4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and maleimide

BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-

Topics: Boron Compounds; Boron Neutron Capture Therapy; Coloring Agents; Drug Delivery Systems; Fluorescent Dyes; Homocysteine; Humans; Lactones; Maleimides; Molecular Probes; Neoplasms; Precision Medicine; Serum Albumin, Human; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet

Design and synthesis of novel water-soluble polymers bearing reactive side chains are actively pursued due to their increasing demand in areas such as bioconjugation and drug delivery. This study reports the fabrication of poly(ethylene glycol) methacrylate based thiol-reactive water-soluble polymeric supports that can serve as targeted drug delivery vehicles. Thiol-reactive maleimide units were incorporated into polymers as side chains by use of a furan-protected maleimide containing monomer. Atom transfer radical polymerization (ATRP) was employed to obtain a family of well-defined copolymers with narrow molecular weight distributions. After the polymerization, the maleimide groups were activated to their reactive form, ready for conjugation with thiol-containing molecules. Efficient functionalization of the maleimide moieties was demonstrated by conjugation of a tripeptide glutathione under mild and reagent-free aqueous conditions. Additionally, hydrophobic thiol-containing dye (Bodipy-SH) and a cyclic peptide-based targeting group (cRGDfC) were sequentially appended onto the maleimide bearing polymers to demonstrate their efficient multifunctionalization. The conjugates were utilized for in vitro experiments over both cancerous and healthy breast cell lines. Obtained results demonstrate that the conjugates were nontoxic, and displayed efficient cellular uptake. The presence of the peptide based targeting group had a clear effect on increasing the uptake of the dye-conjugated polymers into cells when compared to the construct devoid of the peptide. Overall, the facile synthesis and highly efficient multifunctionalization of maleimide-containing thiol-reactive copolymers offer a novel and attractive class of polyethylene glycol-based water-soluble supports for drug delivery.

Topics: Adenocarcinoma; Boron Compounds; Breast; Breast Neoplasms; Cell Survival; Drug Design; Female; Humans; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Maleimides; Methacrylates; Polyethylene Glycols; Polymerization; Polymers

We designed and synthesized a novel thiol-reactive fluorescence probe based on the BODIPY fluorophore. The fluorescence of this probe is strongly quenched by donor-excited photoinduced electron transfer (d-PeT) from BODIPY to maleimide, but after reaction with thiol, the fluorescence of BODIPY is restored, affording a 350-fold intensity increase.

Topics: Boron Compounds; Electrons; Fluorescent Dyes; Maleimides; Molecular Probes; Sulfhydryl Compounds

Structural evidence has demonstrated that P-glycoprotein (P-gp) undergoes considerable conformational changes during catalysis, and these alterations are important in drug interaction. Knowledge of which regions in P-gp undergo conformational alterations will provide vital information to elucidate the locations of drug binding sites and the mechanism of coupling. A number of investigations have implicated transmembrane segment six (TM6) in drug-P-gp interactions, and a cysteine-scanning mutagenesis approach was directed to this segment. Introduction of cysteine residues into TM6 did not disturb basal or drug-stimulated ATPase activity per se. Under basal conditions the hydrophobic probe coumarin maleimide readily labeled all introduced cysteine residues, whereas the hydrophilic fluorescein maleimide only labeled residue Cys-343. The amphiphilic BODIPY-maleimide displayed a more complex labeling profile. The extent of labeling with coumarin maleimide did not vary during the catalytic cycle, whereas fluorescein maleimide labeling of F343C was lost after nucleotide binding or hydrolysis. BODIPY-maleimide labeling was markedly altered during the catalytic cycle and indicated that the adenosine 5'-(beta,gamma-imino)triphosphate-bound and ADP/vanadate-trapped intermediates were conformationally distinct. Our data are reconciled with a recent atomic scale model of P-gp and are consistent with a tilting of TM6 in response to nucleotide binding and ATP hydrolysis.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding Sites; Boron Compounds; Catalysis; Cell Line; Cell Membrane; Codon; Coumarins; Cysteine; Electrophoresis, Polyacrylamide Gel; Hydrolysis; Insecta; Kinetics; Maleimides; Models, Chemical; Models, Molecular; Protein Conformation; Protein Isoforms; Protein Structure, Tertiary; Recombinant Proteins; Sequence Analysis, DNA

A comprehensive two-dimensional (2-D) separation system, coupling capillary reverse-phase liquid chromatography (cRPLC) to capillary isoelectric focusing (CIEF), is described for protein and peptide mapping. cRPLC, the first dimension, provided high-resolution separations for salt-free proteins. CIEF, the second dimension with an orthogonal mechanism to cRPLC afforded excellent resolution capability for proteins with efficient protein enrichment. Since all sample fractions in cRPLC effluents could be transferred to the CIEF dimensions, the combination of the two high-efficiency separations resulted in maximal separation capabilities of each dimension. Separation effectiveness of this approach was demonstrated using complex protein/peptide samples, such as yeast cytosol and a BSA tryptic digest. A peak capacity of more than 10 000 had been achieved. A laser-induced fluorescence (LIF) detector, developed for this system, allowed for high-sensitive detection, with a fmol level of peptide detection for the BSA digest. FITC and BODIPY maleimide were used to tag the proteins, and the latter was found better both for separation and detection in our 2-D system.

Topics: Boron Compounds; Chromatography, Liquid; Cytosol; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Isoelectric Focusing; Lasers; Maleimides; Peptide Mapping; Reproducibility of Results; Serum Albumin, Bovine; Trypsin; Yeasts