Separating the isomers--efficient synthesis of the N-hydroxysuccinimide esters of 5 and 6-carboxyfluorescein diacetate and 5 and 6-carboxyrhodamine B.

Diacetate protection of 5 and 6-carboxyfluorescein followed by synthesis of the N-hydroxysuccinimide esters allowed ready separation of the two isomers on a multi-gram scale. The 5 and 6-carboxyrhodamine B N-hydroxysuccinimide esters were also readily synthesised and separated.

Highly specific, multi-branched fluorescent reporters for analysis of human neutrophil elastase.

Human neutrophil elastase (HNE) is a serine protease implicated in the pathogenesis of acute and chronic inflammatory disease. Here a series of, internally quenched, single fluorophore fluorescent reporters were synthesised that allowed the rapid, highly specific and sensitive analysis of HNE activity over closely related proteases.

Enhanced avidity from a multivalent fluorescent antimicrobial peptide enables pathogen detection in a human lung model.

Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected. We have previously described an antimicrobial peptide (AMP) Ubiquicidin (fragment UBI29-41) labelled with an environmentally sensitive fluorophore that optically detected bacteria in vitro but not ex vivo. Here, we describe further chemical development of this compound and demonstrate that altering the secondary structure of the AMP to generate a tri-branched dendrimeric scaffold provides enhanced signal in vitro and ex vivo and consequently allows the rapid detection of pathogens in situ in an explanted human lung. This compound (NBD-UBIdend) demonstrates bacterial labelling specificity for a broad panel of pathogenic bacteria and Aspergillus fumigatus. NBD-UBIdend demonstrated high signal-to-noise fluorescence amplification upon target engagement, did not label host mammalian cells and was non-toxic and chemically robust within the inflamed biological environment. Intrapulmonary delivery of NBD-UBIdend, coupled with optical endomicroscopy demonstrated real-time, in situ detection of bacteria in explanted whole human Cystic Fibrosis lungs.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A.

Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

Optical molecular imaging of lysyl oxidase activity - detection of active fibrogenesis in human lung tissue.

Aberrant fibrogenesis is a feature of many diseases in multiple organ systems. The lysyl oxidase family of enzymes are central to tissue homeostasis and elevated lysyl oxidase activity is implicated in fibroproliferation as well as in cancer stroma. We have synthesised a novel fluorogenic reporter for monitoring lysyl oxidase activity that generates a 3-5 fold increase in fluorescence following probe activation in ventilating fibrotic ex vivo asinine lung and ex vivo human lung tissue. The probe termed "oLOX" can provide real-time measurement of lysyl oxidase activity in a number of biological settings and is tractable from an in vitro setting to man.

Development of inositol-based antagonists for the D-myo-inositol 1,4,5-trisphosphate receptor.

The syntheses of four D-myo-inositol 1,4,5-trisphosphate (InsP(3)) derivatives, incorporating phosphate bioisosteres at the 5-position, are reported. The methyl phosphate ester and sulfate derivatives retain InsP(3) receptor (InsP(3)R) agonist activity; the compounds that possess a methylphosphonate or a carboxymethyl moiety are InsP(3)R antagonists.

Synthesis and biological action of novel 4-position-modified derivatives of D-myo-inositol 1,4,5-trisphosphate.

The design of a range of 4-position-modified D-myo-inositol 1,4,5-trisphosphate derivatives is described. The enantioselective synthesis of these compounds is reported, along with initial biological analysis, which indicates that these compounds do not act as D-myo-inositol 1,4,5-trisphosphate receptor agonists or antagonists.