Heptamethine cyanines
are broadly used for a range of near-infrared
imaging applications. As with many fluorophores, these molecules are
prone to forming nonemissive aggregates upon biomolecule conjugation.
Prior work has focused on persulfonation strategies, which only partially
address these issues. Here, we report a new set of peripheral substituents,
short polyethylene glycol chains on the indolenine nitrogens and a
substituted alkyl ether at the C4′ position, that provide exceptionally
aggregation-resistant fluorophores. These symmetrical molecules are
net-neutral, can be prepared in a concise sequence, and exhibit no
evidence of H-aggregation even at high labeling density when
appended to monoclonal antibodies or virus-like particles. The resulting
fluorophore–biomolecule conjugates exhibit exceptionally bright in vitro and in vivo signals when compared
to a conventional persulfonated heptamethine cyanine. Overall, these
efforts provide a new class of heptamethine cyanines with significant
utility for complex labeling applications.
Due to its inherent reactivity, HNO must be generated in situ through the use of donor compounds. One of the primary strategies for the development of new HNO donors has been modifying hydroxylamines with good leaving groups. A recent example of this strategy is the (hydroxylamino)barbituric acid (HABA) class of HNO donors. In this case, however, an undesired intramolecular rearrangement pathway to the corresponding hydantoin derivative competes with HNO formation, particularly in the absence of chemical traps for HNO. This competitive non-HNO-producing pathway has restricted the development of the HABA class to examples with fast HNO release profiles at physiological pH and temperature (t(1/2) < 1 min). Herein, the factors that favor the rearrangement pathway have been examined and two independent strategies that protect against rearrangement to favor HNO generation have been developed. The timecourse and stoichiometry for the in vitro conversion of these compounds to HNO (trapped as a phosphine aza-ylide) and the corresponding barbituric acid (BA) byproduct have been determined by (1)H NMR spectroscopy under physiologically relevant conditions. These results confirm the successful extension of the HABA class of pure HNO donors with half-lives at pH 7.4, 37 °C ranging from 19 to 107 min.
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