Fluorophore-Tagged Poly-Lysine RAFT Agents: Controlled Synthesis of Trackable Cell-Penetrating Peptide-Polymers.

The conjugation of a fluorophore and a variety of cell-penetrating peptides onto a RAFT agent allowed for the synthesis of polymers of defined sizes with quantifiable cell-uptake. Each peptide-RAFT agent was used to polymerize acrylamide, acrylate, and styrene monomers to form high or low molecular weight polymers (here 50 or 7.5 kDa) with the peptide having no influence on the RAFT agent's control. The incorporation of a single fluorophore per polymer chain allowed cellular analysis of the uptake of the size-specific peptide-polymers via flow cytometry and confocal microscopy. The cell-penetrating peptides had a direct effect on the efficiency of polymer uptake for both high and low molecular weight polymers, demonstrating the versatility of the strategy. These "all-in-one", synthetically accessible RAFT agents allow highly controlled preparation of synthetic peptide-polymer conjugates and subsequent quantification of their delivery into cells

Moving into the red - a near infra-red optical probe for analysis of human neutrophil elastase in activated neutrophils and neutrophil extracellular traps.

Neutrophils are the first immune cells recruited for defence against invading pathogens; however, their dysregulated activation and subsequent release of the enzyme human neutrophil elastase is associated with several, inflammation-based, diseases. Herein, we describe a FRET-based, tri-branched (one quencher, three fluorophores) near infrared probe that provides an intense OFF/ON amplified fluorescence signal for specific detection of human neutrophil elastase. The probe allowed selective detection of activated neutrophils and labelling of neutrophil extracellular traps

Bioorthogonal Swarming: In Situ Generation of Dendrimers

With the aid of bioorthogonal chemistry, we demonstrate the fabrication of synthetic dendrimers in situ around living cells. Using tetrazine dienophile and aminooxyl/hydrazide aldehyde chemistries, the density of functional groups on the dendrimers exponentially amplified intensities of fluorescent markers in antibody-targeted live cell imaging. This novel “swarming” approach highlights the power of bioorthogonal chemistry and provides a route to non-natural chemical structures on cells, paving the way for the generation of various artificial cellular nanostructures and scaffolds

Fluorescent Formazans and Tetrazolium Salts - Towards Fluorescent Cytotoxicity Assays

Published in Combinatorial Chemistry and High Throughput Screening 2016, vol 19 part 5 pp 384-391Formazan-based colorimetric cytotoxicity assays, such as the MTT assay, are typically used to assess cell viability with only metabolically active cells reducing tetrazolium salts into the formazans, which is then quantified by absorbance. Fluorescence offers several advantages compared to colorimetric assays and would enable techniques such as flow cytometry and confocal microscopy to be used for analysis. Here, fluorescent formazans 10, 11 and 12, and their corresponding tetrazolium salts 13, 16 and 24, respectively, were synthesised by incorporation of a known fluorophore backbone (coumarin, fluorescein and rhodol) with disruption of the conjugated system preventing or reducing fluorescence of the tetrazolium salts. The tetrazolium moiety was able to quench the fluorescence of the incorporated fluorescein and O-methyl rhodol, whereas with the coumarin-based tetrazolium salt the fluorescence was only quenched under acidic conditions. These tetrazolium salts were successfully reduced to the fluorescent formazans with cells and offer a step forward in the development of fluorescent cytotoxicity assays

Thermoresponsive hydrogel maintains the mouse embryonic stem cell "naïve" pluripotency phenotype

A chemically defined hydrogel HG21, which allows enzyme-free passaging, is a substitute for gelatin allowing standardised and inexpensive mESC culture.</p

Hydroxybenzothiazoles as New Nonsteroidal Inhibitors of 17β-Hydroxysteroid Dehydrogenase Type 1 (17β-HSD1)

17β-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17β-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17β-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17β-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17β-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC50-values in the nanomolar range for the transformation of E1 to E2 by 17β-HSD1, reasonable selectivity against 17β-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17β-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17β-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17β-HSD1 inhibitors for the development of potential therapeutics

Fluorogenic Substrates for In Situ Monitoring of Caspase-3 Activity in Live Cells

The in situ detection of caspase-3 activity has applications in the imaging and monitoring of multiple pathologies, notably cancer. A series of cell penetrating FRET-based fluorogenic substrates were designed and synthesised for the detection of caspase-3 in live cells. A variety of modifications of the classical caspase-3 and caspase-7 substrate sequence Asp-Glu-Val-Asp were carried out in order to increase caspase-3 affinity and eliminate caspase-7 cross-reactivity. To allow cellular uptake and good solubility, the substrates were conjugated to a cationic peptoid. The most selective fluorogenic substrate 27, FAM-Ahx-Asp-Leu-Pro-Asp-Lys(MR)-Ahx, conjugated to the cell penetrating peptoid at the C-terminus, was able to detect and quantify caspase-3 activity in apoptotic cells without cross-reactivity by caspase-7.This work was supported by the Ramon Areces and Caja Madrid Foundations to AMPL and Spanish Ministry of Economy and Competitiveness to MLSG (graduate student fellowships FPI BES-2010-030257 and EEBB-I-13-07131)

A fluorogenic, peptide-based probe for the detection of Cathepsin D in macrophages.

Cathepsin D is a protease that is an effector in the immune response of macrophages, yet to date, only a limited number of probes have been developed for its detection. Herein, we report a water soluble, highly sensitive, pH insensitive fluorescent probe for the detection of Cathepsin D activity that provides a strong OFF/ON signal upon activation and with bright emission at 515 nm. The probe was synthesised using a combination of solid and solution-phase chemistries, with probe optimisation to increase its water solubility and activation kinetics by addition of a long PEG chain (5 kDa) at the C-terminus. A BODIPY fluorophore allowed detection of Cathepsin D across a wide pH range, important as the protease is active both at the low pH found in lysosomes and also in higher pH phagolysosomes, and in the cytosol. The probe was successfully used to detect Cathepsin D activity in macrophages challenged by exposure to bacteria