Ultrasensitive detection of cellular protein interactions using bioluminescence resonance energy transfer quantum dot-based nanoprobes.

Sensitive detection of protein interactions is a critical step toward understanding complex cellular processes. As an alternative to fluorescence-based detection, Renilla reniformis luciferase conjugated to quantum dots results in self-illuminating bioluminescence resonance energy transfer quantum dot (BRET-Qdot) nanoprobes that emit red to near-infrared bioluminescence light. Here, we report the development of an ultrasensitive technology based on BRET-Qdot conjugates modified with streptavidin ([BRET-Qdot]-SA) to detect cell-surface protein interactions. Transfected COS7 cells expressing human cell-surface proteins were interrogated with a human Fc tagged protein of interest. Specific protein interactions were detected using a biotinylated anti-human Fc region specific antibody followed by incubation with [BRET-Qdot]-SA. The luciferase substrate coelenterazine activated bioluminescence light emission was detected with an ultra-fast and -sensitive imager. Protein interactions barely detectable by the fluorescence-based approach were readily quantified using this technology. The results demonstrate the successful application and the flexibility of the BRET-Qdot-based imaging technology to the ultrasensitive investigation of cell-surface proteins and protein-protein interactions.

Self-illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano-particle.

Autofluorescence arising from normal tissues can compromise the sensitivity and specificity of in vivo fluorescence imaging by lowering the target-to-background signal ratio. Since bioluminescence resonance energy transfer quantum dot (BRET-QDot) nano-particles can self-illuminate in near-infrared in the presence of the substrate, coelenterazine, without irradiating excitation lights, imaging using BRET-QDots does not produce any autofluorescence. In this study, we applied this BRET-QDot nano-particle to the in vivo lymphatic imaging in mice in order to compare with BRET, fluorescence or bioluminescence lymphatic imaging. BRET-QDot655, in which QDot655 is contained as a core, was injected at different sites (e.g. chin, ear, forepaws and hind paws) in mice followed by the intravenous coelenterazine injection, and then bioluminescence and fluorescence imaging were serially performed. In all mice, each lymphatic basin was clearly visualized in the BRET imaging with minimal background signals. The BRET signal in the lymph nodes lasted at least 30 min after coelenterazine injections. Furthermore, the BRET signal demonstrated better quantification than the fluorescence signal emitting from QDot655, the core of this BRET particle. These advantages of BRET-QDot allowed us to perform real-time, quantitative lymphatic imaging without image processing. BRET-Qdots have the potential to be a robust nano-material platform for developing optical molecular imaging probes.

Advances in organic synthesis using polymer-supported reagents and scavengers under microwave irradiation.

Microwave-assisted and polymer-supported organic syntheses have emerged independently as versatile tools for rapid generation of organic molecules. Chemists are increasingly looking for a combination of both techniques for efficient organic synthesis. This review covers the recent literature on organic synthesis using microwave heating in conjunction with polymer-supported reagents and scavengers.

New developments in polymer-supported reagents, scavengers and catalysts for organic synthesis.

The ever-increasing demand for efficient syntheses of novel organic compounds with therapeutic potential remains the major driving force for the pharmaceutical industry to adopt novel technologies for organic synthesis. The use of polymer-supported reagents and scavengers is a powerful technique for integrated organic synthesis and purification. Polymer-supported reagents and scavengers can be used to selectively remove excess reagents and byproducts through simple filtration, rather than liquid-liquid extraction and chromatographic separation. In addition, polymer-supported reagents offer advantages that include the reaction of active intermediates by 'catch and release', selectivity, and immobilization of toxic reagents and byproducts. This review summarizes significant new advances in polymer-assisted solution-phase synthesis, and advances in microwave-assisted organic synthesis utilizing polymer-supported reagents and catalysts are also discussed. In view of previous review publications on this topic, only the literature from 2003 to date is included.

Use of statistical design of experiments in the optimization of amide synthesis utilizing polystyrene-supported N-hydroxybenzotriazole resin.

Two fields that routinely perform reaction optimization studies are chemical development (prior to scale-up) and medicinal or combinatorial chemistry (prior to analogue synthesis or library production). To date, the use of statistical design of experiments (DoE) in conjunction with automated synthesizers has been applied in process research to a greater extent than in the medicinal or combinatorial laboratories. We have applied DoE in conjunction with an automated synthesizer to optimize the synthesis of amides employing resin-bound N-hydroxybenzotriazole (PS-HOBt) active esters as intermediates. This methodology allowed the rapid development of an improved protocol for the parallel synthesis of amides by conversion of carboxylic acids to PS-HOBt esters followed by treatment with appropriate amines. Product isolation involved only simple filtration and evaporation.