Here we describe the design, synthesis, and characterization of new, metal-functionalized, amphiphilic diblock copolymers for molecular recognition. Polybutadiene-block-polyethylenoxide copolymers were synthesized by living anionic polymerization and end group functionalized with nitrilotriacetic acid and tris(nitrilotriacetic acid). After complexation with nickel and copper, these groups are known to selectively bind to oligohistidine residues of proteins. The polymers were characterized by 1H NMR spectroscopy, size exclusion chromatography, electron paramagnetic resonance, and UV-vis spectroscopy. Mixtures of these polymers with the respective nonfunctionalized block copolymers self-assemble in aqueous solution into vesicular structures with a controlled density of the metal complex end-groups on their surface. The accessibility of these binding sites was tested using maltose binding protein carrying a terminal decahistidine moiety and His-tagged enhanced green fluorescent protein as model systems. Fluorescence correlation spectroscopy clearly showed a significant and selective binding of these proteins to the vesicle surface.

The radical polymerization of three monomers bearing nucleobases 1-(4vinylbenzyl)thymine (VBT), 1-(4-vinylbenzyl)uracil (VBU) and 9-(4-vinylbenzyl)adenine (VBA) was investigated. The corresponding homopolymers could be prepared in high yields via conventional radical polymerization. However, the resulting polymers were found to be only soluble in a few polar solvents. On the other hand, copolymers of dodecyl methacrylate (DMA) with either VBT or VBA could be prepared via both free radical polymerization and atom transfer radical polymerization and could be dissolved in a large variety of organic solvents. Moreover, the formed complementary copolymers P(VBT-co-DMA) and P(VBA-co-DMA) were found to self-assemble in dilute solutions in dioxane or chloroform via base recognition, as evidenced by a significant hypochromicity effect in UV spectroscopy. Nevertheless, at higher concentrations in chloroform, both dynamic light scattering and optical microscopy indicate that P(VBT-co-DMA), P(VBA-co-DMA), or P(VBT-co-DMA)/P(VBA-co-DMA) mixtures spontaneously self-assemble into micron size spherical aggregates. 1 H NMR and FTIR studies confirmed that the self-assembly process is driven in all cases via H-bond formation. V V C 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: [4805][4806][4807][4808][4809][4810][4811][4812][4813][4814][4815][4816][4817][4818] 2005

No abstract

The development of nanocarriers for drug/protein delivery is in focus today, as they can serve to both decrease dosages and improve localization to a desired biological compartment. A powerful tool to functionalize these carriers is specific affinity tagging supported by molecular recognition, a key principle in biology. However, the geometry of the binding region in a molecular recognition process, and thus its conformation and specificity, are in many cases poorly understood. Here, we demonstrate that short, model peptides, His(6)-tags, selectively recognize Cu(II)-trisnitrilotriacetic acid moieties (Cu(II)-trisNTA) when exposed at the surfaces of polymer vesicles designed to serve as nanocarriers or as surfaces for proteins binding. A mixture of poly(butadiene)-b-poly(ethylene oxide) (PB-b-PEO) and Cu(II)-trisNTA-functionalized PB-b-PEO diblock copolymers (10:1) self-assembles in aqueous solution, generating vesicles with a hydrodynamic radius of approximately 100 nm, as established by light scattering and TEM. Fluorescently labeled His(6) tags specifically bind to metal centers exposed on vesicles' surface, with a dissociation constant of 0.6 ± 0.2 μM, as determined by fluorescence correlation spectroscopy. The significant rearrangement in the geometry of the metal center upon peptide binding was characterized by a combination of CW-EPR, pulse-EPR, and DFT computations. Understanding the binding configuration around the metal center inside NTA pocket exposed at the surface of vesicles supports further development of efficient targetable nanocarriers that can be recognized selectively by molecular recognition in a biological environment and facilitates their immobilization on solid supports and their use in two-dimensional protein arrays.

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall C enrichment in fatty acids derived from theC labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M) of each labeled FA by one or more mass units (M, M, M, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.