Wenqi Liu

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

A macrocyclic tetralactam host is threaded by a highly fluorescent squaraine dye that is flanked by two polyethylene glycol (PEG) chains with nanomolar dissociation constants in water. Furthermore, the rates of bimolecular association are very fast with k(on) ≈ 10(6)-10(7) M(-1) s(-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 those 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, macrocycle threading by a dye conjugate with two appended PEG2000 chains is only three times slower than threading by a conjugate with triethylene glycol chains that are 20 times shorter. The results are a promising advance toward synthetic mimics of streptavidin/biotin.

Sensitive Structural Control of Macrocycle Threading by a Fluorescent Squaraine Dye Flanked by Polymer Chains

A macrocyclic tetralactam is threaded by a complementary squaraine dye that is flanked by two polyethylene glycol chains to produce a pseudorotaxane complex with favorable near-infrared fluorescence properties. The association thermodynamics and kinetics were measured for a homologous series of squaraines with different N-alkyl substituents at both ends of the dye. The results show that subtle changes in substituent steric size have profound effects on threading kinetics without greatly altering the very high association constant.

Fluorescent Neuraminidase Assay Based on Supramolecular Dye Capture After Enzymatic Cleavage

A conceptually new type of enzymatic cleavage assay is reported that utilizes in situ supramolecular capture of the fluorescent product. A squaraine-derived substrate with large blocking groups at each end of its structure cannot be threaded by a tetralactam macrocycle until the blocking groups are removed by enzyme cleavage. A prototype design responds to viral neuraminidase, an indicator of influenza infection, and also measures susceptibility of the sample to neuraminidase inhibitor drugs. The substrate structure incorporates three key features: (a) a bis(4-amino-3-hydroxyphenyl)squaraine core with bright deep-red fluorescence and excellent photostability, (b) an N-methyl group at each end of the squaraine core that ensures fast macrocycle threading kinetics, and (c) sialic acid blocking groups that prevent macrocycle threading until they are removed by viral neuraminidase. The enzyme assay can be conducted in aqueous solution where dramatic colorimetric and fluorescence changes are easily observed by the naked eye. Alternatively, affinity capture beads coated with macrocycle can be used to immobilize the liberated squaraine and enable a range of heterogeneous analysis options. With further optimization, this new type of neuraminidase assay may be useful in a point of care clinic to rapidly diagnose influenza infection and also determine which of the approved antiviral inhibitor drugs is likely to be the most effective treatment for an individual patient. The assay design is generalizable and can be readily modified to monitor virtually any type of enzyme-catalyzed cleavage reaction.

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

Croconaine dyes have narrow and intense absorption bands at ∼800 nm, very weak fluorescence, and high photostabilities, which combine to make them very attractive chromophores for absorption-based imaging or laser heating technologies. The physical supramolecular properties of croconaine dyes have rarely been investigated, especially in water. This study focuses on a molecular threading process that encapsulates a croconaine dye inside a tetralactam macrocycle in organic or aqueous solvent. Macrocycle association and rate constant data are reported for a series of croconaine structures with different substituents attached to the ends of the dye. The association constants were highest in water (Ka ∼ 10(9) M(-1)), and the threading rate constants (kon) increased in the solvent order H2O > MeOH > CHCl3. Systematic variation of croconaine substituents located just outside the croconaine/macrocycle complexation interface hardly changed Ka but had a strong influence on kon. A croconaine dye with N-propyl groups at each end of the structure exhibited a desirable mixture of macrocycle threading properties; that is, there was rapid and quantitative croconaine/macrocycle complexation at relatively high concentrations in water, and no dissociation of the preassembled complex when it was diluted into a solution of fetal bovine serum, even after laser-induced photothermal heating of the solution. The combination of favorable near-infrared absorption properties and tunable mechanical stability makes threaded croconaine/macrocycle complexes very attractive as molecular probes or as supramolecular composites for various applications in absorption-based imaging or photothermal therapy.

Structural Control of Kinetics for Macrocycle Threading by Fluorescent Squaraine Dye in Water

While the general concept of steric speed bumps has been demonstrated in rotaxane shuttles and macrocycle threading systems, the sensitivity of speed bump effects has not been evaluated as a function of structural geometry. Values of Ka and kon for macrocycle threading in water are reported for a series of homologous squaraine dyes with different substituents (speed bumps) on the flanking chains and two macrocycles with different cavity sizes. Sensitivity to a steric speed bump effect was found to depend on (a) structural location, being lowest when the speed bump was near the end of a flanking chain, and (b) macrocycle cavity size, which was enhanced when the cavity was constricted. This new insight is broadly applicable to many types of molecular threading systems.

Guest Back-Folding: A Molecular Design Strategy That Produces a Deep-Red Fluorescent Host/Guest Pair with Picomolar Affinity in Water

One of the major goals of modern supramolecular chemistry, with important practical relevance in many technical fields, is the development of synthetic host/guest partners with ultrahigh affinity and selectivity in water. Currently, most association pairs exhibit micromolar affinity or weaker, and there are very few host/guest systems with Ka > 109 M-1, apparently due to a barrier imposed by enthalpy/entropy compensation. This present study investigated the threading of a water-soluble tetralactam cyclophane by a deep-red fluorescent squaraine guest with flanking polyethylene glycol chains, an association process that is dominated by a highly favorable enthalpic driving force. A squaraine structure was rationally designed to permit guest back-folding as a strategy to greatly expand the hydrophobic surface area that could be buried upon complexation. Guided by computational modeling, an increasing number of N-benzyl groups were appended to the squaraine core, so that, after threading, the aromatic rings could fold back and stack against the cyclophane periphery. The final design iteration exhibited an impressive combination of fluorescence and supramolecular properties, including ratiometric change in deep-red emission, picomolar affinity ( Ka = 5.1 × 1010 M-1), and very rapid threading ( kon = 7.9 × 107 M-1 s-1) in water at 25 °C. Similar excellent behavior was observed in serum solution. A tangible outcome of this study is a new cyclophane/squaraine association pair that will be a versatile platform for many different types of fluorescence-based imaging and diagnostics applications. From a broader perspective, guest back-folding of aromatic groups is a promising new supramolecular stabilization strategy to overcome enthalpy/entropy compensation and produce ultrahigh affinity [2]pseudorotaxane complexes in water and biological media.

Non-Covalent Assembly Method that Simultaneously Endows a Liposome Surface with Targeting Ligands, Protective PEG Chains, and Deep-Red Fluorescence Reporter Groups

A new self-assembly method is used to rapidly functionalize the surface of liposomes without perturbing the membrane integrity or causing leakage of the aqueous contents. The key molecule is a cholesterol-squaraine-PEG conjugate with three important structural elements: a cholesterol membrane anchor, a fluorescent squaraine docking station that allows rapid and high-affinity macrocycle threading, and a long PEG-2000 chain to provide steric shielding of the decorated liposome. The two-step method involves spontaneous insertion of the conjugate into the outer leaflet of pre-formed liposomes followed by squaraine threading with a tetralactam macrocycle that has appended targeting ligands. A macrocycle with six carboxylates permitted immobilization of intact fluorescent liposomes on the surface of cationic polymer beads, whereas a macrocycle with six zinc(II)-dipicolylamine units enabled selective targeting of anionic membranes, including agglutination of bacteria in the presence of human cells.

Macrocyclic Receptor for Precious Gold, Platinum, or Palladium Coordination Complexes

Two macrocyclic tetralactam receptors are shown to selectively encapsulate anionic, square-planar chloride and bromide coordination complexes of gold(III), platinum(II), and palladium(II). Both receptors have a preorganized structure that is complementary to its precious metal guest. The receptors do not directly ligate the guest metal center but instead provide an array of arene π-electron donors that interact with the electropositive metal and hydrogen-bond donors that interact with the outer electronegative ligands. This unique mode of supramolecular recognition is illustrated by six X-ray crystal structures showing receptor encapsulation of AuCl4-, AuBr4-, PtCl4-2, or Pd2Cl6-2. In organic solution, the 1:1 association constants correlate with specific supramolecular features identified in the solid state. Technical applications using these receptors are envisioned in a wide range of fields that involve precious metals, including mining, recycling, catalysis, nanoscience, and medicine.

Fluorescent Thienothiophene-Containing Squaraine Dyes and Threaded Supramolecular Complexes with Tunable Wavelengths between 600–800 nm

A new family of fluorescent thiophene and thienothiophene-containing squaraine dyes is described with tunable wavelengths that cover the absorption/emission range of 600–800 nm. The deep-red and near-infrared fluorescent compounds were easily prepared by simple synthesis and purification methods. Spectral studies showed that each squaraine was rapidly encapsulated by a tetralactam macrocycle, with nanomolar affinity in water, to produce a threaded supramolecular complex with high chemical stability, increased fluorescence quantum yield, and decreased fluorescence quenching upon dye self-aggregation. Energy transfer within the supramolecular complex permitted multiplex emission. That is, two separate dyes with fluorescence emission bands that match the popular Cy5 and Cy7 channels, could be simultaneously excited with a beam of 375 nm light. A broad range of practical applications is envisioned in healthcare diagnostics, microscopy, molecular imaging, and fluorescence-guided surgery.

Non-Covalently Pre-Assembled High-Performance Near-Infrared Fluorescent Molecular Probes for Cancer Imaging

New fluorescent molecular probes, which can selectively target specific cell surface receptors, are needed for microscopy, in vivo imaging, and image guided surgery. The preparation of multivalent probes using standard synthetic chemistry can be a laborious process due to low reaction yields caused by steric effects. In this study, fluorescent molecular probes were prepared by a programmed non-covalent pre-assembly process that used a near-infrared fluorescent squaraine dye to thread a macrocycle bearing a cyclic arginine-glycine-aspartate peptide antagonist (cRGDfK) as a cancer targeting unit. Cell microscopy studies using OVCAR-4 (ovarian cancer) and A549 (lung cancer) cells that express high levels of the integrin αvβ3 or αvβ5 receptors, respectively, revealed a multivalent cell targeting effect. That is, there was comparatively more cell uptake of a pre-assembled probe equipped with two copies of the cRGDfK antagonist than a pre-assembled probe with only one appended cRGDfK antagonist. The remarkably high photostability and low phototoxicity of these near-infrared probes allowed for acquisition of long-term fluorescence movies showing endosome trafficking in living cells. In vivo near-infrared fluorescence imaging experiments compared the biodistribution of a targeted and untargeted probe in a xenograft mouse tumor model. The average tumor-to-muscle ratio for the pre-assembled targeted probe was 3.6 which matches the tumor targeting performance reported for analogous cRGDfK-based probes that were prepared entirely by covalent synthesis. The capability to excite these pre-assembled near-infrared fluorescent probes with blue or deep-red excitation light makes it possible to determine if a target site is located superficially or buried in tissue, a probe performance feature that is likely to be very helpful for eventual applications such as fluorescence guided surgery.