Instability of Surface-Grafted Weak Polyacid Brushes on Flat Substrates

We study the stability of weak polyacid brush (WPAB) gradients in aqueous media covering a range in grafting density (σ) spanning 0.05–0.5 chains/nm² using two analogous surface-anchored bromoisobutyrate-based initiators for atom transfer radical polymerization (ATRP) bearing either an ester or amide linker. Variations in dry thickness of ester-based WPABs as a function of time and pH are consistent with WPAB degrafting via linker hydrolysis catalyzed by mechanical tension in the grafted chains. Sources of tension considered include high σ, as well as swelling and electrostatic repulsion associated with increasing degree of deprotonation (α) of repeat units in the WPAB. Normalized thickness of the WPAB decreases by a maximum amount at intermediate σ between ∼0.05−0.15 chains/nm², implying that contributions to tension by α are counterbalanced by charge regulation in the WPAB at high σ. Amide-based WPABs are more stable up to 264 h incubation, suggesting that commonly used ester-bearing ATRP initiators are more susceptible to hydrolysis over the time scales examined

New Fluorescent Substrate Enables Quantitative and High-Throughput Examination of Vesicular Monoamine Transporter 2 (VMAT2)

Vesicular monoamine transporter 2 (VMAT2) is an essential component of the monoaminergic neurotransmission system in the brain as it transports monoamine neurotransmitters from the neuronal cytosol into the synaptic vesicles and thus contributes to modulation of neurotransmitter release. Considering the continuing interest in VMAT2 as a drug target, as well as a target for the design of imaging probes, we have developed a fluorescent substrate well suited for the study of VMAT2 in cell culture. Herein, we report the synthesis and characterization of a new fluorescent probe, FFN206, as an excellent VMAT2 substrate capable of detecting VMAT2 activity in intact cells using fluorescence microscopy, with subcellular localization to VMAT2-expressing acidic compartments without apparent labeling of other organelles. VMAT2 activity can also be measured via microplate reader. The apparent <i>K</i>_m of FFN206 at VMAT2 was found to be 1.16 ± 0.10 μM, similar to that of dopamine. We further report the development and validation of a cell-based fluorescence assay amenable to high-throughput screening (HTS) using VMAT2-transfected HEK cells (Z′-factor of 0.7–0.8), enabling rapid identification of VMAT2 inhibitors and measurement of their inhibition constants over a broad range of affinities. FFN206 thus represents a new tool for optical examination of VMAT2 function in cell culture