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.
A new class of nitrosocarbonyl precursors, O-substituted hydroxamic acids with pyrazolone leaving groups (OHPY), is described.These compounds generate nitrosocarbonyl intermediates, which upon hydrolysis release nitroxyl (azanone, HNO) under physiologically relevant conditions. Pyrazolones have been used to confirm the generation of nitrosocarbonyls by competitive trapping to form isomeric N-substituted hydroxamic acids (NHPY) via an N-selective nitrosocarbonyl aldol reaction. The rate of nitrosocarbonyl release from OHPY donors is impacted by donor substituents, including the pyrazolone leaving group.
A novel class of nitrosocarbonyl precursors, N-substituted hydroxamic acids with pyrazolone leaving groups (NHPY), has been synthesized. Under physiological conditions, these compounds generate nitrosocarbonyl intermediates, which upon hydrolysis release nitroxyl (azanone, HNO) in excellent yields. The amount and rate of nitrosocarbonyl generation are dependent on the nature of the pyrazolone leaving groups and significantly on the structural properties of the NHPY donors. Pyrazolones have been found to be efficient nitrosocarbonyl traps, undergoing an N-selective nitrosocarbonyl aldol reaction. This trapping reaction has been used to confirm the involvement of nitrosocarbonyl intermediates in NHPY aqueous decomposition. In addition, NHPY compounds are shown to generate nitrosocarbonyls efficiently under mild basic conditions in organic solvent and may therefore also enjoy synthetic utility.
We demonstrate concomitant release of HNO and small molecule organics from amphiphilic poly-(norbornene)-based copolymers. This key function was achieved by incorporation of thermally labile oxazine units within random and block copolymer architectures. Upon thermolysis, we observed generation of HNO and release of a small molecule conjugate. Importantly, the release kinetics of HNO and a UV-active small molecule (4-nitroaniline) were found to be 1:1, signifying an ability to monitor HNO production indirectly, or to simultaneously release organic therapeutics (e.g., nonsteroidal anti-inflammatory drugs) along with HNO. To our knowledge, these are the first reported polymeric materials demonstrating HNO release from covalently attached HNO donors.
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