The absorption and emission wavelengths of group 14 pyronines and rhodamines, which contain silicon, germanium, or tin at the 10 position of the xanthene chromophore, showed large bathochromic shifts compared to the original rhodamines, owing to stabilization of the LUMO energy levels by σ*-π* conjugation between group 14 atom-C (methyl) σ* orbitals and a π* orbital of the fluorophore. These group 14 pyronines and rhodamines retain the advantages of the original rhodamines, including high quantum efficiency in aqueous media (Φ(fl) = 0.3-0.45), tolerance to photobleaching, and high water solubility. Group 14 rhodamines have higher values of reduction potential than other NIR light-emitting original rhodamines, and therefore, we speculated their NIR fluorescence could be controlled through the photoinduced electron transfer (PeT) mechanism. Indeed, we found that the fluorescence quantum yield (Φ(fl)) of Si-rhodamine (SiR) and Ge-rhodamine (GeR) could be made nearly equal to zero, and the threshold level for fluorescence on/off switching lies at around 1.3-1.5 V for the SiRs. This is about 0.1 V lower than in the case of TokyoGreens, in which the fluorophore is well established to be effective for PeT-based probes. That is to say, the fluorescence of SiR and GeR can be drastically activated by more than 100-fold through a PeT strategy. To confirm the validity of this strategy for developing NIR fluorescence probes, we employed this approach to design two kinds of novel fluorescence probes emitting in the far-red to NIR region, i.e., a series of pH-sensors for use in acidic environments and a Zn(2+) sensor. We synthesized these probes and confirmed that they work well.

A far-red to near-infrared (NIR) fluorescence probe, MMSiR, based on Si-rhodamine, was designed and synthesized for sensitive and selective detection of HOCl in real time. MMSiR and its oxidized product SMSiR have excellent properties, including pH-independence of fluorescence, high resistance to autoxidation and photobleaching, and good tissue penetration of far-red to NIR fluorescence emission. The value of MMSiR was confirmed by real-time imaging of phagocytosis using a fluorescence microscope. wsMMSiR, a more hydrophilic derivative of MMSiR, permitted effective in vivo imaging of HOCl generation in a mouse peritonitis model. This probe is expected to be a useful tool for investigating the wide range of biological functions of HOCl.

Background and Objectives Antimicrobial photodynamic therapy (APDT) employs a nontoxic photosensitizer (PS) and visible light, which in the presence of oxygen produce reactive oxygen species (ROS), such as singlet oxygen (1O2, produced via Type II mechanism) and hydroxyl radical (HO•, produced via Type I mechanism). This study examined the relative contributions of 1O2 and HO• to APDT killing of Gram-positive and Gram-negative bacteria. Study Design/Materials and Methods Fluorescence probes, 3'-(p-hydroxyphenyl)-fluorescein (HPF) and singlet oxygen sensor green reagent (SOSG) were used to determine HO• and 1O2 produced by illumination of two PS: tris-cationic-buckminsterfullerene (BB6) and a conjugate between polyethylenimine and chlorin(e6) (PEI–ce6). Dimethylthiourea is a HO• scavenger, while sodium azide (NaN3) is a quencher of 1O2. Both APDT and killing by Fenton reaction (chemical generation of HO•) were carried out on Gram-positive bacteria (Staphylococcus aureus and Enteroccoccus fecalis) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa. Results Conjugate PEI-ce6 mainly produced 1O2 (quenched by NaN3), while BB6 produced HO• in addition to 1O2 when NaN3 potentiated probe activation. NaN3 also potentiated HPF activation by Fenton reagent. All bacteria were killed by Fenton reagent but Gram-positive bacteria needed a higher concentration than Gram-negatives. NaN3 potentiated Fenton-mediated killing of all bacteria. The ratio of APDT killing between Gram-positive and Gram-negative bacteria was 2 or 4:1 for BB6 and 25:1 for conjugate PEI-ce6. There was a NaN3 dose dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN3 almost failed to inhibit killing of Gram-positive bacteria. Conclusion Azidyl radicals may be formed from NaN3 and HO•. It may be that Gram-negative bacteria are more susceptible to HO• while Gram-positive bacteria are more susceptible to 1O2. The differences in NaN3 inhibition may reflect differences in the extent of PS binding to bacteria (microenvironment) or differences in penetration of NaN3 into cell walls of bacteria.