Chemical and bacterial reduction of azo-probes: monitoring a conformational change using fluorescence spectroscopy

• Published in: Tetrahedron 2013-04, Vol.69 (13), p.2758-2766
• Main Authors: Rattray, Nicholas J.W., Zalloum, Waleed A., Mansell, David, Latimer, Joe, Jaffar, Mohammed, Bichenkova, Elena V., Freeman, Sally
• Format: Article
• Language: English
• Subjects: AMBER; Azo-reductase; Conformational lock; Dimer; Escherichia coli; Fluorescent probe
• ISSN: 0040-4020; 1464-5416
• DOI: 10.1016/j.tet.2013.01.086

Sterically constrained probes 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene (8) and 2,4-O-bispyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene (9) exhibit specific dimer fluorescent characteristics (λmax 555 nm and 511 nm, respectively), attributed to the 2,4-diaxial arrangement of the dansyl or pyrene groups. Reduction of the azo-conformational locking group in (8) and (9) yielded 1,3-bisdansyl-4,6-diaminocyclohexane (16) and 1,3-bispyrenoyl-4,6-diaminocyclohexane (17) in the tetra-equatorial chair conformation, thus minimising interaction of the bisdansyl or bispyrenoyl groups. This induces a change in fluorescence from a cooperative green emission dimer band to a blue-shifted, monomer type fluorescence, with λmax 448 nm and 396 nm for the reduced forms (16) and (17), respectively. The azo-bond conformational lock can either be reduced under biomimetic conditions (using sodium dithionite) or with bacteria (Clostridium perfringens or Escherichia coli) utilising azo-reductase enzymes. These fluorescent probes have the potential to specifically detect azo-reductase expressing bacteria. [Display omitted] Pathogenic bacterial detection has previously relied upon microscopy techniques that require skilled clinical microbiologists and expensive equipment. Recent advances have seen the use of enzyme specific chromogenic and fluorogenic substrates that, although less expensive, still require comparatively long culture times. With certain bacterial strains known to express azo-reductase enzymes (Azo-1/Azo-2), this work describes the synthesis, characterisation and biological evaluation of two fluorescent probes, 2,4-O-bisdansyl-6,7-diazabicyclo[3.2.1]oct-6-ene and 2,4-O-bispyrenoyl-6,7-diazabicyclo[3.2.1]oct-6-ene. These probes can potentially be used for the rapid clinical evaluation of low levels of pathogenic strains of bacteria that express Azo-1/Azo-2.