Dynamic Approach to Synthetic Lectin for Glucose with Boosted Binding Affinity through C-H Hydrogen Bonds.

A biomimetic receptor for glucose has been developed with high affinity and selectivity. The receptor was efficiently synthesized in three steps through dynamic imine chemistry followed by imine-to-amide oxidation. The receptor features two parallel durene panels, forming a hydrophobic pocket for [CH⋅⋅⋅π] interactions, and two pyridinium residues directing four amide bonds towards the pocket. These pyridinium residues not only improve solubility but also provide polarized C-H bonds for hydrogen bonding. Experimental data and DFT calculations show that these polarized C-H bonds significantly enhance substrate binding. These findings demonstrate the power of dynamic covalent chemistry for creating molecular receptors and using polarized C-H bonds for boosted carbohydrate recognition in water, providing a foundation for developing glucose-responsive materials and sensors.

Single-molecule characterization of a bright and photostable deep-red fluorescent squaraine-figure-eight (SF8) dye.

Squaraine Figure Eight (SF8) dyes are a unique class of deep-red fluorescent dyes with self-threaded molecular architecture that provides structural rigidity while simultaneously encapsulating and protecting the emissive fluorochrome. Previous cell microscopy and bulk phase studies of SF8 dyes indicated order of magnitude enhancements in photostability over conventional pentamethine cyanine dyes such as Cy5. Studies conducted at the single molecule level now reveal that these ensemble level enhancements carry over to the single molecule level in terms of enhanced emission quantum yields, longer times to photobleaching, and enhanced total photon yields. When compared to Cy5, the SF8-based dye SF8(D4)2 possesses a three-fold larger single molecule emission quantum yield, exhibits order of magnitude longer average times before photobleaching, and exhibits twenty times larger photon yields. Additional features such as water solubility, fluorochrome encapsulation to protect it against nucleophilic attack, and selective biomarker targeting capability make SF8-based dyes promising candidates for biological labeling and microscopy applications and single molecule tracking.

Synthesis and Immunological Evaluation of Pentamannose-Based HIV-1 Vaccine Candidates.

Dense glycosylation and the trimeric conformation of the human immunodeficiency virus-1 (HIV-1) envelope protein limit the accessibility of some cellular glycan processing enzymes and end up with high-mannose-type N-linked glycans on the envelope spike, among which the Man5GlcNAc2 structure occupies a certain proportion. The Man5GlcNAc2 glycan composes the binding sites of some potent broadly neutralizing antibodies, and some lectins that can bind Man5GlcNAc2 show HIV-neutralizing activity. Therefore, Man5GlcNAc2 is a potential target for HIV-1 vaccine development. Herein, a highly convergent and effective strategy was developed for the synthesis of Man5 and its monofluoro-modified, trifluoro-modified, and S-linked analogues. We coupled these haptens to carrier protein CRM197 and evaluated the immunogenicity of the glycoconjugates in mice. The serological assays showed that the native Man5 conjugates failed to induce Man5-specific antibodies in vivo, while the modified analogue conjugates induced stronger antibody responses. However, these antibodies could not bind the native gp120 antigen. These results demonstrated that the immune tolerance mechanism suppressed the immune responses to Man5-related structures and the conformation of glycan epitopes on the synthesized glycoconjugates was distinct from that of native glycan epitopes on gp120.

New Synthetic Route to Water-Soluble Prism[5]arene Hosts and Their Molecular Recognition Properties.

We report that the direct macrocyclization of naphthalene monomers bearing ethyl ester functional groups delivers prism[5]arene derivatives, which can be deprotected to yield water-soluble prism[5]arenes (H1 and H3). 1 H NMR spectroscopy showed that dicationic guests bind with the hydrophobic cores buried inside the anisotropic magnetically shielding cavity. Isothermal titration calorimetry measurements showed that H1 and H3 are high-affinity hosts in PBS-buffered water with Ka values exceeding 109  M-1 for a select guest. The complexation events are driven by the non-classical hydrophobic effect, CH⋅⋅⋅π interactions, and electrostatic interactions. Host H1 displays somewhat higher affinity toward a common guest than pillar[6]arene bearing carboxylic acid functional groups but is significantly less potent than pillar[6]arene bearing sulfate groups. H1 and H3 should be considered alongside other high affinity hosts for a variety of chemical and biological applications.

Chiral figure-eight molecular scaffold for fluorescent probe development.

Targeted fluorescent molecular probes are useful for cell microscopy, diagnostics, and biological imaging. An emerging discovery paradigm is to screen libraries of fluorescent molecules and identify hit compounds with interesting targeting properties. However, a current limitation with this approach is the lack of fluorescent molecular scaffolds that can produce libraries of probe candidates with three dimensional globular shape, chiral centers, and constrained conformation. This study evaluated a new probe scaffold called squaraine figure-eight (SF8), a self-threaded molecular architecture that is comprised of an encapsulated deep-red fluorescent squaraine dye, surrounding tetralactam macrocycle, and peripheral loops. Easy synthetic variation of the loops produced four chiral isomeric SF8 probes, with the same log P values. Cell microscopy showed that subtle changes in the loop structure led to significant differences in intracellular targeting. Most notably, a comparison of enantiomeric probes revealed a large difference in mitochondrial accumulation, very likely due to differences in affinity for a chiral biomarker within the organelle. A tangible outcome of the research is a probe candidate that can be: (a) developed further as a bright and photostable, deep-red fluorescent probe for mitochondrial imaging, and (b) used as a molecular tool to identify the mitochondrial biomarker for selective targeting. It will be straightforward to expand the SF8 probe chemical space and produce structurally diverse probe libraries with high potential for selective targeting of a wide range of biomarkers.

Paired Agent Fluorescence Imaging of Cancer in a Living Mouse Using Preassembled Squaraine Molecular Probes with Emission Wavelengths of 690 and 830 nm.

New methods are described for the construction of targeted fluorescence probes for imaging cancer and the assessment of tumor targeting performance in a living mouse model. A novel noncovalent assembly process was used to fabricate a set of structurally related targeted fluorescent probes with modular differences in three critical assembly components: the emission wavelength of the squaraine fluorochrome, the number of cRGDfK peptide units that target the cancer cells, and the length of the polyethylene glycol chains as pharmacokinetic controllers. Selective targeting of cancer cells was proven by a series of cell microscopy experiments followed by in vivo imaging of subcutaneous tumors in living mice. The mouse imaging studies included a mock surgery that completely removed a fluorescently labeled tumor. Enhanced tumor accumulation due to probe targeting was first evaluated by conducting Single Agent Imaging (SAI) experiments that compared tumor imaging performance of a targeted probe and untargeted probe in separate mouse cohorts. Although there was imaging evidence for enhanced tumor accumulation of the targeted probe, there was moderate scatter in the data due to tumor-to-tumor variability of the vasculature structure and interstitial pressure. A subsequent Paired Agent Imaging (PAI) study coinjected a binary mixture of targeted probe (with emission at 690 nm) and untargeted probe (with emission at 830 nm) into the same tumor-burdened animal. The conclusion of the PAI experiment also indicated enhanced tumor accumulation of the targeted probe, but the statistical significance was much higher, even though the experiment required a much smaller cohort of mice. The imaging data from the PAI experiment was analyzed to determine the targeted probe's Binding Potential (BP) for available integrin receptors within the tumor tissue. In addition, pixelated maps of BP within each tumor indicated a heterogeneous spatial distribution of BP values. The results of this study show that the combination of fluorescent probe preassembly and PAI is a promising new way to rapidly develop targeted fluorescent probes for tumors with high BP and eventual use in clinical applications such as targeted therapy, image guided surgery, and personalized medicine.

Fluorescent Self-Threaded Peptide Probes for Biological Imaging.

A general synthetic method creates a new class of covalently connected, self-threaded, fluorescent molecular probes with figure-eight topology, an encapsulated deep-red fluorophore, and two peripheral peptide loops. The globular molecular shape and rigidified peptide loops enhance imaging performance by promoting water solubility, eliminating probe self-aggregation, and increasing probe stability. Moreover, the peptide loops determine the affinity and selectivity for targets within complex biological samples such as cell culture, tissue histology slices, or living subjects. For example, a probe with cell-penetrating peptide loops targets the surface of cell plasma membranes, whereas, a probe with bone-targeting peptide loops selectively stains the skeleton within a living mouse. The unique combination of bright deep-red fluorescence, high stability, and predictable peptide-based targeting is ideal for photon intense fluorescence microscopy and biological imaging.

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 αvß3 or αvß5 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.

Charge-assisted hydrogen bonding in a bicyclic amide cage: an effective approach to anion recognition and catalysis in water.

Hydrogen bonding is prevalent in biological systems, dictating a myriad of life-sustaining functions in aqueous environments. Leveraging hydrogen bonding for molecular recognition in water encounters significant challenges in synthetic receptors on account of the hydration of their functional groups. Herein, we introduce a water-soluble hydrogen bonding cage, synthesized via a dynamic approach, exhibiting remarkable affinities and selectivities for strongly hydrated anions, including sulfate and oxalate, in water. We illustrate the use of charge-assisted hydrogen bonding in amide-type synthetic receptors, offering a general molecular design principle that applies to a wide range of amide receptors for molecular recognition in water. This strategy not only revalidates the functions of hydrogen bonding but also facilitates the effective recognition of hydrophilic anions in water. We further demonstrate an unconventional catalytic mechanism through the encapsulation of the anionic oxalate substrate by the cationic cage, which effectively inverts the charges associated with the substrate and overcomes electrostatic repulsions to facilitate its oxidation by the anionic MnO4 -. Technical applications using this receptor are envisioned across various technical applications, including anion sensing, separation, catalysis, medical interventions, and molecular nanotechnology.

Pillar[6]MaxQ: A Potent Supramolecular Host for In Vivo Sequestration of Methamphetamine and Fentanyl.

Pillar[6]MaxQ (P6AS) functions as an in vivo sequestration agent for methamphetamine and fentanyl. We use 1H NMR, isothermal titration calorimetry, and molecular modelling to deduce the geometry and strength of the P6AS•drug complexes. P6AS forms tight complexes with fentanyl (Kd=9.8 nM), PCP (17.1 nM), MDMA (25.5 nM), mephedrone (52.4 nM), and methamphetamine (101 nM). P6AS has good in vitro biocompatibility according to MTS metabolic, Adenylate Kinase cell death, and hERG ion channel inhibition assays, and the Ames fluctuation test. The no observed adverse effect level for P6AS is 45 mg/kg. The hyperlocomotion of mice treated with methamphetamine (0.5 mg/kg) can be ameliorated by treatment with P6AS (35.7 mg/kg) 5-minutes later, whereas the hyperlocomotion of mice treated with fentanyl (0.1 mg/kg) can be controlled by treatment with P6AS (5 mg/kg) up to 15-minutes later. P6AS has significant potential for development as a broad spectrum in vivo sequestration agent.

Structural Engineering of Fluorescent Self-Threaded Peptide Probes for Targeted Cell Imaging.

Squaraine figure-eight (SF8) molecules are a new class of deep-red fluorescent probes that are well suited for fluorescence cell microscopy due to their very high fluorescence brightness and excellent stability. Three homologous SF8 probes, with peptidyl loops that differ by very minor changes in the peptide sequence, were synthesized and assessed for probe uptake by cancer cells. One of probes included the RGD motif that is recognized by many classes of integrin receptors that reside on the surface of the cancer cells, and it permeated the cells by receptor-mediated endocytosis. In contrast, cell microscopy showed that there was negligible cell uptake of the two homologous SF8 probes indicating differences in probe targeting capability. The synthetic method allows for easy alteration of the peptide sequence; thus, it is straightforward to develop new classes of peptidyl SF8 probes with loop sequences that target other cancer biomarkers.

Silica nanoparticle remodeling under mild conditions: versatile one step conversion of mesoporous to hollow nanoparticles with simultaneous payload loading.

A binary mixture of mesoporous silica nanoparticles plus organic polyammonium additive (dye or drug) is cleanly converted upon mild heating into hollow nanoparticles. The remodeled nanoparticle shell is an organized nanoscale assembly of globular additive/silica subunits and cancer cell assays show that a loaded drug additive is bioavailable.

Anthracene-Walled Acyclic CB[n] Receptors: in†vitro and in†vivo Binding Properties toward Drugs of Abuse.

We report studies of the interaction of six acyclic CB[n]-type receptors toward a panel of drugs of abuse by a combination of isothermal titration calorimetry and 1 H NMR spectroscopy. Anthracene walled acyclic CB[n] host (M3) displays highest binding affinity toward methamphetamine (Kd =15 nM) and fentanyl (Kd =4 nM). Host M3 is well tolerated by Hep G2 and HEK 293 cells up to 100 µM according to MTS metabolic and adenylate kinase release assays. An in vivo maximum tolerated dose study with Swiss Webster mice showed no adverse effects at the highest dose studied (44.7 mg kg-1 ). Host M3 is not mutagenic based on the Ames fluctuation test and does not inhibit the hERG ion channel. In vivo efficacy studies showed that pretreatment of mice with M3 significantly reduces the hyperlocomotion after treatment with methamphetamine, but M3 does not function similarly when administered 30 seconds after methamphetamine.

Synthesis and immunological evaluation of N-acyl modified Globo H derivatives as anticancer vaccine candidates.

Globo H is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target for antitumor vaccine or cancer immunotherapies. However, most TACAs are T-cell-independent, and they cannot induce powerful immune response due to their poor immunogenicity. To address this problem, herein, several Globo H analogues with modification on the N-acyl group were prepared through a preactivation-based glycosylation strategy from the non-reducing end to the reducing end. These modified Globo H derivatives were then conjugated with carrier protein CRM197 to form glycoconjugates as anticancer vaccine candidates, which were used in combination with adjuvant glycolipid C34 for immunological studies. The immunological effects of these synthetic vaccine candidates were evaluated on Balb/c mice. The results showed that the fluorine-modified N-acyl Globo H conjugates can induce higher titers of IgG antibodies that can recognize the naturally occurring Globo H antigen on the surface of cancer cells and can eliminate cancer cells in the presence of a complement, indicating the potential of these synthetic glycoconjugates as anticancer vaccine candidates.

High affinity threading of a new tetralactam macrocycle in water by fluorescent deep-red and near-infrared squaraine dyes.

A new tetralactam macrocycle was prepared and found to encapsulate deep-red and near-infrared squaraine and croconaine dyes in water with tunable threading kinetics. The new supramolecular paradigm of guest back-folding was used to increase macrocycle/squaraine affinity by 370-fold and achieve an association constant of 2.8 × 109 M-1.

Altering the Specificity of the Antibody Response to HIV gp120 with a Glycoconjugate Antigen.

Some conserved glycans on the HIV envelope protein are targets of broadly neutralizing antibodies (bnAbs) of HIV. BnAbs provide a precise definition of broadly neutralizing epitopes on the envelope protein of HIV. These epitopes are promising for vaccine design. Many glycan-related antigens with high affinity to bnAbs have been tested as immunogens in vivo. However, it was found that no bnAb-like antibodies were induced. Vaccination with different immunogens containing the same neutralizing epitope may enhance the affinity maturation of antibodies which focus on the shared epitope. This combined immunization strategy showed great potential in peptide epitope-based vaccine design. However, it has not yet been explored on glycan-related epitopes to date. Herein, we take 2G12 as a model to validate this strategy on glycan-related epitopes. A high-affinity antigen of 2G12 was constructed by conjugating the D1 arm tetramannoside to bovine serum albumin. Then, the glycoconjugate was coimmunized with a recombinant gp120, which was expected to selectively benefit the induction of antibodies recognizing the neutralizing epitope of 2G12 on gp120. Mice were inoculated with the two antigens simultaneously or alternately to determine the suitable regimen for this strategy. The serological assays demonstrated that the antibody titers and subtypes responded to the whole gp120 were not improved, and the proportion of antibodies competitively bound to the 2G12 epitope was not enhanced significantly either. However, the coimmunized glycoconjugate selectively raised the proportion of antibodies recognizing D1 arm tetramannoside-related structures on gp120. These results provide important experience for the design of glycan-dependent bnAb-based vaccines.