
and 6a–c, we added 4-dimethylaminopyridine (DMAP) to
the dissolved lipid–HCl salts and purified this mixture by
column chromatography (see the Experimental Section,
method b, yield: 97–99%). This technique eliminates the
washing steps, which are connected with enormous loss of
substance due to the high hydrophilicity of lipids 5a–c and
6
a–c (see the Experimental Section, method a, yield: 21–
27%). In this procedure, DMAP acts as a strong base and
ised, although the R values of the two substances indicated
f
yields the free amino groups through transprotonation.
During column chromatography (by using the gradient tech-
nique and chloroform, methanol, and ammonia as eluents),
the resulting DMAP–HCl was eluted first as a lipophilic ion
pair, followed by the lipids 5a–c and 6a–c.
a distinct separation. Surprisingly, the extraction of one dia-
stereomer from the TLC plate resulted again in two spots in
a second TLC run, which reflected the “in situ” formation
of the pair of diastereomers (see the Supporting Informa-
tion). This indicates that the stereocentre at the a carbon of
the malonic acid unit racemises in the alkaline milieu of the
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12827

B. Dobner et al.
dition, we observed a strong
solvent dependency of the
chemical shift and the number
1
3
of C NMR signals. In Figure 2,
1
3
two regions of C NMR spectra
of lipid 1c are shown with vari-
ous solvents and lipid concen-
trations. Figure 2F (39–56 ppm:
CH NHCO, CH NH , COCHR-
Scheme 4. Postulated mechanism for the stereocentre racemisation at the malonic acid moiety of the lipids.
2
2
2
mobile phase during chromatography, which is also an ex-
planation for our inability to separate the mixture. Scheme 4
shows a possible mechanism of the diastereomer transfor-
mation, including an enol as the intermediate structure.
For further investigations with respect to detailed physico-
chemical properties, polynucleotide binding properties, and
gene transfer efficiencies (results will be published else-
where), we will take into account the presence of two dia-
stereomers.
CO and lysine a carbon) shows the signal broadening as a
result of the formation of a gel. Furthermore, the solvent de-
pendency of the number of peaks and chemical shifts is
clearly shown (see Figure 2E and F, four peaks in the range
between 39 and 43 ppm related to 2ꢁCH NHCO and 2ꢁ
2
CH NH carbon atoms in CDCl , and Figure 2D, three
2
2
3
peaks resulting from the same carbon atoms in the CDCl3/
CD OD mixture). The last observation also applies to the
3
carbonyl region of the spectrum (Figure 2A–C); two signals,
which correspond to three carbonyl carbon atoms, appeared
in CDCl , whereas six signals appeared in the CDCl /
Nuclear magnetic resonance investigations: All novel lipids
3
3
1
13
(
lipids 1–6a–c) were investigated by H and C NMR meas-
CD OD mixture for the same carbonyl atoms. The reason
3
1
urements. The general H NMR spectra of our lipids are
similar to those obtained from polymers. This is due to the
complex molecular structures exhibiting a large number of
protons with comparable chemical shifts. The NMR charac-
teristics of the amide groups, such as anisotropy and cis–
for this behaviour is the coexistent presence and absence of
hydrogen bonds between the carbonyl functions and the sol-
[27]
vent methanol.
This behaviour makes the interpretation
of spectra quite demanding and further complicates the
comparability of spectra of different lipids. Hence, the
choice of NMR solvent is a compromise between solubility
and comparability.
[26]
trans isomerism, further complicate the spectra. Hence, it
1
is quite demanding to interpret H NMR spectra on the
basis of signal splitting and coupling constants. However,
with the use of H,H COSY measurements, we were able to
interpret the H NMR spectra (examples of H NMR and
H,H COSY spectra are shown in the Supporting Informa-
tion). For NMR measurements, the lipids were normally dis-
Differential scanning calorimetry: The influence of alkyl
chain and head group variations on the temperature-de-
pendent aggregation behaviour of aqueous dispersions of
the lipids synthesised in this work was investigated to
extend the lipid characterisation. For this purpose, we used
DSC measurements to examine possible phase transitions.
We are aware that our substances are diastereomers and ac-
count for this in the analysis of the DSC curves.
1
1
solved in CDCl . Surprisingly, some of our lipids showed the
3
formation of a transparent organo-gel, which is further con-
nected with an extensive broadening of the proton signals.
A decrease of the lipid concentration or the addition of
small amounts of CD OD (which is able to break the hydro-
The DSC investigations were carried out at two different
pH values. At first pH 5 was used to achieve full protona-
tion of the amino groups leading to cationic lipids applicable
for gene transfer studies. At this pH value, the lipids form a
transparent suspension, which is an indication of the forma-
tion of micellar aggregates. In acetate buffer at pH 5, the
DSC curves of dispersions of lipids 1–6c and lipids 2,4a
show no transition between 2 and 958C (see the Supporting
Information). DLS data and TEM images reveal the forma-
tion of micelles (data not shown).
3
gen bonds between the lipids) led to the breakdown of the
gel. However, the signal intensity and the comparability of
the spectra were affected; for example, methanol can shift
1
some H NMR signals to higher/lower ppm values and
1
almost all NH H NMR signals are lost with the use of
methanol. Surprisingly, the lipids 6a–c and lipid 5b, which
have a large head group, are insoluble in common NMR sol-
vents, except D O. Therefore, we had to carry out the
2
1
H NMR spectroscopy in D O at higher temperatures, at
2
which the signals are narrower, because the lipid structures
in water become more fluid.
Secondly, pH 10 was used for DSC measurements. At this
pH value the lipids form a turbid suspension, which is a first
indicator for the formation of lamellar or vesicular aggre-
gates (see the Supporting Information for TEM images).
Figure 3 summarises the DSC heating curves obtained for
aqueous suspensions of all 18 lipids. The DSC scans were
performed in a temperature range between 2 and 958C in
carbonate buffer at pH 10. We chose the alkaline conditions
to ensure deprotonation of the amino groups. X-ray scatter-
ing data show that lipids 1–4a–c build up lamellar phases at
13
C NMR measurements were carried out in CDCl3/
CD OD mixtures (except the insoluble lipids, for which
3
1
3
D O was used). As the C NMR spectra of our lipids are
2
also very complex, we could only assign related carbon
NMR signal groups with carbon-atom groups bearing a
related chemical shift (e.g., carbonyl carbon atoms; see
1
3
the Experimental Section; examples for C NMR and
C,H COSY are shown in the Supporting Information). In ad-
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Peptide-Mimic Amino-Functionalised Lipids
FULL PAPER
1
3
Figure 2. Details of the C NMR spectrum of lipid 1c measured in different solvents and at different concentrations. A–C) Carbonyl region between
70.0 and 172.3 ppm; D–F) methylene carbon atoms next to amide nitrogen atoms and carbon atoms next to amine moieties in the region between 38
1
and 56 ppm. A,D) Lipid dissolved in CDCl
concentration forming an organo-gel.
3
/CD
3
OD (2:1, v/v); B,E) lipid dissolved in CDCl
3
at low concentration; C,F) lipid dissolved in CDCl
3
at high
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12829

B. Dobner et al.
transition temperatures of lipids
1
–3a–c, which include an ethyl-
enediamine-containing head
group, to determine the de-
pendence of the variation in the
alkyl chain (Figure 3A). For
the smallest lipid head group
(
lipids 1a–c), the main transi-
tion temperature increases from
lipid 1a (92.98C) to lipid 1b (>
9
58C) due to the longer alkyl
chain that is bound to the ma-
lonic acid moiety (TT versus
TH). In contrast, an unsaturat-
1
ed alkyl chain as residue R
(
lipid 1c, see Figure 1) decreas-
es the transition temperature
to 82.58C. A similar effect of
changes in chain structure on
ꢀ
1
Figure 3. DSC heating curves of aqueous dispersions of the lipids (c=1 mgmL in carbonate buffer 10 mm at
pH 10). A) Lipids with a head group of the ethylenediamine type (lipids 1–3a–c); B) lipids with a head group
ꢀ1
of the tris(2-aminoethyl)amine type (lipids 4–6a–c). The heating rate was 60 Kh , except for lipids 5a–c,
which were measured with a heating rate of 20 Kh . Curves are shifted vertically for clarity.
ꢀ1
the thermotropic behaviour was
observed for the lipids 2a–c, in-
cluding a larger head group
with one additional lysine residue and also for the lipids 3a–
c consisting of two lysine moieties in the head group. The
[
28]
this pH value.
The broadening of the phase transition
peaks for lipids with an oleyl chain (OT; see Figure 1) was
expected because the oleylamine used contains up to 30%
of other alkyl amines with different chain length and satura-
main transition temperatures follow the sequence: T (lipid
m
2,3b)>T (lipid 2,3a)>T (lipid 2,3c). If we compare the T
m
m
m
[11b]
tion.
values of lipids with different head groups, while keeping
the alkyl chains constant, we see that for all three alkyl-
chain combinations (TT, TH, OT) the main transition tem-
perature decreases by roughly 10 K after the insertion of the
first lysine moiety and then stays nearly constant after the
addition of the second lysine residue to the head group.
The DSC curves of lipids 4–6a–c, which contain a tris(2-
aminoethyl)amine structure in
The peak maxima (main transition temperature, T ) of
m
the main endothermic transition peaks and the correspond-
ing enthalpies (DH) for the lipids 1–6a–c are summarised in
Table 1. Other DSC peaks, which appear at lower tempera-
tures than the main DSC peak, are termed herein as pre-
transitions (Tp as peak maximum). First, we compare the
the head group, differ from
Table 1. Main transition temperatures (T
urements (heating rate of 60 Kh ) of the lipids.
m
) and corresponding transition enthalpies (DH) of the DSC meas-
those for the lipids 1–3a–c de-
scribed above (Figure 3B). On
examining the dependence of
ꢀ
1
Lipid Alkyl
chains
Head group
Type
Number
of primary
T
p
[8C]
T
m
[8C]
ACHTUNGTRENNUNG
[
a]
ꢀ1
ꢀ1
Number
A
H
U
G
R
N
U
G
A
H
U
G
R
N
N
A
H
U
G
R
N
N
T on the chain variation of the
m
of lysines amino groups
lipids 4 a–c and lipids 5a–c,
both the exchange of a tetra-
decyl chain by a hexadecyl
1
2
3
1
2
3
1
2
3
4
5
6
4
5
6
4
5
6
a
a
a
b
b
b
c
TT
TH
OT
TT
TH
OT
ethylenediamine
1
2
3
1
2
3
1
2
3
1
3
4
1
3
4
1
3
4
2
3
4
2
3
4
2
3
4
3
5
6
3
5
6
3
5
6
35.1 (1.7)
–
69.4 (2.8)
69.9 (0.6)
92.9 (20.0)
84.2 (47.9)
83.4 842.4)
[
b]
–
chain and by an oleyl chain re-
sults in a decrease of the main
transition temperature for lipid
4 [T (lipid 4a)>T (lipid 4b)>
–
–
–
–
–
–
–
–
–
–
–
–
91.4 (41.3)
86.9 (43.7)
82.5 (24.2)
70.0 (51.7)
72.7 (29.8)
77.6 (52.9)
57.4 (59.1)
–
c
c
m
m
T (lipid 4c)], as well as for
m
a
a
a
b
b
b
c
tris(2-aminoethyl)amine
lipid 5 [T (lipid 5a)>T (lipid
[
c]
m
m
5
b)]. The dependence of Tm on
74.1 (44.1)
the head group is similar to
that observed for the lipids
[
c]
27.6 (9.2)
–
1
–3a–c. The introduction of
60.0 (11.8)
[
d]
[d]
two additional lysine residues
decreases the main transition
c
c
–
–
–
–
temperature [T (lipid 4a,b)>
T (lipid 5a,b)].
m
m
[
a] TT=tetradecyl-/tetradecyl-; TH=tetradecyl-/hexadecyl-; OT=oleyl-/tetradecyl-. [b] The main transition
appears at the end of the observed temperature range and could not be determined. [c] The heating rate was
2
ꢀ
1
0 Kh . [d] Data could not be determined because of irreproducible DSC curves.
12830
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 12824 – 12838

Peptide-Mimic Amino-Functionalised Lipids
FULL PAPER
The lipids 5a and 5b only show reproducible DSC curves
4a–c)>T (lipids 5a–c)]. The transition temperatures of the
m
ꢀ
1
at a heating rate of 20 Kh . If we used a heating rate of
lipids 1–4a–c present a further head-group effect: all Tm
values are equal to or higher than 608C. These values are
very high taking into account the presence of tetradecyl and
oleyl chains. The comparison of the Tm values of lipids 1–
4a–c with values from glycerol-based phospholipids or cati-
onic lipids containing the same alkyl-chain combination (see
ꢀ
1
6
0 Kh , we observed an irreproducible, slow decrease of
the heat capacity, C , over a wide temperature range, fol-
p
lowed by a rapid increase of C (see, for example, lipid 5a
p
in the Supporting Information). The lipid 5c shows no re-
producible DSC curves at either heating rate (see the Sup-
porting Information). The DSC curves of lipids 6a–c show
no transition between 2 and 958C, in either the heating or in
the cooling scans. This probably indicates that no lamellar
phases are formed if the head group becomes too large.
The Tm values of the series of lipids (see Table 1) show
clear structure dependence. As mentioned for the class of
lipids with an ethylenediamine-containing head group (lipids
[31]
data from the literature in Table 2) clearly illustrates the
m
Table 2. Main transition temperature (T ) values from glycerol-based
phospholipids or cationic lipids in different media taken from the litera-
ture.
[
a]
Lipid
Medium
T
m
[8C]
ꢀ1
A
H
U
G
E
N
N
ACHTUNGTRENNUNG
1
–3a–c), we found the following relationships for the lipids
DMPC
DMPC
DMPE
DMPE
DMTAP
MPPC
PMPC
MOPC
OMPC
H
H
H
2
2
2
O, pH 7
O, pH 12
O, pH 7
24.0
23.6
51.3
21.5
38.4
34.0 (33.8)
26.0 (32.6)
with equal head groups: based on the lipids with two tetra-
decyl chains (TT), the elongation of the malonic acid bound
alkyl chain to a hexadecyl chain increases Tm and, on the
other hand, the exchange of the lysine-bound alkyl chain by
an oleyl chain decreases the main transition temperature. In
contrast, the lipids 4a–c and lipids 5a–c (with tris(2-aminoe-
thyl)amine-containing head groups) show for both alkyl
H O, pH 12
Na
H
H
H
2
2
HPO
4
(50 mm), NaH
2
PO
4
, pH 7.4
2
2
2
O
O
O, ethylene glycol
ꢀ19.0 (20.1)
H O, ethylene glycol
2
ꢀ27.0
chain variations (TT!TH and TT!OT) a decrease in T .
m
[a] DMPC=1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPE=1,2-di-
myristoyl-sn-glycero-3-phosphoethanolamine, DMTAP=1,2-dimyristoyl-
3-trimethylammonio propane chloride, MPPC=1-myristoyl-2-palmitoyl-
sn-glycero-3-phosphocholine, PMPC=1-palmitoyl-2-myristoyl-sn-glycero-
The decrease of Tm after the introduction of an oleyl chain
is in line with results found for phospholipids; for example,
cis double bonds reduce the effective alkyl chain length and
3
-phosphocholine, MOPC=1-myristoyl-2-oleoyl-sn-glycero-3-phospho-
[29]
disturb the chain packing that results in a decrease of Tm.
choline, OMPC=1-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine.
For the exchange of one tetradecyl chain by a hexadecyl
chain we would expect an increase of T based on principles
m
[29]
found for phospholipids. However, only the lipids of the
ethylenediamine type show the expected Tm dependencies
significantly higher transition temperatures. In addition, a
group of amino acid based cationic lipids with lysine as head
group, two tetradecyl chains as the lipophilic part of the
molecule and glutamate as the backbone show lower transi-
(
T (TH)>T (TT)). The lipids of the tris(2-aminoethyl)-
m m
amine type exhibit the opposite behaviour (T (TT)>
m
T (TH)). We cannot explain this different behaviour at the
m
moment, but assume a head-group-dependent difference in
the molecular conformation, resulting in different packing
properties and, as a consequence, deviations from the ex-
pected variations of Tm with alkyl-chain length. Further
physicochemical investigations such as X-ray scattering are
necessary to prove this assumption.
tion temperatures (T between 25.5 and 41.08C) than the
m
[32]
lipids 1–4a. This observation indicates a very strong hy-
drogen-bonding interaction between the head group and/or
lipid-backbone region in addition to the van der Waals inter-
[30,33]
action between the alkyl chains.
Another piece of evi-
dence that supports this assumption is the high transition en-
As mentioned before, the attachment of one or two lysine
residues to the head group decreases the transition tempera-
ture of the lipids 1–5a–c on comparing the lipids with the
same alkyl chain combination. The lipids 1a–c exhibit the
smallest head group (see Figure 1), resulting in a cylindrical
lipid shape: a structure that allows dense packing of the
lipids. The attachment of a lysine molecule to the head
group increases the conical shape of the lipids by enlarging
the head group. This leads to a decrease of Tm due to a dis-
tortion of the lipid packing. Furthermore, we assume a
better head-group hydration because of the hydrophilic
lysine moieties. This hydration is associated with changes in
the properties of the bilayer/solute interface and, hence, also
thalpy of the main transition of lipids 2–5a (42.4–
59.1 kJmol ; see Table 1). These values exceed the range of
ꢀ
1
the L /L phase transition enthalpies of 1,2-diacyl-sn-glyc-
a
b
ero-3-phospholipids and glycolipid bilayers of comparable
hydrocarbon-chain length (published DH values ꢁ21–
ꢀ
1
[31c]
25 kJmol ).
Furthermore, high enthalpy values are also
observed for lipids 2b, 3b, 4b and 2c (see Table 1). The en-
thalpy change that occurs during a transition in lamellar
phases is caused by conformational changes within the lipid
hydrocarbon chains, by changes in van der Waals interac-
tions due to an increase in the molecular area at the bilayer/
water interface, and also by contributions from the changes
in heat of hydration of the head group and the breaking of
hydrogen bonds in the backbone and/or head-group region.
At present, we have no clear evidence for the assumption of
better chain packing and additional hydrogen-bonding inter-
actions in the head-group region. Additional experiments in-
[30]
influences the transition temperature.
Moreover, the at-
tachment of two lysine residues at the tris(2-aminoethyl)a-
mine head group results in a more conical lipid shape, which
explains the observed decrease of the Tm value [T (lipids
m
Chem. Eur. J. 2013, 19, 12824 – 12838
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12831

B. Dobner et al.
[
35]
cluding X-ray diffraction and infrared spectroscopy are nec-
essary to prove this notion.
ical shape, which prefers the formation of micelles. Fur-
thermore, a second species of aggregate with a larger radius
(about 1–2% of all aggregates in the mass-weighted particle
size distribution) was observed (see Table 3). These aggre-
gates, with radii in the range between 35 and 155 nm, are
too large for micellar structures. It is conceivable that these
aggregates are agglomerates of micelles. To visualise the
shape of the aggregates, TEM investigations were per-
formed.
Comparing the lipid structures of lipids 6a–c, they only
vary in the length and degree of saturation of the alkyl
chains (see Figure 1). These lipids show no transitions in the
DSC scans, presumably due to the formation of micellar ag-
gregates. Based on the chemical structure (Figure 1), we can
assume two explanations for the absence of transition peaks
in the DSC curves: 1) lipids with six primary amino groups
are very hydrophilic and, hence, soluble in carbonate buffer
at pH 10; and 2) the lipids self-assemble into micelle-like ag-
gregates that do not show endothermic transitions. To
obtain further information about the shape and size of the
aggregates of lipids 6a–c formed in aqueous dispersions, we
investigated the aggregate size by using DLS and visualised
the aggregates with TEM.
Particle size measurements: The results of the DLS meas-
urements (intensity and mass-weighted radii of the particles)
are summarised in Table 3. Besides the intensity-weighted
ꢀ
1
Table 3. Particle sizes of aggregates formed of lipids 6a–c (c=1 mgmL
,
in carbonate buffer at pH 10).
Lipid
Peak 1
r [nm] area [%])
Peak 2
r [nm] (area [%])
[
a]
[a]
intensity weighted:
lipid 6a
lipid 6b
4.4ꢂ0.1 (22.2ꢂ1.7)
4.4ꢂ0.1 (17.9ꢂ0.6)
6.1ꢂ0.2 (10.7ꢂ0.5)
196.2ꢂ160.3 (68.8ꢂ14.4)
46.9ꢂ2.1 (76.1ꢂ4.5)
85.9ꢂ1.3 (85.7ꢂ1.4)
lipid 6c
mass weighted:
lipid 6a
lipid 6b
3.8ꢂ0.3 (99.1ꢂ0.3)
4.1ꢂ0.2 (99.2ꢂ0.1)
5.4ꢂ0.2 (97.7ꢂ0.1)
155.6ꢂ76.2 (0.9ꢂ0.3)
34.6ꢂ2.8 (0.7ꢂ0.1)
95.6ꢂ3.6 (2.2ꢂ0.1)
lipid 6c
ꢀ1
[
a] The averages of the particle radii and the corresponding peak areas
Figure 4. TEM images of lipid dispersions (c=0.05 mgmL in carbonate
buffer at pH 10): A) lipid 6a; B) lipid 6b; C) lipid 6c. The samples were
stained with uranyl acetate. Black arrows point to small round aggregates
are given with the corresponding standard deviations (n=3).
(
micelles). White arrows point to larger agglomerates.
particle size, we also used the mass-weighted particle size
distribution because this is more meaningful for polydis-
[
34]
perse samples with different particle species. All investi-
gated lipids reproducibly showed two species of particles
with different radii in aqueous dispersions (carbonate buffer
Transmission electron microscopy: Figure 4 shows TEM
ꢀ
1
images of lipid dispersions containing 0.05 mgmL of lipid
(lipids 6a–c, Figure 4A–C, respectively) in carbonate buffer
at pH 10. The samples were negatively stained with uranyl
acetate. All three images show two species of aggregates
with different particle sizes that are roughly in agreement
with the DLS results mentioned above. We observed nearly
round aggregates with radii of about 10 nm (see Figure 4,
black arrows) and some larger agglomerates, which consist
of small spherical aggregates (micelles) with radii smaller
than 4 nm (see Figure 4, white arrows). These observations
are in agreement with the micelle hypothesis proposed
above for aqueous dispersions of lipids 6a–c.
ꢀ
1
at pH 10, c=1 mgmL ). This observation refutes the
theory that the lipids 6a–c are molecularly dissolved in
aqueous media. The dominant species has a particle size
(
radius) of between 4 and 6 nm for all investigated lipid dis-
persions. This particle size indicates that aggregates of a mi-
cellar type are probably present in the aqueous suspension.
The theoretical length of the lipid molecules, which is in the
range between 2.5 and 3.0 nm (see the Supporting Informa-
tion), supports this hypothesis. Under the assumption that
the radius of a micelle equals approximately the length of
one lipid molecule and bearing in mind that DLS deter-
mines the hydrodynamic radius, the radii determined by
DLS measurements are in line with the theoretical values.
Another supporting fact is the shape of our lipid molecules:
the large hydrophilic head group of lipids 6a–c with three
lysine molecules within the head group causes a distinct con-
Conclusion
A new structure type of amino-functionalised lipids has
been synthesised. These lipids exhibit a complex malonic
12832
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 12824 – 12838

Peptide-Mimic Amino-Functionalised Lipids
FULL PAPER
acid diamide backbone and represent the second generation
of the malonic acid diamides. The lipids contain six head-
group types of variable size and different numbers of amino
moieties, as well as three different types of alkyl chain. The
use of lysine as a major component of the lipids and the
high number of amide bonds determine the peptide-mimic
character of the lipids. The modular nature of the synthesis
of the lipid backbone, as well as the head-group attachment,
results in a wide variation in the lipid structures. The combi-
nation of different alkyl chains with head groups of variable
size and number of amino groups allow a detailed investiga-
tion of structure dependencies for subsequent research ac-
tivities.
Lipid precursor synthesis: The synthesis of the monoalkylated malonic
acid diethyl esters (5a and b) and monoethyl esters (6a and b) was de-
scribed previously.
[
10a,17a,b,f]
Synthesis of orthogonally protected alkyl lysine amides (2a and b): Tetra-
decylamine or oleylamine (10 mmol) and DIPEA (10 mmol, 1.29 g) were
2 2
dissolved in CH Cl (10 mL). A solution of Fmoc-Lys ACHUTGNRNEUNG( Boc)-OH (1;
10 mmol, 4.69 g) and PyBOP (10 mmol, 5.20 g) in CH Cl (50 mL) was
2
2
added under an argon atmosphere whilst stirring, which was continued
for 3 h at room temperature. Afterwards, the reaction mixture was dilut-
ed with CHCl
layer was washed with brine, dried over Na
was evaporated in vacuo. The crude compounds 2a and b were purified
3
and washed with saturated aqueous NaHCO
3
. The organic
2
SO , filtered and the solvent
4
by column chromatography using silica gel 60 and CHCl
the gradient technique.
3
/MeOH with
(
2S)-6-[(tert-Butoxycarbonyl)]amino-2-[(9H-fluoren-9-yl)methoxycarbo-
nyl]amino-N-tetradecylhexanamide (2a), C40 : Yield: 82%; R
.81 (CHCl /MeOH, 9:1, v/v); m.p. 129–1328C; H NMR (500 MHz,
CDCl , 278C): d=0.88 (t, J
39H; CH (CH ) CH , (CH ) CH NHBoc, OC
H
61
N
3
O
5
1
f
=
Head-group dependent and alkyl-chain dependent influ-
ences on the main transition temperatures of the lipids 1–
0
3
3
3
A
H
U
G
R
N
U
G
2 3
CH ), 1.23–1.84 (m,
5
a–c at pH 10 were found: the introduction of lysine in the
A
H
U
G
R
N
U
G
ACHTUNGTRENNUNG
2
2
12
3
2
3
2
3 3
CH
2
3
NHCO, CH
2
NHBoc), 4.10–4.11 (m, 1H; COCHNHFmocCH
(H,H)=7.0 Hz, 1H; H9-fluorene), 4.38–4.39 (m, 2H; CH -fluorene),
.63 (s, 1H; NHBoc), 5.58 (s, 1H; NHFmoc), 6.20 (s, 1H; NHCO), 7.30
2
), 4.20
head group decreases the Tm value. The alkyl-chain effect
was different between lipids of the ethylenediamine type
(
t, J
A
H
U
T
E
U
G
2
4
(
T (TH)>T (TT)>T (OT)) and tris(2-aminoethyl)amine
3
3
m
m
m
(
t, J
ACTHUNGTRENNUNG( H,H)=7.5 Hz, 2H; H3,H6-fluorene), 7.39 (t, J ACHTUGNTRENNNUG
type (T (TT)>T (TH)>T (OT)). The observed high tran-
3
m
m
m
H2,H7-fluorene), 7.58 (d,
7.75 ppm (d,
[
JACHTUNGTRENNUNG
3
sition temperature and enthalpy values indicate strong inter-
actions in the head group and backbone region. This as-
sumption is supported by observations from NMR measure-
ments.
JACHTUNGTRENNUNG
+
61 3 5
H N O
(
67 3 5
H N O
Special thermal behaviour was observed for lipids 6, bear-
ing the largest head group with six primary amino moieties.
These lipids exhibit no phase transition in the observed tem-
perature region. DLS and TEM investigations indicate that
these lipids form micellar aggregates in an aqueous environ-
ment. The micelles tend to agglomerate, resulting in the for-
mation of larger aggregates.
First results suggest that the gene-transfer efficiencies of
these lipids are very promising. Further physicochemical in-
vestigations with respect to the complexation ability of poly-
nucleotides (as pure lipid or in a mixture with other lipids),
including other scattering techniques and transfection ex-
periments to test the transfection efficiency of the amino-
functionalised lipids, are currently under way.
7
R
f
3
1
3
3
JACHTUNGTRENNUNG
CH
2
CH
CH CH
.10–3.23 (m, 4H; CH
), 4.20 (t,
3
), 1.27–1.86 (m, 39H; CH
2
A
H
U
G
R
N
N
(CH
2
)
6
CH
2
2
A
H
U
G
E
U
G
2
)
6
3
,
(
3
2
)
3
2
NHBoc, OC(CH
A
H
U
T
E
N
N
3
)
3
2
2
2
2
3
COCHNHFmocCH
.40 (m, 2H; CH
CH=CH), 5.54 (s, 1H; NHFmoc), 6.14 (s, 1H; NHCO), 7.30 (t, J-
2
J
A
H
U
G
R
N
U
4
2
3
3
A
H
U
G
R
N
U
G
JACHTUNGTRENNUNG
3
H2,H7-fluorene), 7.58 (d,
JACHTUNGTRENNUNG
3
7
.76 ppm (d,
JACHTUNGTRENNUNG
+
[
67 3 5
H N O
Fmoc cleavage (3a and b): Alkyl lysine amides 2a and b (10 mmol) and
piperidine (64.6 mmol, 5.5 g) were dissolved in DMF (55 mL) and stirred
for 3 h at room temperature. Afterwards, the reaction batch was diluted
with water and extracted three times with heptane. The organic layers
were combined, dried over Na
2 4
SO , filtered and the solvent was evapo-
rated in vacuo. The crude compounds 3a and b were purified by column
chromatography using silica gel 60 and CHCl
dient technique.
3 3
/MeOH/NH with the gra-
Experimental Section
(
(
2S)-2-Amino-6-[(tert-butoxycarbonyl)]amino-N-tetradecylhexanamide
3a), C25 : Yield: 99%; R =0.82 (CHCl /MeOH/NH , 90:10:0.5,
, 278C): d=0.87 (t, J-
), 1.25–1.87 (m, 39H; CH (CH
), 3.11–3.12 (m, 2H; CH NHBoc), 3.22 (dd,
(H,H)=6.7 Hz, 2H; CH NHCO), 3.33 (dd, J
(H,H)=4.3 Hz, 1H; COCHNH CH ), 4.56 (s, 1H; NHBoc),
H
51
N
3
O
3
f
3
3
General: All materials and reagents were purchased from Sigma Aldrich
Co. Ltd. unless stated otherwise. All solvents were analytically pure and
dried before use. Thin-layer chromatography was carried out on alumini-
um sheets pre-coated with silica gel 60 F254 (Merck, Darmstadt) and de-
veloped with bromothymol blue dip. For column chromatography under
normal pressure, silica gel 60 (0.063–0.200 mm) was used. Mass spectrom-
etry analyses were performed with a Finnigan MAT 710C instrument
1
3
v/v/v); m.p. 67–698C; H NMR (500 MHz, CDCl
(H,H)=6.9 Hz, 3H; CH CH
CH CH NHBoc, OC(CH
(H,H)=13.5, J
3
A
H
U
G
R
N
U
G
2
3
2
A
H
U
G
E
N
N
2 3
)12CH ,
(
2
)
3
2
A
H
N
T
E
N
N
3
)
3
2
3
3
3
J
1
A
H
N
T
E
N
N
2
A
T
N
R
N
G
2
1
ACHTUNGTRENNUNG( H,H)=
3
7
.9,
J
2
A
H
U
G
R
N
U
2
2
+
+
7
.25 ppm (s, 1H; NHCO); MS: m/z: 442 [M+H] , 883 [2M+H] , 464
+
+
[
M+Na] , 905 [2M+Na] ; elemental analysis calcd (%) for C25
C 67.98, H 11.64, N 9.51; found: C 68.24, H 11.45, N 9.38.
(2S)-2-Amino-6-[(tert-butoxycarbonyl)]amino-N-[(9Z)-octadec-9-enyl]-
hexanamide (3b), C29 : Yield: 94%; R =0.54 (CHCl /MeOH/
NH , 90:10:0.5, v/v/v); m.p. 37–388C; H NMR (500 MHz, CDCl , 278C):
d=0.86 (t, ), 1.24–1.87 (m, 39H; CH
(CH CH CH=CHCH CH NHBoc, OC(CH ), 1.95–
2.02 (m, 4H; CH CH=CHCH (H,H)=
6.4 Hz, 2H; CH NHBoc), 3.21 (dd, (H,H)=6.4 Hz,
2H; CH NHCO), 3.31 (dd, (H,H)=4.4 Hz, 1H;
51 3 3
H N O :
(
Thermoseparation Products, San Jose, CA) for ESI-MS, and with an
LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen) for
1
13
HRMS. H and C NMR spectra were recorded on Varian Gemini 2000
and Varian Inova 500 instruments. In general, we used CDCl for
OD mixture (2:1, v/v) for
C NMR spectroscopy. In some cases, we had to vary the solvent compo-
sition and the solvent (by using D O) due to solubility problems. Elemen-
H
57
N
3
O
3
f
3
3
1
1
H NMR spectroscopy and
a
CDCl
3
/CD
3
3
3
1
3
3
J
A
H
U
G
R
N
N
2
CH
3
2
-
A
H
U
G
E
N
N
2
)
6
2
2
A
H
N
T
E
N
N
2
)
6
3
2
)
3
2
A
H
U
G
R
N
U
G
3 3
)
J ACHTUNGTRENNUNG
2
2
3
3
tal analyses were performed with a CHNS-932 apparatus (Leco Corpora-
tion, St. Joseph, MI). MilliQ water was produced with a Milli-Q Advant-
age A10 pure-water system (Millipore, Billerica, MA).
2
2
J
1
A
H
U
G
R
N
U
G
3
3
2
1 2
J ACHTUNGERTNNU(GN H,H)=6.9, J ACHNUTGTREUNNGN
(H,H)=8.0,
3
3
2
J
1
A
H
U
G
R
N
U
G
2
J ACHTUNRTGENNUNG
Chem. Eur. J. 2013, 19, 12824 – 12838
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12833

B. Dobner et al.
COCHNH
2
CH
2
), 4.59 (s, 1H; NHBoc), 5.31–5.36 (m, 2H; CH=CH),
1.25–1.88 (m, 65H; NHCH
OC(CH ), 3.02–3.26 (m, 5H; CH
cyl)CO), 4.42–4.43 (m, 1H; COCH
NHBoc), 6.65–6.83 (m, 1H; NHCO), 7.34–7.44 ppm (m, 1H; NHCO);
2
A
H
N
T
E
N
N
2
)
12CH
3
, (CH
NHBoc, CH
(NHCO)RCH
2
)
13CH
3
, (CH
NHCO, COCH(tetrade-
), 4.73 (s, 1H;
2 3 2
) CH NHBoc,
+
+
7
.25 ppm (s, 1H; NHCO); MS: m/z: 496 [M+H] , 991 [2M+H] , 518
A
T
G
R
N
U
G
3
)
3
2
2
+
+
[
M+Na] , 1013 [2M+Na] ; elemental analysis calcd (%) for
: C 70.25, H 11.59, N 8.48; found: C 70.04, H 11.27, N 8.51.
A
H
U
G
R
N
U
G
2
29 57 3 3
C H N O
+
+
+
ꢀ
MS: m/z: 747 [M+Na] , 1471 [2M+Na] , 1488 [2M+K] , 723 [MꢀH] ,
Coupling of the lipid backbone (7a–c): The appropriate lysine amide 3a
and b (1 mmol) and DIPEA (2 mmol, 250.5 mg) were dissolved in
ꢀ
1
1
446 [2MꢀH] ; elemental analysis calcd (%) for C42
81 3 6
H N O : C 69.66, H
1.27, N 5.80; found: C 69.36, H 10.99, N 5.78.
CH
ethyl ester 6a and b (1 mmol) and PyBOP (1 mmol, 520,4 mg) in CH
15 mL) was added under an argon atmosphere whilst stirring, which was
continued for 16 h. Afterwards, the reaction mixture was diluted with
2
Cl
2
(3 mL). A solution of the appropriate alkyl malonic acid mono-
2
Cl
2
2-[(N-{6-[(tert-Butoxycarbonyl)amino]-1-oxo-1-[(N-tetradecyl)amino]hex-
an-(2S)-2-yl}amino)carbonyl]octadecanoic acid (8b), C H N O : Yield:
(
4
4
85
3
6
1
94%; R =0.30 (CHCl /MeOH, 9:1 v/v); m.p. 102–1058C; H NMR
f
3
3
CHCl
3
and washed with aqueous NaHCO
3
(5%). Then, the organic layer
, filtered and the solvent was
3 2 3
(500 MHz, CDCl , 278C): d=0.88 (t, J ACHNUTRTGENNUNG( H,H)=6.8 Hz, 6H; 2ꢁCH CH ),
was washed with brine, dried over Na
2
SO
4
2 2 12 3 2 15 3 2 3 2
1.25–1.91 (m, 69H; NHCH ACHTUNGTRENNGNU( CH ) CH , (CH ) CH , (CH ) CH NHBoc,
evaporated in vacuo. The crude compounds 7a–c were purified by iso-
OC
cyl)CO), 4.37–4.39 (m, 1H; COCH
6.32–6.41 (m, 1H; NHCO), 7.07–7.16 ppm (m, 1H; NHCO); MS: m/z:
3 3 2 2
ACHTUNGTRNENUG( CH ) ), 3.03–3.25 (m, 5H; CH NHBoc, CH NHCO, COCH(hexade-
cratic column chromatography using silica gel 60 and CHCl
v) as eluent.
3
/Et
2
O (1:1, v/
2
ACHTUNGTRENNUNG( NHCO)CH ), 4.70 (s, 1H; NHBoc),
+
+
+
ꢀ
7
75 [M+Na] , 1526 [2M+Na] , 790 [M+K] , 751 [MꢀH] ; elemental
analysis calcd (%) for C44 : C 70.26, H 11.39, N 5.59; found: C
9.88, H 11.24, N 5.51.
2-[(N-{6-[(tert-Butoxycarbonyl)amino]-1-{N-[(9Z)-octadec-9-enyl]ami-
no}-1-oxohexan-(2S)-2-yl}amino)carbonyl]hexadecanoic acid (8c),
C H N O : Yield: 95%; R =0.33 (CHCl /MeOH, 9:1, v/v); m.p. 75–
2
-[(N-{6-[(tert-Butoxycarbonyl)amino]-1-oxo-1-[(N-tetradecyl)amino]hex-
an-(2S)-2-yl}amino)carbonyl]hexadecanoic acid ethyl ester (7a),
: Yield: 97%; R =0.47 (CHCl /Et O, 1:1, v/v); m.p. 84–878C;
H NMR (500 MHz, CDCl , 278C): d=0.87 (t, J
), 1.25–1.92 (m, 68H; NHCH (CH
NHBoc, OCH CH OC(CH ), 3.03–3.14 (m, 2H;
NHCO, COCH(tetradecyl)CO),
), 4.32–4.37 (m, 1H; COCH(NHCO)CH ),
.61 (s, 1H; NHBoc), 6.26/6.36 (2ꢁs, 1H; CH NHCO), 6.91 ( J(H,H)=
(H,H)=7.7 Hz) (2ꢁd, 1H; COCH(NHCO)CH );
85 3 6
H N O
6
C
44
H
85
N
3
O
6
f
3
2
1
3
3
ACHTUNGTRENNUNG
(
CH
2
)
)
2
3
CH
3
2
A
H
U
G
R
N
U
G
2
)
12CH
3
,
2 3
)13CH ,
(
CH
2
CH
2
2
3
,
A
H
U
G
E
N
N
3 3
)
4
6
87
3
6
f
3
1
3
CH
2
NHBoc), 3.19–3.24 (m, 3H; CH
2
3
788C; H NMR (500 MHz, CDCl , 278C): d=0.88 (t, JAHCTNUGTRENUNGN
4
4
8
.15–4.22 (m, 2H; OCH CH
2
3
A
H
U
G
R
N
U
G
2
2 3 2
6H; 2ꢁCH CH ), 1.25–1.90 (m, 65H; NHCH ACHUTNRTGENNUNG
2
6
2
2
3
2
A
H
U
T
E
N
N
2 6 3 2 13 3 2 3 2 3 3
ACHTUNGERTNNGNU( CH ) CH , (CH ) CH , (CH ) CH NHBoc, OC ACUTHNGTRENNGU( CH ) ), 1.94–2.02 (m,
4H; CH CH=CHCH ), 3.05–3.27 (m, 5H; CH NHBoc, CH NHCO,
2 2 2 2
3
.0 Hz)/6.96 ppm ( J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
H
N
R
N
N
2
+
+
+
MS: m/z: 750 [M+H] , 774 [M+Na] , 1526 [2M+Na] ; elemental analy-
sis calcd (%) for C44 : C 70.26, H 11.39, N 5.59; found: C 70.34, H
COCH(tetradecyl)CO), 4.38–4.46 (m, 1H; COCH
2
ACHTUNGTNERNUN(G NHCO)CH ), 4.72 (s,
85 3 6
H N O
1H; NHBoc), 5.33–5.38 (m, 2H; CH=CH), 6.53–6.63 (m, 1H; NHCO),
+
ꢀ
1
1.25, N 5.58.
7.30–7.35 ppm (m, 1H; NHCO); MS: m/z: 801 [M+Na] , 777 [MꢀH] ,
ꢀ
2
-[(N-{6-[(tert-Butoxycarbonyl)amino]-1-oxo-1-[(N-tetradecyl)amino]hex-
1554 [2MꢀH] ; elemental analysis calcd (%) for C46
87 3 6
H N O : C 71.00, H
an-(2S)-2-yl}amino)carbonyl]octadecanoic acid ethyl ester (7b),
: Yield: 96%; R =0.48 (CHCl /Et O, 1:1, v/v); m.p. 87–898C;
H NMR (500 MHz, CDCl , 278C): d=0.88 (t, J
), 1.25–1.92 (m, 72H; NHCH (CH
NHBoc, OCH CH OC(CH ), 3.03–3.15 (m, 2H;
NHCO, COCH(hexadecyl)CO),
), 4.32–4.37 (m, 1H; COCH(NHCO)CH ),
.61 (s, 1H; NHBoc), 6.25/6.36 (2ꢁs, 1H; CH NHCO), 6.91 ( J(H,H)=
(H,H)=7.7 Hz) (2ꢁd, 1H; COCH(NHCO)CH );
11.27, N 5.40; found: C 70.78, H 11.12, N 5.47.
C
46
H
89
N
3
O
6
f
3
2
Coupling with ethylene diamine (9a–c) or tris(2-aminoethyl)amine (13a–
c): Ethylene diamine (20 mmol, 1.20 g) or tris(2-aminoethyl)amine
(20 mmol, 2.93 g), respectively, and TEA (2 mmol, 202 mg) were dis-
solved in CH Cl (2 mL). A solution of the appropriate acid 8a–c
1
3
3
ACHTUNGTRENNUNG
(
(
CH
CH
2
)
)
2
3
CH
CH
3
2
A
H
U
G
R
N
U
G
2
)12CH
3
,
2 3
)15CH ,
2
2
2
3
,
A
H
U
G
R
N
U
G
3 3
)
2
2
CH
2
NHBoc), 3.18–3.24 (m, 3H; CH
2
2 2
(1 mmol) and PyBOP (1 mmol, 520.4 mg) in CH Cl (50 mL) was added
4
4
7
.14–4.24 (m, 2H; OCH CH
2
3
A
H
U
G
R
N
N
2
slowly to the reaction mixture under argon atmosphere whilst stirring,
which was continued for further 16 h. Afterwards, the dispersion was fil-
tered and the filtrate was diluted with CHCl3 and washed with aqueous
K CO (15%). The organic layer was washed with brine, dried over
3
2
ACHTUNGTRENNUNG
3
.9 Hz)/7.00 ppm ( J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
H
U
G
R
N
N
2
+
ꢀ
ꢀ
MS: m/z: 802 [M+Na] , 778 [MꢀH] , 814 [M+Cl] ; elemental analysis
2
3
calcd (%) for C46 : C 70.81, H 11.50, N 5.39; found: C 71.01, H
H
89
N
3
O
6
2 4
Na SO , filtered, and solvent was evaporated. The crude product was pu-
1
1.19, N 5.39.
3
rified by column chromatography using silica gel 60 and CHCl /MeOH/
2
-[(N-{6-[(tert-Butoxycarbonyl)amino]-1-{N-[(9Z)-octadec-9-enyl]ami-
NH
Boc-Lys
15a–c, 16a–c): The appropriate amount of amino-functionalised lipid,
Boc-Lys(Boc)-OSu, and TEA were dissolved in CH Cl (20 mL) and stir-
3
with gradient technique.
no}-1-oxohexan-(2S)-2-yl}amino)carbonyl]hexadecanoic acid ethyl ester
ACHTUNGTR(EUNNNG Boc)-OSu coupling with primary amino groups (11a–c, 12a–c,
(
7c), C48
H
91
N
3
O
6
: Yield: 93%; R
f
=0.44 (CHCl
, 278C): d=0.88 (t,
), 1.25–1.92 (m, 68H; NHCH (CH
(CH CH NHBoc, OCH
CH=CHCH
NHCO, COCH(tetradecyl)CO), 4.15–4.24 (m, 2H; OCH
.38 (m, 1H; COCH(NHCO)CH ), 4.61 (s, 1H; NHBoc), 5.32–5.38 (m,
H; CH=CH), 6.22/6.36 (2ꢁs, 1H; CH
3 2
/Et O, 1:1, v/v); m.p. 70–
1
3
7
18C; H NMR (500 MHz, CDCl
3
J
A
H
U
G
R
N
U
G
ACHTUNGTRENNUNG
2
2
6
H; 2ꢁ(CH
CH
.93–2.05 (m, 4H; CH
CH
2
)
2
CH
3
2
A
H
U
G
E
N
N
2
)
6
CH
2
CH=CHCH
OC(CH
NHBoc,
CH ), 4.31–
2
-
red for 16 h at room temperature. Following, the mixture was diluted
with CHCl (20 mL) and washed with aqueous K CO (15%). The organ-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(CH
2
)
6
3
,
(CH
2
)
13CH
3
,
2
)
3
2
2
CH
3
,
A
H
U
G
R
N
U
G
3 3
) ),
3
2
3
1
2
2
), 3.02–3.25 (m, 5H; CH
2
2 4
ic layer was separated, washed with brine, dried over Na SO , and solvent
2
2
3
was evaporated. The crude product was purified by column chromatogra-
phy using silica gel 60 and CHCl /MeOH with gradient technique. Reac-
tants and stoichiometric ratios for the reaction with regard to the re-
quired product: 11a–c: 9a–c (0.5 mmol), Boc-Lys(Boc)-OSu (0.5 mmol),
TEA (0.5 mmol); 12a–c: 10a–c (0.5 mmol), Boc-Lys(Boc)-OSu (1 mmol),
TEA (1 mmol); 15a–c: 13a–c (0.5 mmol), Boc-Lys(Boc)-OSu (1 mmol),
(Boc)-OSu (1.5 mmol),
4
2
6
8
1
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
3
3
2
NHCO), 6.91 ( J
ACHTUNGTRENNUNG
3
.98 ppm ( J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=7.6 Hz) (2ꢁd, 1H; COCH(NHCO)CH
A
H
U
G
R
N
U
G
2
AHCTUNGTRENNUNG
+
29 [M+Na] ; elemental analysis calcd (%) for C48
91 3 6
H N O
AHCTUNGTRENNUNG
1.38, N 5.21; found: C 71.28, H 11.28, N 5.03.
AHCTUNGTRENNUNG
Saponification of 7a–c (8a–c): The appropriate ester 7a–c (1 mmol) was
dissolved in a mixture of THF (15 mL) and water (5 mL), and potassium
hydroxide (1.5 mmol, 84.2 mg) was added. The mixture was stirred for
4
7
ice bath. The mixture was stirred for 15 min, followed by extraction with
CHCl . The organic layer was washed with brine, dried over Na SO , fil-
tered and the solvent was evaporated. The crude compounds 8a–c were
purified by column chromatography using silica gel 60 and CHCl
with the gradient technique.
TEA (1 mmol); 16a–c: 14a–c (0.5 mmol), Boc-LysCAHTUNGTRENNNUG
TEA (1.5 mmol).
Removal of the Boc protective group with trifluoroacetic acid (lipids 1a–
c, lipids 2a–c, lipids 3a–c, and lipids 4a–c): The appropriate amount of
6 h at room temperature. Then,
2 4
a solution of H SO (0.75 mmol,
3.6 mg) in water (13 mL) was added while stirring and cooling with an
Boc-protected lipid (0.5 mmol) was dissolved in CH
quired amount of trifluoroacetic acid (9a–c 4 mL; 11a–c 6 mL; 12a–c
mL; 13a–c 6 mL) was added stepwise while stirring at room tempera-
ture. Then, the solvent was evaporated at 258C. The residue was dis-
solved in CHCl (20 mL) and ammonia solution (10 mL, 10%) was
added dropwise while stirring, with an ice bath. Afterwards, CHCl
(20 mL) and aqueous K CO (10 mL, 10%) were added to the dispersion.
The organic layer was separated and the water layer was extracted twice
with CHCl . The combined organic layers were washed with brine, dried
over Na SO and the solvent was evaporated. The crude product was pu-
2 2
Cl (10 mL). The re-
3
2
4
8
3
/MeOH
3
3
2
-[(N-{6-[(tert-Butoxycarbonyl)amino]-1-oxo-1-[(N-tetradecyl)amino]hex-
an-(2S)-2-yl}amino)carbonyl]hexadecanoic acid (8a), C42 : Yield:
9%; =0.39 (CHCl /MeOH, 9:1, v/v); m.p. 107–1108C; H NMR
500 MHz, CDCl , 278C): d=0.88 (t, J
2
3
81 3 6
H N O
1
8
(
R
f
3
3
3
3
A
H
U
T
E
N
N
(H,H)=6.9 Hz, 6H; 2ꢁCH
2
CH
3
),
2
4
12834
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 12824 – 12838

Peptide-Mimic Amino-Functionalised Lipids
FULL PAPER
ꢀ
ꢀ
+
rified by column chromatography using silica gel 60 and CHCl
3
/MeOH/
[M+Cl] , 792.6 [MꢀH] ; HRMS calcd for
C
45
H
92
N
7
O
4
[M+H] :
NH
3
with the gradient technique.
794.7205; found: 794.7208.
N-(2-Aminoethyl)-N’-[6-amino-1-oxo-1-[(N-tetradecylamino)hexan-(2S)-
N-[6-Amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N’-[2-(N-{(2S)-
2,6-diamino-1-oxohexyl}amino)ethyl]-2-hexadecylpropandiamide (lipid
, 65:35:5, v/v/
2
0
2
-yl]-2-tetradecylpropandiamide (lipid 1a), C39
H
79
N
5
O
3
: Yield: 94%; R
f
=
1
.36 (CHCl
3
/MeOH/NH
3
, 80:20:2, v/v/v); H NMR (500 MHz, CDCl
3
,
2b), C47
H
95
N
7
O
4
: Yield: 70%; R
f
=0.18 (CHCl
3
/MeOH/NH
3
3
1
3
78C): d=0.88 (t,
(CH
.5 Hz, 2H; (CH
NHCH CH NH ), 3.00 (t,
.18–3.36 (m, 4H; 2ꢁCH
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=6.9 Hz, 6H; 2ꢁCH
(CH (CH CH NH
CH ), 2.83 (t,
(H,H)=7.6 Hz, 1H; COCH(tetradecyl)CO),
NHCO), 4.30–4.34 (m, 1H; COCH-
3
), 1.25–1.88 (m, 56H;
v); H NMR (500 MHz, CDCl
3
, 278C): d=0.88 (t, J
A
H
U
G
E
N
N
3
NHCH
6
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
)
12CH
3
,
2
)
13CH
3
,
2
)
3
2
2
), 2.70 (t,
J
A
H
N
T
E
N
N
2ꢁCH
(CH
3
), 1.25–1.88 (m, 66H; NHCH
2
A
H
N
T
E
N
N
(CH
2
)
12CH
3
,
(CH
2
)
15CH
3
3
, 2ꢁ
3
2
)
3
2
NH
2
J
A
H
U
G
R
N
U
G
2
)
3
CH NH ), 2.69–2.74 (m, 4H; 2ꢁCH
2
2
2
NH
2
), 2.99 (t, J ACHTUNGTRENNUNG( H,H)=
3
2
2
2
J
A
H
U
G
R
N
U
G
7.5 Hz, 1H; COCH(hexadecyl)CO), 3.16–3.46 (m, 7H; 3ꢁCH
2
NHCO,
3
2
CHNH CH ), 4.26–4.32 (m, 1H; COCH(NHCO)CH ), 6.47–6.49/6.62–
2
2
A
H
U
G
R
N
U
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(NHCO)CH
NHCH CH NH
(NHCO)CH
2
), 6.30–6.32 (m, 1H; tetradecyl-NHCO), 6.85–6.87 (m, 1H;
6.64/7.56–7.58/7.66–7.74 (4ꢁm, 3H; 3ꢁNHCO), 7.20–7.31 ppm (m, 1H;
3
13
2
2
2
1
), 7.16 ppm (d,
); C NMR (125 MHz, CDCl
J
A
H
U
T
N
U
G
COCH
A
H
U
G
R
N
N
(NHCO)CH
2
); C NMR (125 MHz, CDCl
3
/CD
3
OD, 278C): d=
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
3
13.7, 22.1, 22.3, 22.4, 26.7, 27.2, 29.0, 29.09, 29.19, 29.26, 29.36, 29.40,
29.45, 29.6, 29.7, 30.6, 31.19, 31.29, 31.7, 34.24, 38.57, 38.68, 38.78, 38.82,
39.3, 39.4, 40.02, 40.08, 49.5, 53.1, 53.2, 54.35, 54.40, 170.82, 170.87, 171.0,
2
2
5
[
C
2.5, 26.7, 27.2, 27.27, 27.29, 29.0, 29.07, 29.09, 29.2, 29.3, 29.36, 29.39,
9.41, 29.43, 31.0, 31.31, 31.35, 31.7, 39.3, 40.52, 40.57, 41.60, 41.64, 53.3,
3.2, 53.3, 54.7, 171.0, 171.1, 171.2, 171.3, 171.6, 171.7 ppm; MS: m/z: 666
+
171.3, 171.9, 172.0, 175.9, 176.0 ppm; MS: m/z: 822.6 [M+H] , 844.7
+
2+
+
ꢀ
+
ꢀ
ꢀ
M+H] , 334 [M+2H] , 688 [M+Na] ,664 [MꢀH] ; HRMS calcd for
[M+Na] , 856.6 [M+Cl] , 820.6 [MꢀH] ; HRMS calcd for C47
H
96
N
7
O
4
+
+
39
H
80
N
5
O
3
[M+H] : 666.6256; found: 666.6255.
[M+H] : 822.7518; found: 822.7518.
N-(2-Aminoethyl)-N’-[6-amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-
N-{6-Amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N’-
[2-(N-{(2S)-2,6-diamino-1-oxohexyl}amino)ethyl]-2-tetradecylpropandi-
yl]-2-hexadecylpropandiamide (lipid 1b), C41
83 5 3 f
H N O : Yield: 71%; R =
1
0
.27 (CHCl
3
/MeOH/NH
3
, 80:20:2, v/v/v); H NMR (500 MHz, CDCl
3
,
A
H
U
G
R
N
U
G
H
97
N
7
O
4
: Yield: 81%; R
65:35:5, v/v/v); H NMR (500 MHz, CDCl
6.9 Hz, 6H; 2ꢁCH ), 1.23–1.86 (m, 62H; NHCH
CHCH (CH CH , (CH
CH CH=CHCH ), 2.69–2.74 (m, 4H; 2ꢁCH
f
=0.09 (CHCl
, 278C): d=0.88 (t, J
3
/MeOH/NH
3
3
1
3
2
78C): d=0.88 (t,
(CH
.6 Hz, 2H; (CH
NHCH CH NH ), 3.01 (t,
.16–3.35 (m, 4H; 2ꢁCH
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=6.9 Hz, 6H; 2ꢁCH
(CH (CH CH NH
CH ), 2.82 (t,
(H,H)=7.7 Hz, 1H; COCH(hexadecyl)CO),
NHCO), 4.28–4.35 (m, 1H; COCH-
3
), 1.25–1.90 (m, 60H;
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=
3
NHCH
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
)
12CH
3
,
2
)
15CH
3
,
2
)
3
2
2
), 2.70 (t,
J
A
H
U
G
R
N
U
G
3
2
A
H
U
G
E
N
N
(CH
2
)
6
CH CH=
2
3
6
2
)
3
2
3
NH
2
J
A
H
U
G
E
N
N
2
)
13CH
3
, 2ꢁ(CH
2
)
3
CH
2
NH
NH
2
), 1.95–2.02 (m, 4H;
), 3.01 (t,
3
2
2
2
J
A
H
U
G
R
N
U
G
2
2
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=
NHCO,
), 5.33–5.38 (m, 2H;
CH=CH), 6.58–6.60/7.62–7.63/7.73–7.75 (3ꢁm, 3H; 3ꢁNHCO), 7.29–
3
2
7.4 Hz, 1H; COCH(tetradecyl)CO), 3.13–3.46 (m, 7H; 3ꢁCH
CHNH CH ), 4.27–4.32 (m, 1H; COCH(NHCO)CH
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(NHCO)CH
NHCH CH NH
(NHCO)CH
2
), 6.33–6.37 (m, 1H; tetradecyl-NHCO), 6.87–6.90 (m, 1H;
A
H
U
G
R
N
U
G
2
3
2
2
2
1
), 7.20 ppm (d,
); C NMR (125 MHz, CDCl
JACHTUNGTRENNUNG
3
13
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
3
3
7.30 ppm (m, 1H; COCH
2 3
ACHTUNGTRENNGU( NHCO)CH ); C NMR (125 MHz, CDCl /
2
2
4
1
2.2, 22.3, 22.42, 26.48, 27.1, 27.1, 28.82, 28.90, 28.93, 29.0, 29.15, 29.17,
9.19, 29.22, 29.24, 30.9, 31.0, 31.15, 31.17, 31.3, 31.5, 39.1, 40.34, 40.39,
0.42, 41.52, 52.8, 53.1, 53.4, 53.8, 170.8, 170.9, 171.2, 171.3, 171.6,
CD OD, 278C): d=13.7, 22.4, 26.7, 26.9, 27.2, 29.1, 29.2, 29.4, 31.25,
3
31.32, 31.57, 31.64, 31.8, 32.1, 32.3, 34.6, 38.5, 38.6, 38.8, 39.0, 39.3, 39.4,
40.8, 40.9, 53.1, 53.2, 53.7, 54.0, 54.7, 129.4, 129.7, 170.8, 171.0, 171.2,
+
2+
ꢀ
+
71.8 ppm; MS: m/z: 694 [M+H] , 345 [M+2H] , 728 [M+Cl] , 693
171.4, 171.6, 171.8, 176.0, 176.2 ppm; MS: m/z: 848.6 [M+H] , 425.0
ꢀ
+
2+
ꢀ
ꢀ
[
MꢀH] ; HRMS calcd for
94.6567.
N-(2-Aminoethyl)-N’-{6-amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohex-
C
41
H
84
N
5
O
3
[M+H] : 694.6569; found:
[M+2H] , 882.6 [M+Cl] , 846.7 [MꢀH] ; HRMS calcd for C49
98 7 4
H N O
+
6
[M+H] : 848.7675; found: 848.7671.
N-[2-(N-{(2S)-2,6-Diamino-1-oxohexyl}amino)ethyl]-N’-[6-(N-{(2S)-2,6-
diamino-1-oxohexyl}amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-yl]-2-
an-(2S)-2-yl}-2-tetradecylpropandiamide (lipid 1c), C43
85 5 3
H N O : Yield:
, 80:20:2, v/v/v); H NMR (500 MHz,
1
9
2%; R
CDCl
6H;
CH
(H,H)=6.7 Hz, 2H; (CH
f
=0.20 (CHCl
3
/MeOH/NH
3
tetradecylpropandiamide (lipid 3a), C51
H
103
N
9
O
5
: Yield: 31%; R
, 278C):
), 1.24–1.86 (m, 68H; NHCH
NH , (CH CH NHCO), 2.64–
), 3.02–3.07 (m, 1H; COCH(tetradecyl)CO),
3.18–3.49 (m, 10H; 4ꢁCH NHCO, 2ꢁCHNH CH ), 4.27–4.31 (m, 1H;
COCH(NHCO)CH ), 6.62–6.64/7.40–7.50/7.62–7.64/7.71–7.73 (4ꢁm, 4H;
4ꢁNHCO), 7.34–7.36 ppm (d,
f
=0.24
3
1
3
, 278C): d=0.88 (t,
NHCH (CH
NH ), 1.95–2.02 (m, 8H; 2ꢁCH
CH NH
J
A
H
U
G
R
N
U
G
3
), 1.24–1.89 (m,
(CHCl
3
/MeOH/NH
3
, 50:50:10, v/v/v); H NMR (500 MHz, CDCl
3
3
5
(
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
)
6
CH
2
2
A
T
G
R
N
N
2
)
6
3
,
(CH
), 2.70 (t, J-
2
)
13CH
3
3
,
d=0.88 (t,
(CH
2.69 (m, 4H; 2ꢁCH
J
A
H
U
G
R
N
U
G
3
2
-
2
)
3
CH
2
2
2
CH=CHCH
2
A
H
U
G
R
N
N
2
)
12CH
3
, (CH
2
)
13CH
3
2
)
3
2
2
2
)
3
2
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
3
)
3
2
2
J
A
H
U
G
R
N
U
G
2
2
NHCH
.14–3.33 (m, 4H; 2ꢁCH
(NHCO)CH ), 5.33–5.38 (m, 2H; CH=CH), 6.45–6.46 (m, 1H; oleyl-
NHCO), 7.02–7.03 (m, 1H; NHCH CH NH ), 7.33–7.40 ppm (m, 1H;
); C NMR (125 MHz, CDCl /CD OD, 278C): d=
3.4, 22.1, 22.27, 22.35, 26.4, 26.7, 26.98, 27.03, 28.76, 28.83, 28.87, 29.0,
2
CH
2
NH
2
), 3.04 (t,
J
A
H
U
G
R
N
N
2
2
2
3
2
A
H
U
G
R
N
N
2
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
JACHTUNGTRENNUNG
1
3
2
2
2
A
H
U
G
R
N
N
(NHCO)CH
2
3
3
1
3
COCH
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(NHCO)CH
2
3
3
21.8, 22.03, 22.08, 22.09, 22.2, 22.3, 26.14, 26.15, 26.69, 26.70, 27.96, 28.07,
28.48, 28.50, 28.54, 28.56, 28.61, 28.69, 28.81, 28.84, 28.88, 29.28, 30.37,
30.7, 30.8, 30.9, 31.0, 31.1, 34.0, 34.08, 34.17, 34.20, 37.96, 38.02, 38.07,
38.3, 38.4, 38.71, 38.76, 40.06, 40.13, 40.14, 40.16, 52.7, 52.8, 54.1, 54.2,
170.36, 170.40, 170.8, 171.6, 171.7, 171.8, 175.5, 175.9, 176.0 ppm; MS: m/
1
2
3
1
9.1, 29.17, 29.22, 30.1, 30.76, 30.83, 30.99, 31.06, 31.1, 31.2, 31.4, 32.0,
9.0, 40.29, 40.32, 41.4, 52.8, 53.0, 53.3, 129.2, 129.4, 170.7, 170.8, 171.1,
71.2, 171.6, 171.7 ppm; MS: m/z: 720.5 [M+H] , 361.1 [M+2H] , 754.5
+
2+
ꢀ
ꢀ
+
+
2+
+
ꢀ
[
7
M+Cl] , 718.6 [MꢀH] ; HRMS calcd for
20.6725; found: 720.6750.
N-[6-Amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N’-[2-(N-{(2S)-
,6-diamino]-1-oxohexyl}amino)ethyl]-2-tetradecylpropandiamide (lipid
C
43
H
86
N
5
O
3
[M+H] :
z: 922.5 [M+H] , 461.9 [M+2H] , 944.6 [M+Na] , 956.5 [M+Cl] ,
ꢀ
+
920.5 [MꢀH] ; HRMS calcd for C51
104 9 5
H N O [M+H] : 922.8155; found:
9
22.8152.
2
N-(2-{N-[(2S)-2,6-Diamino-1-oxohexyl]amino}ethyl)-N’-[(2S)-2,6-diami-
no-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-2-hexadecylpropandi-
2
a), C45
H
91
N
7
O
4
: Yield: 89%; R
, 278C): d=0.88 (t, J
), 1.25–1.88 (m, 62H; NHCH (CH
CH NH ), 2.69–2.74 (m, 4H; 2ꢁCH
f
=0.15 (CHCl
3
/MeOH/NH
3
, 65:35:5, v/v/
1
3
v); H NMR (500 MHz, CDCl
2
3
A
H
U
G
E
U
G
A
H
U
G
R
N
U
amide (lipid 3b), C53
H
107
N
9
O
5
: Yield: 95%; R
f
=0.26 (CHCl
/CD OD, 278C): d=
), 1.24–1.96 (m, 68H; NHCH
CH NH , (CH CH NHCO), 2.93–
), 3.08–3.56 (m, 9H; COCH(hexadecyl)CO, 4ꢁ
NHCO), 3.87–3.99 (m, 2H; 2ꢁCHNH CH ), 4.22–4.28 ppm (m, 1H;
); C NMR (125 MHz, CDCl /CD OD, 278C): d=
3
/MeOH/
1
ꢁCH
3
2
A
H
U
G
R
N
U
G
2
)
12CH
3
,
(CH
2
)
13CH
3
3
,
2ꢁ
NH
0.85 (t,
(CH
2.98 (m, 4H; 2ꢁCH
CH
COCH
3
, 50:50:10, v/v/v); H NMR (400 MHz, CDCl
3
3
3
(
CH
2
)
3
2
2
2
NH
2
), 2.99 (t,
J
A
H
U
G
R
N
U
G
J
A
H
U
G
R
N
N
3
2
-
7
.5 Hz, 1H; COCH(tetradecyl)CO), 3.16–3.46 (m, 7H; 3ꢁCH
CH ), 4.26–4.32 (m, 1H; COCH(NHCO)CH ), 6.47–6.49/6.62–
.64/7.56–7.58/7.66–7.74 (4ꢁm, 3H; 3ꢁNHCO), 7.20–7.31 ppm (m, 1H;
2
NHCO,
A
T
N
T
E
N
N
2
)
12CH
3
2
)
15CH
3
2
)
3
2
2
2
)
3
2
CHNH
6
2
2
A
H
U
G
R
N
U
2
2 2
NH
2
2
2
1
3
13
COCH
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(NHCO)CH
2
); C NMR (125 MHz, CDCl
3
/CD
3
OD, 278C): d=
A
H
U
G
R
N
N
(NHCO)CH
2
3
3
1
3
4
1
3.5, 22.3, 22.4, 22.5, 26.6, 27.1, 28.89, 28.94, 29.1, 29.20, 29.24, 29.3, 31.2,
13.1, 20.9, 21.1, 22.0, 22.3, 25.9, 26.0, 26.3, 26.4, 26.76, 26.83, 27.7, 27.8,
28.7, 29.0, 29.9, 30.2, 30.9, 31.3, 38.0, 38.3, 38.4, 38.5, 38.9, 39.0, 52.4, 52.5,
52.8, 53.1, 53.4, 168.1, 168.5, 168.7, 170.3, 170.6, 171.0, 172.1, 172.2 ppm,
1.4, 31.5, 31.8, 32.0, 34.4, 34.5, 38.4, 38.5, 38.6, 38.8, 39.17, 39.20, 40.7,
0.8, 49.2, 52.9, 53.1, 54.5, 54.6, 170.7, 170.8, 171.0, 171.5, 171.6, 171.9,
+
2+
+
+
ꢀ
75.9, 176.0 ppm; MS: m/z: 794.7 [M+H] , 398.1 [M+2H] , 828.5
MS: m/z: 950.7 [M+H] , 972.8 [M+Na] , 984.7 [M+Cl] , 948.7
Chem. Eur. J. 2013, 19, 12824 – 12838
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12835

B. Dobner et al.
ꢀ
+
ꢀ
+
[
9
MꢀH] ; HRMS calcd for
C
53
H
108
N
9
O
5
[M+H] : 950.8468; found:
[MꢀH] ; HRMS calcd for
47 96 7 3
C H N O [M+H] : 806.7569; found:
50.8470.
806.7567.
N-(2-{N-[(2S)-2,6-Diamino-1-oxohexyl]amino}ethyl)-N’-[6-(N-{(2S)-2,6-
diamino-1-oxohexyl}amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-
Removal of the Boc protective group with hydrochloric acid (lipid 5a–c
and lipid 6a–c):
(
8
2S)-2-yl]-2-tetradecylpropandiamide (lipid 3c),
2%; R =0.24 (CHCl /MeOH/NH , 50:50:10, v/v/v); H NMR (500 MHz,
CDCl , 278C): d=0.88 (t, (H,H)=6.9 Hz, 6H; 2ꢁCH ), 1.24–1.88 (m,
8H; NHCH (CH CH CH=CHCH (CH CH (CH
CH CH NH (CH CH NHCO), 1.96–2.03 (m, 4H; CH
CHCH ), 2.70–2.74 (m, 4H; 2ꢁCH NH
tradecyl)CO), 3.17–3.46 (m, 10H; 4ꢁCH
55 109 9 5
C H N O : Yield:
Method a: The Boc-protected lipid (0.2 mmol) was suspended in acetic
acid ethyl ester (15 mL). HCl (4 mL, 36%) was added stepwise within
1
f
3
3
3
3
J
A
H
U
G
R
N
U
3
2
h and the reaction mixture was stirred for 6 h. Then, the solvent was
6
(
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
)
6
2
2
A
T
N
T
E
N
N
2
)
6
3
,
2
)
13CH
3
,
2ꢁ
separated in vacuo. Ammonia (10 mL, 15%) was added carefully under
stirring while cooling with an ice bath, and subsequently brine with
2
)
3
2
2
,
2
)
3
2
2
CH=
2
2
2
), 3.01–3.06 (m, 1H; COCH(te-
NHCO, 2ꢁCHNH CH ]), 4.26–
), 5.32–5.38 (m, 4H; CH=CH), 6.58–
.63/7.41–7.48/7.61–7.63/7.71–7.73 (4ꢁm, 4H; 4ꢁNHCO), 7.32 ppm (d,
K
CO
tracted three times with CHCl
washed with brine, dried over Na SO , filtered and the solvent was
(20%; 10 mL) was added to the suspension. The mixture was ex-
2
3
2
2
2
. The combined organic layers were
3
4
.30 (m, 1H; COCH ACHTUNGTRENNUNG( NHCO)CH
2
2
4
6
evaporated. The crude lipid was purified by column chromatography
using silica gel 60 (2 g for 150 mg crude product) and CHCl /MeOH/NH
3
13
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=7.8 Hz, 1H; COCH
/CD OD, 278C): d=13.5, 22.13, 22.17, 22.23, 22.52, 22.57, 26.56,
6.58, 26.8, 27.06, 27.08, 28.2, 28.3, 28.89, 28.93, 29.05, 29.08, 29.1, 29.24,
A
H
U
G
R
N
U
G
2
);
C NMR (125 MHz,
3
3
CDCl
3
3
with the gradient technique.
2
2
3
4
1
9
Method b: The Boc-protected lipid (0.2 mmol) was suspended in acetic
acid ethyl ester (15 mL). Over 2 h, HCl (4 mL, 36%) was added stepwise.
The reaction mixture was stirred for 6 h, then the solvent was evaporated.
9.27, 29.33, 29.36, 29.68, 29.75, 30.11, 30.18, 30.3, 30.7, 31.2, 31.48, 31.50,
2.16, 34.18, 34.22, 38.2, 38.3, 38.4, 38.5, 38.7, 38.8, 39.18, 39.23, 40.0,
0.14, 40.17, 40.19, 53.1, 53.2, 54.3, 54.4, 129.3, 129.5, 170.65, 170.73,
70.9, 171.3, 171.9, 172.0, 175.59, 175.63, 175.9, 176.1 ppm; MS: m/z:
The residue was dissolved in a mixture of CHCl
1 mL), and DMAP (1.2 mmol, 146 mg) was added. This mixture was
separated by column chromatography using silica gel 60 (2 g for 150 mg
crude product) and CHCl /MeOH/NH with the gradient technique to
achieve the pure lipid as the free base.
3
(4 mL) and methanol
(
+
2+
ꢀ
ꢀ
76.8 [M+H] , 489.0 [M+2H]
,
1010.7 [M+Cl] , 974.8 [MꢀH] ;
+
HRMS calcd for C55
110 9 5
H N O
[M+H] : 976.8624; found: 976.8628.
3
3
N-[6-Amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N’-{2-[N,N-
bis(2-aminoethyl)amino]ethyl}-2-tetradecylpropandiamide (lipid 4a),
N-[6-Amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N’-{2-[N,N-
bis(2-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}ethyl)amino]ethyl}-2-tetra-
C
43
H
89
N
7
O
3
: Yield: 97%; R
H NMR (500 MHz, CDCl , 278C): d=0.89 (t, J
CH ), 1.24–1.92 (m, 56H; NHCH (CH
CH CH NH ), 2.48–2.61 (m, 6H; CH N(CH CH
(H,H)=6.9, CH NH
CH N(CH CH ), 3.03–3.41 (m, 5H; 2ꢁCH NHCO, COCH(tetrade-
cyl)CO), 4.31–4.41 (m, 1H; COCH(NHCO)CH ), 6.69–6.71/7.38–7.42/
.18–8.20/8.32–8.33 ppm (4ꢁm, 3H; 3ꢁNHCO); C NMR (125 MHz,
CDCl /CD OD, 278C): d=13.6, 22.3, 22.4, 26.6, 27.13, 27.19, 28.92, 28.94,
8.98, 29.03, 29.05, 29.13, 29.15, 29.2, 29.3, 31.1, 31.3, 31.4, 31.6, 36.9, 38.3,
8.4, 39.18, 39.22, 40.5, 53.08, 53.12, 53.28, 55.34, 170.6, 170.8, 171.3,
f
=0.09 (CHCl
3
/MeOH/NH
3
, 65:35:5, v/v/v);
1
3
3
A
H
U
G
R
N
U
G
decylpropandiamide (lipid 5a), C55
=0.18 (CHCl /MeOH/NH 35:65:15, v/v/v); H NMR (500 MHz,
CDCl , 278C): d=0.86 (t, (H,H)=6.7 Hz, 6H; 2ꢁCH ), 1.23–1.81 (m,
8H; NHCH (CH , (CH , 3ꢁ(CH CH NH
H; CH (CH CH ), 2.66–2.74 (m, 6H; 3ꢁCH NH
1H; 4ꢁCH NHCO, COCH(tetradecyl)CO, 2ꢁCHNH
(NHCO)CH ), 7.14–7.21/7.46–7.86 ppm (2ꢁm, 5H; 5ꢁ
NHCO); C NMR (125 MHz, CDCl /CD OD, 278C): d=13.2, 22.06,
2.13, 22.3, 26.4, 26.94, 27.00, 28.76, 28.88, 28.92, 28.98, 29.1, 29.7, 30.1,
113 11 5
H N O : Yield: 69% (method a);
A
H
U
G
R
N
U
2
)
12CH
3
,
(CH
), 2.70 (dt, J-
2
)
13CH
3
3
,
1
3
2
R
3
,
f
3
(
2
)
3
2
2
2
2
2
NH
2
)
2
3
3
J
A
H
U
G
R
N
U
3
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=14.0 Hz, 2H; (CH
2
)
3
2
2
), 2.75–2.79 (m, 4H;
6
6
1
2
A
H
U
G
R
N
U
G
2
)
12CH
3
2
)
13CH
3
2
)
3
2
2
), 2.48–2.58 (m,
2
), 3.11–3.48 (m,
CH ), 4.28–4.31
2 2
2
2
2
NH
2
)
2
2
N
A
H
U
G
R
N
N
NH)
2
2
2
2
2
A
H
U
G
R
N
N
2
2
1
3
8
(
m, 1H; COCH
A
H
U
G
R
N
U
G
2
13
3
3
3
3
2
3
1
2
3
5
1
1
0.5, 30.6, 31.26, 31.34, 31.40, 34.2, 37.0, 37.1, 37.3, 39.0, 39.77, 39.83, 40.0,
2.8, 52.9, 53.3, 53.4, 53.5, 53.6, 54.1, 170.5, 170.7, 171.0, 171.6, 171.7,
+
2+
71.7, 171.8 ppm; MS: m/z: 752.8 [M+H] , 377.1 [M+2H] , 774.8
+
+
[
7
M+Na] ; HRMS calcd for
52.7100.
C
43
H
90
N
7
O
3
[M+H] : 752.7100; found:
+
+
72.1, 175.6, 175.7 ppm; MS: m/z: 1008.6 [M+H] , 1030.7 [M+Na] ,
ꢀ
+
042.6 [M+Cl] ; HRMS calcd for C55
114 11 5
H N O [M+H] : 1008.8999;
N-[6-Amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N’-{2-[N,N-
bis(2-aminoethyl)amino]ethyl}-2-hexadecylpropandiamide (lipid 4b),
found: 1008.9027.
N-[6-Amino-1-oxo-1-(N-tetradecylamino)hexan-(2S)-2-yl]-N’-{2-[N,N-
bis(2-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}ethyl)amino]ethyl}-2-hexa-
C
45
H
93
N
7
O
3
: Yield: 93%; R
H NMR (500 MHz, CDCl , 278C): d=0.88 (t, J
CH ), 1.24–1.89 (m, 60H; NHCH (CH
CH CH NH ), 2.53–2.60 (m, 6H; CH N(CH CH
H; 3ꢁCH NH (H,H)=7.6 Hz, 1H; COCH(hexadecyl)CO),
.12–3.39 (m, 4H; 2ꢁCH NHCO), 4.30–4.40 (m, 1H; COCH-
), 6.69–6.71/7.36–7.38/8.29–8.30 ppm (3ꢁm, 3H; 3ꢁ
f
=0.09 (CHCl
3
/MeOH/NH
3
, 65:35:5, v/v/v);
(H,H)=7.0 Hz, 6H; 2ꢁ
(CH
), 2.67–2.77 (m,
1
3
3
A
H
U
G
R
N
U
G
decylpropandiamide (lipid 5b), C57
=0.18 (CHCl /MeOH/NH , 35:65:15, v/v/v); H NMR (500 MHz, D
2
08C): d=0.88–0.89 (m, 6H; 2ꢁCH ]), 1.30–2.04 (m, 72H; NHCH -
117 11 5
H N O : Yield: 58% (method a);
A
H
U
G
R
N
U
G
2
)
12CH
3
,
2
)
15CH
3
,
1
3
2
R
2
O,
f
3
3
(
6
3
2
)
3
2
2
2
2
2 2 2
NH )
6
3
3
2
2
), 3.07 (t, J
A
H
U
G
R
N
N
A
H
U
G
R
N
U
G
2
12
3
2
15
3
2
3
2
2
2
2
A
H
U
G
R
N
U
G
2
2
2
2
2
3
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(NHCO)CH
2
CH NHCO, COCH(hexadecyl)CO), 4.47 (t,
J
ACHTUNGTRENNUNG
2
1
3
13
2
NHCO); C NMR (125 MHz, CDCl
3
/CD
3
OD, 278C): d=13.3, 22.1,
CHNH CH ), 4.35–4.40 ppm (m, 1H; COCH
A
H
U
G
E
N
N
(NHCO)CH ); C NMR
2
2
2
3
5
3
7
2.39, 26.42, 26.96, 27.03, 28.76, 28.81, 29.0, 29.1, 29.2, 31.6, 31.3, 31.4,
(125 MHz, D O, 60 8C): d=14.0, 21.9, 22.6, 22.7, 22.8, 26.7, 27.3, 27.5,
27.6, 29.5, 29.67, 29.76, 30.2, 30.89, 30.94, 31.15, 31.20, 31.8, 32.2, 36.8,
2
1.5, 31.65, 31.69, 36.8, 36.9, 38.4, 38.99, 39.02, 40.5, 52.88, 52.92, 52.98,
+
5.8, 170.6, 170.8, 171.1, 171.59, 171.64 ppm; MS: m/z: 780.7 [M+H] ,
37.0, 37.2, 39.6, 39.8, 52.8, 53.0, 53.7, 54.0, 54.4, 170.40, 171.44, 171.6,
2
+
ꢀ
+
+
91.1 [M+2H] , 778.7 [MꢀH] ; HRMS calcd for C45
H
94
N
7
O
3
[M+H] :
171.9, 172.5, 172.9, 173.2 ppm; MS: m/z: 1036.6 [M+H] , 1058.7
+
ꢀ
+
80.7413; found: 780.7413.
57 118 11
[M+Na] , 1070.6 [M+Cl] ; HRMS calcd for C H N O5 [M+H] :
N-{6-Amino-1-[N-(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl}-N’-
2-[N,N-bis(2-aminoethyl)amino]ethyl}-2-tetradecylpropandiamide (lipid
1036.9312; found: 1036.9321.
{
N-(6-Amino-1-{N-[(9Z)-octadec-9-enylamino]-1-oxohexan-(2S)-2-yl})-N’-
{2-[N,N-bis(2-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}ethyl)amino]ethyl}-
2-tetradecylpropandiamide (lipid 5c), C H N O : Yield: 70% (meth-
4
c), C47
H
95
N
7
O
3
: Yield: 81%; R
, 278C): d=0.75 (t, J
), 1.11–1.69 (m, 56H; NHCH (CH CH CH=CHCH
, (CH CH NH ), 1.83–1.88 (m, 4H; CH CH=CHCH
.45 (m, 6H; CH N(CH CH NH ), 2.52–2.56 (m, 6H; 3ꢁCH
.90–3.31 (m, 5H; 2ꢁCH NHCO, COCH(tetradecyl)CO), 4.26–4.31 (m,
H; COCH(NHCO)CH ), 5.20–5.24 (m, 2H; CH=CH), 7.15–7.86/8.02–
.10/8.14–8.24 ppm (3ꢁm, 3H; 3ꢁNHCO); C NMR (125 MHz, CDCl
OD, 278C): d=13.9, 22.4, 22.67, 22.68, 26.71, 26.77, 26.9, 27.4, 27.5,
f
=0.30 (CHCl
3
/MeOH/NH
3
, 65:35:5, v/v/
(H,H)=6.5 Hz, 6H;
(CH CH
), 2.32–
NH ),
1
3
v); H NMR (500 MHz, CDCl
3
ACHTUNGTRENNUNG
5
9
119 11
5
1
2
ꢁCH
3
2
A
H
U
G
R
N
N
2
)
6
2
2
A
H
U
G
E
U
G
2
)
6
3
,
od a); R =0.19 (CHCl /MeOH/NH , 35:65:15, v/v/v);
H NMR
f
3
3
3
(
CH
2
)
13CH
3
2
)
3
2
2
2
2
(500 MHz, CDCl , 278C): d=0.88 (t, J ACHNTUGTREG(NUNN H,H)=6.8 Hz, 6H; 2ꢁCH ),
3
3
2
2
1
8
2
2
2
2
)
2
2
2
1.25–1.88
(m,
68H;
2 6 2 2 2 6 3
NHCH2 ACHTUNGERTNNU(GN CH ) CH CH=CHCH ACHTUNGRTUNNEGN( CH ) CH ,
2
(CH ) CH , 3ꢁ(CH ) CH NH ), 1.95–2.01 (m, 4H; CH CH=CHCH ),
2
13
3
2
3
2
2
2
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
2 2 2 2 2 2
2.50–2.58 (m, 6H; CH N ACHTUNTGRNENG(U CH CH NH) ), 2.67–2.72 (m, 6H; 3CH NH ),
3.12–3.36 (m, 11H; 4ꢁCH NHCO, COCH(tetradecyl)CO, 2ꢁ
2
CHNH CH ), 4.28–4.32 (m, 1H; COCH ACHNUTGTNERNUG( NHCO)CH ]), 5.34–5.38 (m,
2 2 2
1
3
3
/
CD
3
2
3
5
1
8.99, 29.05, 29.13, 29.21, 29.24, 29.36, 29.41, 29.45, 29.48, 31.59, 31.61,
1.68, 31.9, 32.16, 32.29, 36.7, 37.6, 39.2, 39.3, 39.38, 39.44, 41.45, 41.53,
2.8, 52.9, 53.0, 53.9, 54.2, 56.9, 57.1, 129.40, 129.61, 170.0, 170.4, 170.9,
2H; CH=CH), 7.14–7.15/7.65–7.66/7.84–7.85 (3ꢁm, 4H; 4ꢁNHCO),
1
3
7.47–7.49 ppm (m, 1H; COCH
A
H
U
G
R
N
N
(NHCO)CH );
2
C NMR (125 MHz,
CDCl /CD OD, 278C): d=13.8, 22.38, 22.45, 22.51, 22.6, 26.8, 27.0, 27.32,
3
3
+
ꢀ
71.0, 171.2, 171.4 ppm; MS: m/z: 807.4 [M+H] , 840.8 [M+Cl] , 804.9
27.37, 28.6, 28.7, 29.0, 29.1, 29.25, 29.32, 29.5, 31.2, 31.3, 31.4, 31.7, 31.8,
12836
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 12824 – 12838

Peptide-Mimic Amino-Functionalised Lipids
FULL PAPER
3
5
1
2.4, 34.4, 34.7, 37.2, 37.3, 37.4, 37.5, 37.6, 39.3, 39.5, 40.4, 40.5, 40.6, 40.7,
3.1, 53.7, 53.8, 54.0, 54.2, 54.4, 54.5, 129.5, 129.8„ 170.6, 170.8, 171.0,
71.3, 171.7, 171.75, 175.71, 175.79, 175.9, 176.0 ppm; MS: m/z: 1062.7
subtracted from the thermograms of the samples, and the DSC scans
were evaluated by using MicroCal Origin 8.0 software.
DLS: The particle size was measured in carbonate buffer of pH 10 by
photon correlation spectroscopy on a Zetasizer Nano-ZS ZEN3600 (Mal-
vern Instruments Ltd., Malvern, Worcestershire, UK) with a sample re-
fractive index of 1.33 and a viscosity of 0.8872 MPas at 258C. Every
sample was measured three times. Each run consisted of 15 consecutive
scans with a duration of 20 s. Results shown are the average of these
three values. The correlation data were evaluated with ALV-Correlation
software version 3.0 using an exponential regularised fit (see Supporting
Information). We used a regularised exponential fit for the analysis of
the correlation functions obtained because the commonly used cumulants
analysis is restricted to monodisperse systems with radii between 30 and
+
2+
ꢀ
[
M+H] , 531.9 [M+2H]
,
1096.7 [M+Cl] ; HRMS calcd for
+
C H N O [M+H] : 1062.9468; found: 1062.9473.
59 120 11 5
N-{2-[N,N-Bis(2-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}ethyl)ami-
no]ethyl}-N’-(6-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}-1-oxo-1-(N-tetra-
decylamino)hexan-(2S)-2-yl)-2-tetradecylpropandiamide
(lipid
6a),
C
61
H
125
N
13
O
6
:
Yield: 27% (method a), 99% (method b);
R
f
=0.14
1
(
CHCl
d=0.87–0.89 (m, 6H; 2ꢁCH
CH , 3ꢁ(CH CH NH
CH CH NH) ), 3.08 (t,
.42 (m, 14H; 5ꢁCH
3
/MeOH/NH
3
, 35:65:15, v/v/v); H NMR (500 MHz, D
), 1.30–1.91 (m, 74H; NHCH (CH
NHCO), 2.71–2.78 (m, 6H;
NH ), 3.17–
, COCH(tetradecyl)CO),
2
O, 60 8C):
3
2
A
H
U
G
E
N
N
2 3
)12CH ,
(
2
)
13CH
(CH
3
2
)
3
2
2
3
, (CH
2
)
3
CH
2
2
N
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
2
2
J
A
H
U
G
R
N
U
G
2
2
[
36]
1
00 nm.
3
4
2
NHCO, 3ꢁCHNH
(H,H)=6.5 Hz, 3H; 3ꢁCHNH
2
CH
2
3
.04 (t,
(NHCO)CH
3.2, 21.3, 21.6, 22.0, 22.3, 26.05, 26.14, 26.17, 26.4, 26.92, 26.99, 27.9, 28.7,
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
CH
2
); 4.31–4.36 ppm (m, 1H;
TEM: The samples were suspended with 10 mm carbonate buffer (pH 10,
1
3
ꢀ1
COCH
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
); C NMR (125 MHz, CDCl
3
/CD OD, 278C): d=
3
Na
2
CO
3
/NaHCO
3
) to a final concentration of 0.05 mgmL and were so-
1
2
5
1
nicated at 608C for 10 min. The negatively stained samples were pre-
pared by spreading 5 mL of the dispersion onto a Cu grid coated with a
formvar film. After 1 min, excess liquid was blotted off with filter paper
and aqueous uranyl acetate (1%, 5 mL) was placed onto the grid and
drained off after 1 min. The dried specimens were examined with a Zeiss
EM900 transmission electron microscope.
8.9, 29.1, 30.3, 30.4, 31.3, 31.7, 31.8, 32.0, 37.2, 37.3, 37.4, 38.3, 38.5, 39.0,
2.9, 53.0, 53.1, 53.6, 53.7, 170.5, 170.6, 170.7, 171.8, 171.9, 172.0,
+
+
ꢀ
76.4 ppm; MS: m/z: 1136.6 [M+H] , 1158.8 [M+Na] , 1170.5 [M+Cl] ;
+
HRMS calcd for C61
126 13 6
H N O [M+H] : 1136.9949; found: 1136.9958.
N-{2-[N,N-Bis(2-{N-[(2S)-2,6-amino-1-oxohexyl]amino}ethyl)amino]eth-
yl}-N’-(6-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}-1-oxo-1-(N-tetradecyl-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
amino)hexan-(2S)-2-yl)-2-hexadecylpropandiamide
(lipid
6b),
C
63
H
129
N
13
O
6
: Yield: 97% (method b); R
f
=0.10 (CHCl /MeOH/NH ,
3
3
1
3
2
5:65:15, v/v/v); H NMR (500 MHz, D
ꢁCH
2
O, 60 8C): d=0.86–0.88 (m, 6H;
(CH (CH 3ꢁ
NHCO), 2.71–2.74 (m, 6H; CH N-
), 2.83–2.85 (m, 6H; 3ꢁCH NH ), 3.17–3.42 (m, 14H; 5ꢁ
CH , COCH(hexadecyl)CO), 4.34–4.37 ppm (m,
Acknowledgements
3
), 1.31–1.94 (m, 78H; NHCH
CH NH (CH CH
CH NH)
2
A
H
U
G
R
N
U
2
)
12CH
3
,
2 3
)15CH ,
(
CH
(CH
CH
2
)
3
2
2
,
2
)
3
2
2
We thank Dr. Christian Ihling and Prof. Dr. Andrea Sinz (Institute of
Pharmacy, Martin-Luther-University, Halle (Saale), Germany) for the
high-resolution mass spectrometry analyses. The support of Dr. Gerd
Hause (Biocenter, Martin-Luther-University, Halle-Wittenberg) by pro-
viding access to the electron microscope facility is greatly appreciated.
Furthermore, we thank Tino Heimburg (Institute of Pharmacy, Martin-
Luther-University Halle-Wittenberg) for the calculation of the energy
minimized lipid structures.
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
2
2
2
2
2
NHCO, 3ꢁCHNH
H; COCH(NHCO)CH
4.1, 22.7, 22.8, 22.9, 23.3, 27.3, 27.4, 27.5, 27.7, 28.8, 29.7, 29.9, 30.0, 30.2,
0.3, 30.8, 31.5, 32.17, 32.23, 34.7, 37.5, 37.6, 37.8, 37.9, 39.4, 39.7, 40.36,
0.41, 53.4, 53.5, 54.4, 55.1, 171.2, 171.3, 171.4, 173.1, 177.5, 177.6 ppm,
2
2
1
3
1
1
3
4
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
); C NMR (125 MHz, D
2
O, 60 8C): d=14.0,
+
+
ꢀ
MS: m/z: 1165.0 [M+H] , 1187.8 [M+Na] , 1163.1 [MꢀH] , 1199.0
M+Cl] ; HRMS calcd for C63 [M+H] : 1165.0262, found:
165.0272.
ꢀ
+
[
1
130 13 6
H N O
N-{2-[N,N-Bis(2-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}ethyl)ami-
no]ethyl}-N’-(6-{N-[(2S)-2,6-diamino-1-oxohexyl]amino}-1-[N-(9Z)-octa-
dec-9-enylamino]-1-oxohexan-(2S)-2-yl)-2-tetradecylpropandiamide (lipid
[1] a) M. A. Mintzer, E. E. Simanek, Chem. Rev. 2009, 109, 259–302;
b) S. Bhattacharya, A. Bajaj, Chem. Commun. 2009, 4632–4656.
[2] C. R. Dass, P. F. M. Choong, Cancer Cell Int. 2006, 6, 17.
[3] a) C. Lonez, M. Vandenbranden, J.-M. Ruysschaert, Prog. Lipid Res.
2008, 47, 340–347; b) A. Joseph, N. Itskovitz-Cooper, S. Samira, O.
Flasterstein, H. Eliyahu, D. Simberg, I. Goldwaser, Y. Barenholz, E.
Kedar, Vaccine 2006, 24, 3990–4006; c) C. Barnier Quer, A. Elshar-
kawy, S. Romeijn, A. Kros, W. Jiskoot, Eur. J. Pharm. Biopharm.
2012, 81, 294–302.
6
c), C65
H
131
N
13
O
6
: Yield: 21% (method a), 97% (method b); R
f
=0.14
, 278C):
), 1.25–1.88 (m, 74H; NHCH
(CH 3ꢁ(CH CH NH
NHCO), 1.94–2.03 (m, 4H; CH CH=CHCH ), 2.54–2.59 (m,
(H,H)=5.3 Hz, 6H; 3ꢁCH NH ),
NHCO, 3ꢁCHNH CH , COCH(tetradecyl)-
(NHCO)CH ), 5.33–5.39 (m, 2H; CH=
CH), 7.18–7.21/7.41–7.44/7.55–7.58/7.66–7.68/7.83–7.86 ppm (5ꢁm, 6H;
1
(
CHCl
3
/MeOH/NH
3
, 35:65:15, v/v/v); H NMR (500 MHz, CDCl
3
3
d=0.88 (t,
J
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(H,H)=6.6 Hz, 6H; 2ꢁCH
3
2
-
2
,
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(CH
2
)
)
6
CH
CH
2
CH=CHCH
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(CH CH
2
)
6
3
,
2
)13CH
3
,
2
)
3
2
(
CH
2
3
2
2
2
3
6
3
H; CH
2
N
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(CH
2
CH
2
NH)
2
), 2.68 (t,
J
A
H
N
T
E
N
N
2
2
.12–3.36 (m, 14H; 5ꢁCH
2
2
2
[4] A. Kanazawa, T. Ikeda, T. Endo, Antimicrob. Agents Chemother.
1994, 38, 945–952.
CO), 4.28–4.32 (m, 1H; COCH
A
H
U
G
R
N
U
G
2
[
5] a) A. El-Aneed, J. Controlled Release 2004, 94, 1–14; b) T. P.
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1
3
6
2
3
5
1
3 3
ꢁNHCO); C NMR (125 MHz, CDCl /CD OD, 278C): d=13.5, 22.2,
[
2.6, 22.7, 26.6, 26.8, 27.2, 28.5, 28.5, 28.89, 28.94, 29.04, 29.09, 29.14, 29.3,
1.2, 32.15, 32.24, 32.3, 34.59, 34.66, 37.1, 37.3, 38.4, 39.2, 40.9, 52.9, 53.0,
3.3, 53.5, 53.6, 54.5, 54.6, 129.2, 129.4, 170.59, 170.63, 171.3, 171.4, 175.5,
+
2+
75.6, 175.7 ppm; MS: m/z: 1190.9 [M+H] , 596.2 [M+2H] , 397.8
3
+
+
ꢀ
[
M+3H]
,
O
1212.9 [M+Na] , 1224.9 [M+Cl] ; HRMS calcd for
+
C
65
H
132
N
13
6
[M+H] : 1191.0418, found: 1191.0427.
[
[
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Revised: June 25, 2012
8
, 330–340.
Published online: August 9, 2013
12838
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 12824 – 12838