Ratiometric near-infrared fluorescent probe for nitroreductase activity enables 3D imaging of hypoxic cells within intact tumor spheroids.

Fluorescent molecular probes that report nitroreductase activity have promise as imaging tools to elucidate the biology of hypoxic cells and report the past hypoxic history of biomedical tissue. This study describes the synthesis and validation of a "first-in-class" ratiometric, hydrophilic near-infrared fluorescent molecular probe for imaging hypoxia-induced nitroreductase activity in 2D cell culture monolayers and 3D multicellular tumor spheroids. The probe's molecular structure is charge-balanced and the change in ratiometric signal is based on Förster Resonance Energy Transfer (FRET) from a deep-red, pentamethine cyanine donor dye (Cy5, emits ∼660 nm) to a linked near-infrared, heptamethine cyanine acceptor dye (Cy7, emits ∼780 nm). Enzymatic reduction of a 4-nitrobenzyl group on the Cy7 component induces a large increase in Cy7/Cy5 fluorescence ratio. The deep penetration of near-infrared light enables 3D optical sectioning of intact tumor spheroids, and visualization of individual hypoxic cells (i.e., cells with raised Cy7/Cy5 ratio) as a new way to study tumor spheroids. Beyond preclinical imaging, the near-infrared fluorescent molecular probe has high potential for ratiometric imaging of hypoxic tissue in living subjects.

Advances in Optical Sensors of N-Acetyl-ß-d-hexosaminidase (N-Acetyl-ß-d-glucosaminidase).

N-Acetyl-ß-d-hexosaminidases (EC 3.2.1.52) are exo-acting glycosyl hydrolases that remove N-acetyl-ß-d-glucosamine (Glc-NAc) or N-acetyl-ß-d-galactosamine (Gal-NAc) from the nonreducing ends of various biomolecules including oligosaccharides, glycoproteins, and glycolipids. The same enzymes are sometimes called N-acetyl-ß-d-glucosaminidases, and this review article employs the shorthand descriptor HEX(NAG) to indicate that the terms HEX or NAG are used interchangeably in the literature. The wide distribution of HEX(NAG) throughout the biosphere and its intracellular location in lysosomes combine to make it an important enzyme in food science, agriculture, cell biology, medical diagnostics, and chemotherapy. For more than 50 years, researchers have employed chromogenic derivatives of N-acetyl-ß-d-glucosaminide in basic assays for biomedical research and clinical chemistry. Recent conceptual and synthetic innovations in molecular fluorescence sensors, along with concurrent technical improvements in instrumentation, have produced a growing number of new fluorescent imaging and diagnostics methods. A systematic summary of the recent advances in optical sensors for HEX(NAG) is provided under the following headings: assessing kidney health, detection and treatment of infectious disease, fluorescence imaging of cancer, treatment of lysosomal disorders, and reactive probes for chemical biology. The article concludes with some comments on likely future directions.

Enzyme Sensing Using 2-Mercaptopyridine-Carbonitrile Reporters and Surface-Enhanced Raman Scattering.

The high sensitivity and functional group selectivity of surface-enhanced Raman scattering (SERS) make it an attractive method for enzyme sensing, but there is currently a severe lack of enzyme substrates that release SERS reporter molecules with favorable detection properties. We find that 2-mercaptopyridine-3-carbonitrile ( o-MPN) and 2-mercaptopyridine-5-carbonitrile ( p-MPN) are highly effective as SERS reporter molecules that can be captured by silver or gold nanoparticles to give intense SERS spectra, each with a distinctive nitrile peak at 2230 cm-1. p-MPN is a more sensitive reporter and can be detected at low nanomolar concentrations. An assay validation study synthesized two novel substrate molecules, Glc-o-MPN and Glc-p-MPN, and showed that they can be cleaved efficiently by ß-glucosidase (K m = 228 and 162 µM, respectively), an enzyme with broad industrial and biomedical utility. Moreover, SERS detection of the released reporters ( o-MPN or p-MPN) enabled sensing of ß-glucosidase activity and ß-glucosidase inhibition. Comparative experiments using a crude almond flour extract showed that the presence of ß-glucosidase activity could be confirmed by SERS detection in a much shorter time period (>10 time shorter) than by UV-vis absorption detection. It is likely that a wide range of enzyme assays and diagnostic tests can be developed using 2-mercaptopyridine-carbonitriles as SERS reporter molecules.

Structure-Activity Studies of Nitroreductase-Responsive Near-Infrared Heptamethine Cyanine Fluorescent Probes.

Two new classes of near-infrared molecular probes were prepared and shown to exhibit "turn on" fluorescence when cleaved by the nitroreductase enzyme, a well-known biomarker of cell hypoxia. The fluorescent probes are heptamethine cyanine dyes with a central 4'-carboxylic ester group on the heptamethine chain that is converted by a self-immolative fragmentation mechanism to a 4'-caboxylate group that greatly enhances the fluorescence brightness. Each compound was prepared by ring opening of a Zincke salt. The chemical structures have either terminal benzoindolinenes or propargyloxy auxochromes, which provide favorable red-shifted absorption/emission wavelengths and a hyperchromic effect that enhances the photon output when excited by 808 nm light. A fluorescent probe with terminal propargyloxy-indolenines exhibited less self-aggregation and was rapidly activated by nitroreductase with large "turn on" fluorescence; thus, it is the preferred choice for translation towards in vivo applications.

Generalizable synthesis of bioresponsive near-infrared fluorescent probes: sulfonated heptamethine cyanine prototype for imaging cell hypoxia.

Continued advancement in bioresponsive fluorescence imaging requires new classes of activatable fluorescent probes that emit near-infrared fluorescence with wavelengths above 740 nm. Heptamethine cyanine dyes (Cy7) have suitable fluorescence properties but it is challenging to create activatable probes because Cy7 dyes have a propensity for self-aggregation and fluorescence quenching. A new synthetic strategy is employed to create a generalizable class of hydrophilic bioresponsive near-infrared fluorescent probes with appended sulfonates that provide excellent physiochemical properties. A prototype version is triggered by nitroreductase enzyme to undergo self-immolative cleavage with a large enhancement in fluorescence signal at 780 nm and the probe enables microscopic imaging of cell hypoxia with "turn on" fluorescence. Near-infrared fluorescence imaging of hypoxia is potentially useful in many different areas of biomedical research and clinical treatment.

Supramolecular optimization of the visual contrast for colorimetric indicator assays that release resorufin dye.

A tetralactam macrocycle acts as a novel supramolecular adjuvant to capture a released resorufin dye and create a higher contrasting yellow/blue color change for enhanced naked eye interpretation of a colorimetric indicator assay.