lipopeptides
Cavalli, S.
Citation
Cavalli, S. (2007, January 25). Chemistry, structural insight and applications of β-sheet forming lipopeptides. Retrieved from https://hdl.handle.net/1887/9452
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Summary and Perspectives
The research described in this thesis aimed to synthesize and investigate the physical properties of a new series of amphiphilic lipopeptides, ALPs (1a-c, Chart 1). These molecules were composed of an amphiphilic oligopeptide domain, (Leu-Glu)n , interlinked by a succinyl moiety to the phospholipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE).
Chart 1. The amphiphilic lipopeptides, ALPs (1a-c). Three ALPs, each exhibiting a different length of the peptide part, (Leu-Glu)2-,(Leu-Glu)3- and (Leu-Glu)4, termed tetra-, hexa- and octa-ALP, respectively (cf. 1a-c).
The ALPs were designed to form supramolecular assemblies composed of β-sheet arrays decorated by hydrophobic lipid tails. First the ALPs were investigated for the ability to form well-ordered monolayers at the air-water interface. The sequential order of hydrophobic and hydrophilic amino acids was chosen to induce the generation of a β-sheet secondary structure at the air-water interface. The peptide backbones in the β-sheet conformation were expected to lay with their long molecular axes parallel to the interface so that the hydrophobic side groups pointed towards the air and the hydrophilic side chains were distributed regularly on the water interface. Because of the hybrid nature of the ALPs, the possibility that the phospholipid tails would interfere with the β-sheet organization was considered. The proper length of the peptide domain, necessary to generate the β-sheet organization, was therefore investigated and three ALPs were prepared, each exhibiting a different length of the peptide part, (Leu-Glu)2-,(Leu-Glu)3- and (Leu-Glu)4, termed tetra-, hexa- and octa-ALP, respectively (cf. 1a, 1b and 1c in Chart 1). Subsequently, the application of the ALP monolayers for the nucleation of calcium carbonate was shown. Next, the self-assembly properties of the ALPs upon dispersion in aqueous solutions were investigated. The preparation of lipid vesicles decorated by β-sheet peptides was rather difficult. Therefore a new synthetic approach was investigated to functionalize pre-formed liposome surfaces by peptides.
In Chapter 1 an overview of amphiphilic peptide-based supramolecular structures is given. In Chapter 2 a solid phase synthetic strategy, followed by an efficient purification protocol is described for the preparation of highly pure lipid-peptide conjugates (tetra-ALP,
n
HN N H
NH2 O HO O
O O
O O O
O P O OH O
NH O
O 1 (a: n=2; b: n=3; c: n=4)
hexa-ALP and octa-ALP). Structural insight into the organization of the amphiphilic lipopeptide monolayers at the air-water interface was provided by surface pressure versus area isotherms (π-A), circular dichroism spectroscopy (CD), Fourier transform infrared spectroscopy (FTIR) and Brewster angle microscopy (BAM) studies. In situ grazing-incidence X-ray diffraction (GIXD) measurements revealed that the lipopeptides, containing six or eight amino acids residues, formed a new type of two-dimensional self-organized monolayers, which exhibited β-sheet ribbons segregated by lipid tails. The experimental observations have been rationalized by considering the “hybrid” characteristics of the system. As envisaged in the design stage, the peptide length indeed dramatically affected the behaviour of the lipopeptides, especially in relation to film compression. The peptide domain of the tetra-ALP was too small to form an ordered structure at the air-water interface. In contrast, both hexa- and octa-ALPs formed stable β-sheet monolayers at the air-water interface. The β-sheet structure of the hexa-ALP remained unaltered upon compression, as indicated by GIXD. The organization of octa-ALP did change as a function of surface pressure (π): a decrease in the spacing along the β-strand long axes and a pronounced difference in Bragg rod shape was observed upon compression. From this it was concluded that the octa-ALP appeared to be more deformable than the hexa-ALP. It is possible that the eight residue β-sheet in the octa-ALP had a higher tendency, relative to the hexa-ALP, to bend or distort under compression. It is known that β-sheet strands have a natural tendency to twist. Therefore the longer the peptide aligned at the air-water interface, the higher the structural frustration in its backbone. The octa-ALP film collapsed into an ordered multilayer structure at high surface pressure. This is the first example of a system comprising β-sheet assemblies, which exhibits an apparent ordered multilayer structure, probably due to the stabilizing contribution of the lipid tails. The conclusions drawn from the experimental findings were supported by a representative model based on molecular dynamics (MD) simulations of the octa-ALP at the vacuum-water interface. Chapter 3 described the application of the ALP monolayers as well-defined two-dimensional templates for the nucleation of calcium carbonate. The influence of the size of the peptide head group on the biomineralization was investigated. For comparison, the N-acetylated peptide Ac-(Leu-Glu)4-NH2 and the phospholipid 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) were studied as well. The introduction of the phospholipid moiety to the octapeptide (Leu-Glu)4 motif was shown to enhance the amphiphilic behaviour of the molecule but also to increase the flexibility of the monolayer, without compromising the β-sheet structure. This led to a distinct change in templating behavior compared to Ac-(Leu-Glu)4-NH2 and the
discussed. It was demonstrated that the hexa- and the octa-ALP formed stable monolayers with an antiparallel β-sheet conformation on a milli-Q water subphase as well as on Ca2+
aqueous solutions. This enabled a study of the effect of these monolayers on the crystallization of calcium carbonate and also an investigation into the way they interacted with the developing mineral phase. Indeed the formation of habit-modified calcite was observed.
Apart from a small amount of pyramidal {01.l} oriented crystals (l=1-2), the majority of the modifications resulted in a new type of indented {10.0} oriented calcite crystals. The formation of this new morphological form was significantly suppressed when the less adaptable N-acetylated peptide was used. Furthermore, different types of modified calcite or only randomly growing crystals were grown underneath the monolayers of the tetra-ALP and DOPE respectively, mainly attributed to the lack of β-sheet structures. With the present system it was demonstrated that the nucleation of different crystal faces could be achieved depending on the ability of the template to adapt to the structure of the inorganic phase.
Furthermore, these results indicated that stretching of the template in only one direction allowed the reorientation of the template’s functional groups such that the stabilization of different crystal planes could be achieved without the need for an epitaxial relation between the two components. In Chapter 4 the self-assembly behaviour of the ALPs was studied. As the monolayers of the ALPs have been shown to template the mineralization of calcium carbonate at the surface, the preparation of such types of aggregates could lead to new templates for biomineralization in solution. Atomic force microscopy (AFM) studies revealed that the octa-ALP formed multilayered β-sheet tape-like structures at the air-water interface.
A model was tentatively proposed, in which the octa-ALP assembled as bilayer into tapes.
These multilayer structures subsequently formed long twisted fibers. Next, the self-assembly of octa-ALP in solution was investigated. Also mixtures containing different ratios of octa-ALP and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolcholine (DOPC), were studied upon dispersion of the lipid films in aqueous solutions. DOPC was added in order to dilute the amount of peptide in the aggregate with the rational to make the self-assembly process more feasible. Vesicular-type aggregates and fibrous structures were found in all samples. Circular dichroism (CD) analysis revealed that these aggregates clearly displayed a β-pleated sheet folding. This study showed that the formation of well-defined assemblies with a regular size and morphology was rather difficult to achieve. The amphiphilic nature and the tendency to form rigid β-sheet arrays probably complicated the self-assembly process. The complications encountered in the preparation of lipid vesicles decorated by β-sheet peptides could be overcome using a functionalized lipid already inserted in a pre-formed liposome and conjugate the (Leu-Glu)4 peptide motif directly at the surface. An example of this approach is
given in Chapter 5. A generic method for the efficient in situ modification of liposomes, based on copper(I)-mediated [3+2] azide-alkyne cycloaddition (“click” chemistry) was developed. Fluorescence resonance energy transfer (FRET) studies with a model compound demonstrated that the reaction occurred at the surface. Small unilamellar lipid vesicles with terminal alkyne groups and the fluorescent acceptor molecule lissamine rhodamine (LR) at their surface were prepared. As a proof of principle, an azido- and fluorescent probe modified (N3-Lys(NBD)-NH2) was coupled to the liposome using a catalytic amount of CuBr. The donor 7-nitrobenzofurazan (NBD)-modified lysine, N3-Lys(NBD)-NH2, was linked in close proximity to the acceptor (DOPE-LR) upon reaction, allowing the energy transfer (FRET) to occur. The terminal alkyne functionalities in the internal membrane of the liposomes were screened and did not take part in the reaction, thus allowing the energy transfer to occur only with the acceptor molecules available at the outer surface. Furthermore, based on the observation that a color shift occurred after dialysis, a simple colorimetric assay was developed for monitoring the reaction. The reaction proceeded at room temperature and was finished within 4 hours. The generic nature of this approach allows any azido-functionalized peptide to be conjugated in a straightforward way to the outer membrane of liposomes, using a similar synthetic protocol as shown for N3-Lys(NBD)-NH2. Additionally, NBD can be easily introduced in order to follow the reaction using the colorimetric assay. In another example, a random coil to β-sheet transition was observed for the (Leu-Glu)4 motif upon conjugation to the liposome outer membrane using the “click” chemistry protocol. The efficient reaction between the peptide N3-(Leu-Glu)4-NH2 and the liposome did not affect the average diameter of the vesicles, demonstrating the possibility to functionalize the liposome outer membrane with peptide sequences rather than just amino acids. Moreover, the random coil to β-sheet transition allowed the in situ monitoring of the reaction in real time using CD spectroscopy. Product formation was already observed after 15 minutes as demonstrated by the change in the CD signal. The reaction proceeded at room temperature, reaching good conversions already after 45 minutes and was completed within 2 hours. Finally, the versatility and scope of this chemical approach for surface modification of vesicles was investigated also for non-peptidic biologically relevant compounds and the preparation of immunogenic liposomes was given as an example. Toll-like receptor ligands (TLR7Ls) were conjugated to the vesicle outer membrane and the immunogenicity was investigated. Initial biological tests demonstrated that the immunogenecity of one of the ligands (TLR7-L2) increased upon conjugation. These results suggested that further optimization of the ligand properties (i.e. spacer length) could lead to the preparation of potent synthetic liposome-based
Chapter 6 the synthesis of a lipidated Gramicidin S analogue via copper(I)-mediated [3+2]
azide-alkyne cycloaddition was described and the ability of this amphiphilic molecule to form stable monolayers at the air-water interface was investigated. Indeed, lipidation of the GS derivative resulted in a more stable monolayer. However, Grazing Incidence X-ray Diffraction (GIXD) measurements at the air-water interface revealed that lipidated GS did not exhibit a well-ordered crystalline assembly. This is in contrast to the non-lipidated GS derivative, which formed crystalline structures on aqueous 0.14 mM NaCl. In this case, the lipidation of an amphiphilic peptide did interfere with the formation of an organized monolayer, in contrast with what was observed for hexa- and octa-ALP. Transferred monolayers of the lipidated Gramicidin S analogue were evaluated as possible antimicrobial surfaces. These results constitute a pilot study towards the preparation of antimicrobial coated surfaces.
Improvements for a more efficient coating are currently under investigation and take into consideration either the modification of the lipid tail (e.g. chains bearing crosslinkable units) or the use of a different substrate (i.e. “hydrophobic” glass or slides covered by a thin Teflon layer).
