Organocatalytic Synthesis and Evaluation of Polycarbonate Pendant Polymer:β-Cyclodextrin-Based Nucleic Acid Delivery Vectors

A family of mPEG-<i>b</i>-polycarbonate (mPEG-PC) diblock pendant polymers were synthesized from trimethylene carbonate and other cyclic carbonate monomers bearing hydrophobic guest ligands via organocatalytic ring-opening polymerization using 1,4,5-triazabicyclo[4.4.0]­dec-5-ene catalyst or 1,8-diazabicyclo[5.4.0]­undec-7-ene/thiourea cocatalyst. Diblock copolymers composed of a methoxy­poly­(ethylene oxide) (mPEG) block and a polycarbonate block containing either homopolymer or mixed polycarbonates (PC) were prepared by homopolymerization or copolymerization of the cyclic carbonate monomers in the presence of mPEG2000 or mPEG5000 initiator to give materials having a tunable pendant group density along the polycarbonate backbone. Polycarbonate blocks targeting the 2.4–10 kDa range were prepared with good molecular weight control and modest polydispersities (averaging ∼1.3). Complexation of plasmid DNA with β-cyclodextrin–polyethylenimine2.5 kDa produced nanoparticle cores that were then coated with the mPEG–PC diblock copolymers to produce transfection complexes in the 100–250 nm size range. Stable transfection complexes prepared at N/P ratios >10 had slightly positive ζ potentials and showed comparable or modestly better transfection efficiencies in HeLa cells than the commercial transfection agent, Lipofectamine2000. Transfection efficiencies were not dependent on polycarbonate block molecular weights. The mPEG-PC constructs displayed similar efficacy for adamantyl and cholesteryl pendants that strongly bind to β-cyclodextrin; however, slightly better performance was observed for the weakly bound pendant, benzyl. These findings suggest that pDNA release is largely mediated by hydrolysis of the ester-bound pendant ligand within the endolysosomal compartment of the cell, with desorption of the mPEG–PC layer also contributing to plasmid release and activation in the case of weak binding pendant groups. We infer from these results that mPEG-PC may be an effective degradable transfection agent for <i>in vivo</i> applications

Metal-dependent allosteric activation and inhibition on the same molecular scaffold: the copper sensor CopY from Streptococcus pneumoniae

Resistance to copper (Cu) toxicity in the respiratory pathogen Streptococcus pneumoniae is regulated by the Cu-specific metallosensor CopY. CopY is structurally related to the antibiotic-resistance regulatory proteins MecI and BlaI from Staphylococcus aureus, but is otherwise poorly characterized. Here we employ a multi-pronged experimental strategy to define the Spn CopY coordination chemistry and the unique mechanism of allosteric activation by Zn(ii) and allosteric inhibition by Cu(i) of cop promoter DNA binding. We show that Zn(ii) is coordinated by a subunit-bridging 3S 1H2O complex formed by the same residues that coordinate Cu(i), as determined by X-ray absorption spectroscopy and ratiometric pulsed alkylation-mass spectrometry (rPA-MS). Apo- and Zn-bound CopY are homodimers by small angle X-ray scattering (SAXS); however, Zn stabilizes the dimer, narrows the conformational ensemble of the apo-state as revealed by ion mobility-mass spectroscopy (IM-MS), and activates DNA binding in vitro and in cells. In contrast, Cu(i) employs the same Cys pair to form a subunit-bridging, kinetically stable, multi-metallic Cu·S cluster (KCu ≈ 1016 M–1) that induces oligomerization beyond the dimer as revealed by SAXS, rPA-MS and NMR spectroscopy, leading to inhibition of DNA binding. These studies suggest that CopY employs conformational selection to drive Zn-activation of DNA binding, and a novel Cu(i)-mediated assembly mechanism that dissociates CopY from the DNA via ligand exchange-catalyzed metal substitution, leading to expression of Cu resistance genes. Mechanistic parallels to antibiotic resistance repressors MecI and BlaI are discussed