Solid-phase synthesis of arginine-based double-tailed cationic lipopeptides: Potent nucleic acid carriers

Article abstract of DOI:10.1039/c1cc15805h

Herein we report a highly-efficient solid-phase strategy for the modular synthesis of 63 double-tailed lipid-peptide conjugates and their application in DNA delivery.

Full text of DOI:10.1039/c1cc15805h

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COMMUNICATION
Solid-phase synthesis of arginine-based double-tailed cationic
lipopeptides: potent nucleic acid carriersw
Aleksandra Liberska,a Annamaria Lilienkampf,a Asier Unciti-Broceta*bc and Mark Bradley*a
Received 19th September 2011, Accepted 14th October 2011
DOI: 10.1039/c1cc15805h
Herein we report a highly-efficient solid-phase strategy for the
modular synthesis of 63 double-tailed lipid-peptide conjugates
and their application in DNA delivery.
approach and their evaluation as DNA carriers (Fig. 1).
Double-tailed cationic lipids were synthesised on a Rink
amide-functionalised polystyrene resin,14 which generates an
amide group at the C-terminus upon resin cleavage, using the
building blocks shown in Fig. 1. Encouraged by the remarkable
aggregation properties displayed by gemini and gemini-like
surfactants,13 four trifunctional spacers containing symmetrical
polyamines were used (Fig. 1, SP1-4). These were synthesised
from diethylenetriamine and norspermidine using DdeOH to
allow the selective blocking of the primary amines in the presence
of the secondary amine, which could then be selectively coupled
to either succinic or maleic anhydride (Scheme 1). These spacers
were employed to investigate the optimal separation distance
between the lipid tails and the role of spacer flexibility on the
transfection abilities of the compounds.
Solid-phase organic synthesis is a versatile technique for the
preparation of oligomeric compounds such as peptides1 and
peptidomimetics,2 nucleic acids3,4 and analogues,5 oligo-
saccharides,6 dendrimers7 and small molecules.8 Apart from the
well-known advantages of solid-phase chemistry (e.g. forcing
reactions to completion by mass action, simplified purifica-
tion, compatibility with a broad range of solvents),9 it enables
splitting (and mixing when appropriately encoded)5,10 during
the stepwise incorporation of different building blocks.
Introducing DNA into cells, commonly termed transfection,
has become an essential technique for countless biochemical
studies and, in principle, can be used to treat both inherited
and acquired diseases.11 To facilitate DNA transfection, cell
delivery vehicles have been developed, often based on liposomal
formulations formed by self-assembly of cationic lipids.11 To
date, a wide range of cationic lipids has been generated and used
as transfection reagents.11–13 However, the determination of
structure/transfection–activity relationships (STARs) remains
elusive since the families of cationic lipids evaluated do not
typically possess related structural features. In addition, transfec-
tion efficiency is often dependent on the targeted cell type and
DNA size, while variations in media and DNA to transfection
reagent ratios add additional complications. This makes pro-
spective correlation efforts a rather erratic exercise.11f–h In the
absence of defined structural criteria, a parallel approach can be
beneficial to assist in the rapid determination of framework-
specific STARs. To this end, parallel solid-phase synthesis is an
optimal strategy for the systematic modification of the modular
cationic lipid structure:11f–h,12 (i) the cationic headgroup/s, (ii) the
linking moiety, and (iii) the hydrophobic tail/s.
Herein we report a solid-phase strategy for the synthesis
of 63 double-tailed lipid-peptide conjugates using a modular
a School of Chemistry, University of Edinburgh, West Mains Road,
Edinburgh, EH9 3JJ, UK. E-mail: mark.bradley@ed.ac.uk
b Edinburgh Cancer Research Centre, MRC IGMM, University of
Edinburgh, Edinburgh, EH4 2XR, UK. E-mail: asier.ub@ed.ac.uk
c Deliverics Ltd, NB1, Joseph Black Building, Edinburgh, EH9 3JJ, UK
w Electronic supplementary information (ESI) available: Synthesis
and compound characterization, transfection and cytotoxicity studies.
See DOI: 10.1039/c1cc15805h
Fig. 1 General structure of the cationic lipopeptide library and the
building blocks used in its synthesis (M1: the cationic headgroup;
M2: the spacers and M3: the lipid tails).
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12774 Chem. Commun., 2011, 47, 12774–12776
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mono-arginine resin (R1). This protocol was repeated to give di-
and tri-arginine scaffold resins (R2 and R3). Resins R1-3 were
subsequently divided into four, independently coupled to the
four Dde-protected spacers SP1-SP4 and treated with 5%
hydrazine in DMF to cleave the Dde groups.14 The resins were
further split into 5 and coupled to a set of fatty acids (palmitic (P),
stearic (S), oleic (O), arachidic (A) and lignoceric (L) acid) using
DIC/HOBt. Removal of the Pbf protecting groups and cleavage
from the resin was achieved using a mixture of TFA/TIS/H2O
(95 : 2.5 : 2.5) to give the lipopeptides as their TFA salts.
The DNA transfection abilities of the compounds were
evaluated with HeLa cells employing pEGFP-C1 (4.7 kilobases)
as a reporter. As shown in Fig. 2, EGFP expression results were
strongly dependent on the number of arginine residues, with little
transfection detected with the mono-arginine compounds. Over-
all, the presence or absence of unsaturation in the spacer moiety
had a minor impact on transfection. Di-arginine and tri-arginine
compounds with diethylenetriamine-based spacers (SP1, SP2)
gave the highest transfection rates. In particular cationic lipids
containing palmitoyl tails (P), the shortest fatty acid used in
the library, and spacers SP1 and SP2 led to transfection rates
comparable or even higher than Lipofectaminet 2000. Flow
cytometry analysis (Fig. 2b,d) confirmed that di-arginine
compounds P2-SP1-R2 and P2-SP2-R2 gave the highest levels
Scheme 1 Synthesis of Dde-protected spacers SP1-4. Reagents and
conditions: (i) DdeOH (2.1 equiv.), DCM, 6 h; (ii) succinic or maleic
anhydride (1 equiv.), DCM, 8 h, overall yield 91–94% (two steps).
The synthesis of 60 arginine-based lipopeptides was performed
in parallel as described in Scheme 2 (for full description see
ESIw). Microwave-assisted coupling of Fmoc-Arg(Pbf)-OH
using DIC/HOBt was followed by Fmoc deprotection to give
Fig. 2 Transfection screening of HeLa cells with arginine-based
lipopeptides. Lipoplexes were formulated using DOPE as co-lipid
(1 : 1 molar ratio) and pEGFP-C1 at N/P ratios of 5 : 1 and 10 : 1
and tested in triplicate. Lipofectaminet 2000 (L2000) was used as the
positive control and untreated cells as a negative control. (a) Mean
fluorescence (a.u. = arbitrary units) of cell population 48 h after
transfection (measured using a BioTek FLx800microplate reader:
485/20 excitation, 530/25 emission). (b) Percentage of transfected
HeLa cells as measured by flow cytometry (FACSaria). (c) Merge
image (brightfield + fluorescent channels) of HeLa cells transfected
with P2-SP1-R2. Scale bar: 50 mm. (d) Flow cytometry histogram of
untransfected HeLa cells (upper) and cells transfected with P2-SP1-R2
(lower).
Scheme 2 Solid-phase synthesis of arginine-containing double-tailed
cationic lipids. Reagents and conditions: (i) Fmoc-Arg(Pbf)-OH (3 equiv.),
DIC (3 equiv.), HOBt (3 equiv.), DMF/DCM (2 : 1), mW, 60 1C, 20 min;
(ii) 20% piperidine in DMF, 10 min (Â2); (iii) spacer (SP1, SP2, SP3 or
SP4, 3 equiv.), DIC (3 equiv.), HOBt (3 equiv.), DMF/DCM (2 : 1), mW,
60 1C, 20 min; (iv) 5% hydrazine in DMF, 2 h; (v) fatty acid (3 equiv.),
DIC (3 equiv.), HOBt (3 equiv.), DMF/DCM (2 : 1), mW, 60 1C, 20 min
(Â2); (vi) TFA/TIS/H2O (95:2.5:2.5), 2 h.
c
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of transfection and MTT cell viability assays showed no
cytotoxicity at the concentrations used.
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Since P2-SP1-R2 (bearing C16 tails), showed superior trans-
fection effiency compared to conjugates with longer fatty tails
and the presence of double bond did not appear to be required,
SP1-R2 compounds with C14, C15 and C17 saturated fatty
tails were synthesised as described above to investigate optimal
lipid length. These compounds, along with the resynthesised
hit P2-SP1-R2, were evaluated against HeLa and HEK293T
cells. Results showed that compound P2-SP1-R2 (C16) yielded
the highest transfection levels (see ESIw), indicating that 2
arginines, a diethylenetriamine spacer and palmitoyl tails were
optimal for DNA transfection with this class of cationic
conjugates.
In conclusion, a straightforward and highly-efficient solid-
phase strategy has been developed for the parallel synthesis of
double-tailed lipid-peptide conjugates using a modular approach
to rapid generation of comprehensive STARs as DNA carriers.
This method was used to generate 63 lipo-arginines and allowed
the identification of several highly efficient, non-toxic transfection
reagents.
This work was supported by the MRC, SE and the European
Regional Development Fund (PoC 10-CHM-001).
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This journal is The Royal Society of Chemistry 2011