Synthesis, In Vitro Evaluation, and In Vivo Metabolism of Fluor/Quencher Compounds Containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3)

Abstract

Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH(2) (8) and its D-arginine (Darg) analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a "non-cleavable" control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ-3 should be used with caution in vivo, due to instability of its azo bond.

Real-time In Vivo Molecular Detection of Primary Tumors and Metastases with Ratiometric Activatable Cell-Penetrating Peptides

“…Although these strategies have varying levels of efficacy, one inherent problem with all such methods is that fluorescence intensity is highly vulnerable to factors other than MMP activity. For example, the use of a dark quencher such as BHQ-3 has recently been shown to be problematic due to the in vivo instability of BHQ-3, leading to nonspecific dequenching within a few minutes after injection (24). Analogues of RACPPs have been synthesized with pheophorbide (25) or Alexa 750 (22) in place of Cy5 and BHQ-3 instead of Cy7, but these probes were nonratiometric, failed to dissociate completely after linker cleavage (perhaps because the chromophores are relatively hydrophobic and sticky), and were not tested in vivo .…”

Recent Development of Chemosensors Based on Cyanine Platforms

“…Also by taking advantage of BHQ-3 as the quencher, Linder and co-workers developed chemosensor 58 for detection of matrix metalloproteinases (MMPs). 106 Recently, Marx's group synthesized two adenosine triphosphate (ATP) based FRET chemosensors, 59 and 60, for detecting ATP-consuming enzymes. 107 Compound 59, constructed by conjugating sulfo-Cy3 and sulfo-Cy5 to an ATP analogue, served as a ratiometric indicator to phosphodiesterase I (a model enzyme) with the change of fluorescence ratio up to 140-fold through monitoring the fluorescent intensities of sulfo-Cy3 (∼570 nm) and sulfo-Cy5 (∼670 nm) upon excitation at 510 nm.…”

Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma

“…Two recent studies (19, 20) have reported possible non-specific cleavage for probes of similar design that would lead to diminished target specificity of our probe. One study suggested the breakdown of the BHQ3 in blood plasma, rather than the MMP cleavage of the probe, to be a process that leads to the fluorescence emission (20).…”

“…One study suggested the breakdown of the BHQ3 in blood plasma, rather than the MMP cleavage of the probe, to be a process that leads to the fluorescence emission (20). Optical imaging of tumor bearing mice injected with the active and the control probe gave insight into the extent and rate of BHQ3 breakdown in our probe.…”