A Nonaggregating Heptamethine Cyanine for Building Brighter Labeled Biomolecules

Abstract

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.

Development of a drug–device combination for fluorescence-guided surgery in neuroendocrine tumors

“…The reduction in fluorescence at high MMC(IR800)-TOC concentrations is presumably due to dye-conjugate aggregation and quenching, which are known phenomena associated with NIRF dye conjugates. 35 Plotting CNR as a function of drug concentration was in accordance with the qualitative assessment for all optical filters [ Fig. 2(b)].…”

Sterically Shielded Heptamethine Cyanine Dyes for Bioconjugation and High Performance Near‐Infrared Fluorescence Imaging

“…High photostability is also a highly desired, but an elusive heptamethine cyanine dye property . Photobleaching of a heptamethine cyanine dye is primarily caused by a bimolecular reaction of the heptamethine polyene with photogenerated singlet oxygen . The predominant reaction pathway forms a strained dioxetane intermediate followed by a fragmentation cascade.…”

“…A recent advance developed by Schnermann and co‐workers employed a more robust meso C−O alkyl bond, and one example of this fluorochrome is UL766, which exhibits excellent chemical stability and very favorable pharmacokinetics due to its charge balanced structure . However, the heptamethine polyene within UL766, (and its close structural analogues) is quite electron rich which means relatively high fluorochrome reactivity with singlet oxygen, and thus susceptibility to photobleaching . Another way to replace the reactive meso C−O aryl bond in ZW800‐1 is to employ a much more stable C−C linkage as exemplified by 756z with its meso ‐aryl substituent .…”

Multivariate AND-gate substrate probes as enhanced contrast agents for fluorescence-guided surgery

“…However, conjugating ICG to the peptide scaffold caused unexpected reactivity issues possibly caused by aggregation or tertiary folding of the molecule. Therefore, we used a heptamethine cyanine fluorophore FNIR-Tag that was designed by the Schnermann group to resist aggregation and have better water solubility compared to ICG 32 . We conjugated the FNIR dye successfully to the α-amine on the central (D)-Glu linker and subsequently attach two QC-1 quenchers to the lysine side chains to produce AND-Gate-FNIR (Supplementary Scheme 2).…”