Rapid Macrocycle Threading by a Fluorescent Dye–Polymer Conjugate in Water with Nanomolar Affinity

Abstract

A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethyleneglycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with kon ~106–107 M−1s−1. The association is effective under cell culture conditions and produces large changes in dye optical properties including turn-on near-infrared fluorescence that can be imaged using cell microscopy. Association constants in water are ~1000 times higher than in organic solvents and strongly enthalpically favored at 27 °C. The threading rate is hardly affected by the length of the PEG chains that flank the squaraine dye. For example, macrocyle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethyleneglycol chains that are twenty times shorter. The results are a promising advance towards synthetic mimics of streptavidin/biotin.

Pre-Assembly of Near-Infrared Fluorescent Multivalent Molecular Probes for Biological Imaging

“…Previously, we reported the structure and properties of threaded, single-station complex 6C ⊃S . 33 As a fluorescent probe in cell culture, it exhibited no specific cell affinity and was observed to slowly enter living cells over several hours. Thus, in this present mouse imaging study, the threaded complex 6C ⊃S and new two-station version 2(6C) ⊃S3S were expected to act as non-targeted control probes that cleared from the blood stream through the kidneys with little retention in other organs.…”

“…Previously, we showed that mixing S and 6C in water at 20 °C formed the single-station untargeted complex 6C ⊃S with an association constant ( K a ) of 1 × 10 9 M −1 and threading rate k on of 130 M −1 s −1 33 . Essentially the same values of K a and k on were observed when S and 6B were mixed in water to form the single-station targeted complex 6B ⊃S .…”

“…32 The macrocycle is a water-soluble tetralactam with anthracene sidewalls, and previous studies have shown that it can be threaded by a PEGylated squaraine dye to give a highly stable complex with nanomolar dissociation constant. 33–35 Detailed thermodynamic and kinetic measurements have identified the structural factors that favor the very strong association and control the rates of macrocycle threading. In short, the two oxygen atoms on the encapsulated squaraine dye form hydrogen bonds to the four macrocycle NH residues and there is coplanar stacking of the squaraine aromatic surfaces with the anthracene sidewalls of the macrocycle.…”

High Affinity Macrocycle Threading by a Near-Infrared Croconaine Dye with Flanking Polymer Chains

“…The kinetic profiles were fitted to the standard second order kinetic equation using Origin Lab ™ 8.6 software. 15 …”

Synthetic mimics of biotin/(strept)avidin

“…46 The empty macrocycle has a highly preorganized structure and the binding cavity is lined with a synergistic mixture of convergent polar hydrogen bonding sites and hydrophobic surfaces. Most biological association systems, including biotin/(strep)avidin, use similar types of amphiphilic binding pockets to recognize complementary guests with high affinity and shape selectivity.…”