Maleimide-Functionalized Lipids that Anchor Polypeptides to Lipid Bilayers and Membranes

Abstract

Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces.

Aqueous-Soluble, Non-Reversible Lipid Conjugate of Salmon Calcitonin: Synthesis, Characterization and In Vivo Activity

“…These methods inevitably involved the use of noxious organic solvents, such as chloroform and dimethylformide, which are hardly favorable for maintaining peptide drug stability (31,32). In addition, solvent removal is mandatory, requiring complicated post-processing (20). On this basis, the ability to conjugate a lipid to a peptide drug in an aqueous media is highly desirable.…”

Development of lipopolyplexes for gene delivery: A comparison of the effects of differing modes of targeting peptide display on the structure and transfection activities of lipopolyplexes

“…Bioconjugation of targeting moieties to lipids or to liposomes is typically carried out via the reaction of thiol‐functionalised targeting moieties to malemide‐derivatised lipids, using click chemistry, or amide bond formation, although hydrazone linkages and Staudiger ligations have also been reported. The majority of peptide‐targeted liposomes to date have relied on the post‐formulation bioconjugation of thiol‐functionalised targeting moieties to liposomes containing DSPE‐PEG2000‐Mal, although other maleimide‐containing lipids with shorter or no PEG linkages have been reported . For the surface targeted lipopolyplexes two lipids with maleimide moieties at their head group were synthesised, for incorporation into the liposome formulations and then conjugation to peptide targeting sequences terminating in Cys residues.…”

Self-association of zwitterionic polymer–lipid conjugates in water as examined by scattering measurements

“…For example, polypeptide-lipid conjugates can be anchored into phospholipid membranes. Therefore, the conjugates are employed for biological structure and function studies [65] and have the potential to be used in a drug delivery system (DDS). Previously, furthermore, poly(N-isopropylacrylamide) (PNI-PAM) having a hydrophobic end group was prepared using a lipophilic radical initiator and temperature-responsiveness of the polymer was examined in detail using fluorometry [66,67].…”