Lipidic peptides

Superose gel material of Pharmacia Biotech is a highly epichloro-hydrine cross-linked agarose matrix that has a pH range of 3-12 (short term 1-14). Hydrophilic interactions may be noticeable for lipids, peptides, and small aromatic compounds, but such interactions might even improve resolution. Superose medium is available in two different porosities Superose 6 HR 10/ 30 (bead size 13 2 /um maximum pressure 1.5 MPa) and Superose 12 HR 10/30 (bead size 10 2 /um maximum pressure 3.0 MPa). 

The method utilizing ID NMR is simple and eonvenient. Henee the NMR method diseussed here ean be applied to the systematie investigation of the membrane irug inter-aetions, elosely related to the vital function in biomembranes. It is expected that the application can be extended to the lipid-peptide interaction and protein uptake. We are now applying the method to apolipoprotein binding with lipid bilayers and emulsions. Preferential protein binding sites in membranes can be specified by NMR on the molecular level. 

Enzymatic Degradation of H214/03. The degradation experiments were carried out with molecular mixtures of H214/03 or LVP and MO. These were obtained from mixtures of the peptide and MO dissolved in 99.5% ethanol, from which the ethanol was evaporated under reduced pressure at room temperature for about two days. Appropriate amounts of melted mixtures were then weighed in small Erlenmeyer flasks. Care was taken to insure that the bottom of each flask was covered with the mixture, which was then left to freeze. In this way the area and the thickness of the lipid/peptide layer was comparable in the flasks. Typically five flasks were used in each experiment... 

The synthesis of lipidated peptides is implicated by the base-lability of the thioester and the acid sensitivity of the prenyl group double bonds. Thus, new protecting groups are required which can be removed under extremely mild, preferably neutral, conditions. 

C). In addition, enzymes often combine a high specificity for the recognized substrates with a large tolerance for secondary structure. Alternatively, noble metal transformations offer reaction conditions that are also mild enough to be compatible with sensitive, doubly lipidated peptides. 

Acid- and base-sensitive lipidated peptides can be selectively deprotected by enzymatic hydrolysis of choline esters.[13al Choline esters of simple peptides, but also of sensitive peptide conjugates like phos-phorylated and glycosylated peptides,1141 nucleopep-tides1151 and lipidated peptides,113,1631 can be cleaved with acetyl choline esterase (AChE) and butyryl choline esterase (BChE) under virtually neutral conditions with complete chemoselectivity. Acid-labile farnesyl groups and base-sensitive thioesters are not attacked. 

The suitability of the Aloe group for the construction of lipidated peptides is emphasized by the synthesis of the maleimidocaproyl-modified, S-palmi-toylated and farnesylated heptapeptide 16 which corresponds to the N-Ras C-terminus (Scheme 10).1211 In contrast to classical urethane-type protecting groups, the Aloe group can be removed in the presence of additional functional groups and under neutral conditions. It is therefore a very convenient protecting group for the synthesis of very hydro-phobic lipid-modified peptides, which are not soluble in the aqueous media required for enzyme catalyzed transformations. 

For biological assays, lipidated peptides embodying a fluorescent label like the bimanyl- and the NBD-group, are required for determining membrane binding or subcellular distribution by fluorescence spectroscopy and fluorescence microscopy. Also, attachment of a biotin group allows research-... 

Lipidated peptides embodying the characteristic linkage region found in the parent lipoproteins and bearing additional functional groups, which could be traced in biological systems or which allowed for their use in biophysical experiments, were used successfully in model studies. However, such model studies only provide a limited amount of information. In order to approximate the situation in a biological system more precisely, experiments with differently lipidated proteins are required. 

Synthetic Lipidated Peptides and Proteins Biophysical Properties... 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Neutrophils may move at speeds of up to 20 /tm min-1 in response to chemoattractants such as denatured proteins, lipids, peptides or C5a. Movement may be defined either as chemokinesis, which is generalised (non-directional) locomotive activity, or as chemotaxis, which is orientation and directional migration up a concentration gradient. A concentration difference at opposite ends of the cell of only 1% is sufficient to activate such directional movement. However, neutrophils do not respond chemotactical-ly to static gradients of chemoattractants, and both temporal and directional changes in chemoattractant concentrations are required. 

Cullis PR, Hope MJ, Bally MB, Madden TD, Mayer LD, Fenske DB. Influence of pH gradients on the transbilayer transport of drugs, lipids, peptides and metal ions into large unilamellar vesicles. Biochim Biophys Acta 1997 1331 187. 

Friede M, et al. Selective induction of protection against influenza virus infection in mice by a lipid-peptide conjugate delivered in hposomes. Vaccine 1994 12 791. 

As the solubility of this peptide in water is very low, the peptide can be associated with the liposomal membrane. As the peptide is only sparingly soluble in methanol or chloroform, DMSO had to be used as dissolution medium for mixing the peptide with the lipids for liposome formation. A lOmg/mL stock solution in DMSO of the peptide could be obtained. Appropriate amounts of lipid stock solutions and the peptide stock solution were mixed (lipid peptide ratio = 95 5) and processed as thoroughly described in the... 

At a molar ratio of peptide to lipid of 0.002 (0.2 mol%) and 0.005 (0.5 mol%) at process start, all of the peptide was incorporated into the liposomal membrane bilayer. At a 5 mol% value of the peptide in relation to the lipid amount at the start of the experiments, only 33% of the total peptide was recovered in the final liposomal formulation after extrusion a concomitant loss in lipid content was also observed. This led us to the conclusion that at higher TRP-2 peptide ratios lipid-peptide aggregates may be formed, which cannot be extruded. 

Efficient Gene Transfer by Lipid/Peptide Transfection Complexes... 

Finally, it should be considered that additional non-natural functional groups, which are often incorporated in the lipidated peptides for biological studies such as fluorophores or photoactive groups, typically lead to additional restrictions for the synthesis protocols. 

Considering all these features and limitations, it is understandable that currently there is no general procedure available, such as the Fmoc or Boc protocols that are available for nonfunctionalized peptides. However, several approaches have been developed in the last two decades to make lipidated peptides accessible. This chapter describes both the corresponding solution-phase approaches and solid-support approaches for the synthesis of lipidated peptides. In line with the framework sketched above, both the different protecting groups and solid-phase linker systems that have been developed will be reviewed. 

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