Syntheses of Cyclotriveratrylene Analogues and Their Long Elusive Triketone Congeners

Although interest in cyclotriveratrylene and its analogues has been significant, limitations in the ability to adjust its structure fully have hampered studies into their complete range of properties. A unique strategy to synthesize a previously unobtainable cyclotriveratrylene analogue and a procedure which adjusts the inner methylene bridges of that material to a triketone is reported. A second triketone synthesis and computational studies indicate the parameters needed for success

Photoactivated Bioconjugation Between <i>ortho</i>-Azidophenols and Anilines: A Facile Approach to Biomolecular Photopatterning

Methods for the surface patterning of small molecules and biomolecules can yield useful platforms for drug screening, synthetic biology applications, diagnostics, and the immobilization of live cells. However, new techniques are needed to achieve the ease, feature sizes, reliability, and patterning speed necessary for widespread adoption. Herein, we report an easily accessible and operationally simple photoinitiated reaction that can achieve patterned bioconjugation in a highly chemoselective manner. The reaction involves the photolysis of 2-azidophenols to generate iminoquinone intermediates that couple rapidly to aniline groups. We demonstrate the broad functional group compatibility of this reaction for the modification of proteins, polymers, oligonucleotides, peptides, and small molecules. As a specific application, the reaction was adapted for the photolithographic patterning of azidophenol DNA on aniline glass substrates. The presence of the DNA was confirmed by the ability of the surface to capture living cells bearing the sequence complement on their cell walls or cytoplasmic membranes. Compared to other light-based DNA patterning methods, this reaction offers higher speed and does not require the use of a photoresist or other blocking material

<i>ortho</i>-Methoxyphenols as Convenient Oxidative Bioconjugation Reagents with Application to Site-Selective Heterobifunctional Cross-Linkers

The synthesis of complex protein-based bioconjugates has been facilitated greatly by recent developments in chemoselective methods for biomolecular modification. The oxidative coupling of <i>o</i>-aminophenols or catechols with aniline functional groups is chemoselective, mild, and rapid; however, the oxidatively sensitive nature of the electron-rich aromatics and the paucity of commercial sources pose some obstacles to the general use of these reactive strategies. Herein, we identify <i>o</i>-methoxyphenols as air-stable, commercially available derivatives that undergo efficient oxidative couplings with anilines in the presence of periodate as oxidant. Mechanistic considerations informed the development of a preoxidation protocol that can greatly reduce the amount of periodate necessary for effective coupling. The stability and versatility of these reagents was demonstrated through the synthesis of complex protein–protein bioconjugates using a site-selective heterobifunctional cross-linker comprising both <i>o</i>-methoxyphenol and 2-pyridinecarboxaldehyde moieties. This compound was used to link epidermal growth factor to genome-free MS2 viral capsids, affording nanoscale delivery vectors that can target a variety of cancer cell types

Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting

The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody–capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics

Biodistribution of Antibody-MS2 Viral Capsid Conjugates in Breast Cancer Models

A variety of nanoscale scaffolds, including virus-like particles (VLPs), are being developed for biomedical applications; however, little information is available about their <i>in vivo</i> behavior. Targeted nanoparticles are particularly valuable as diagnostic and therapeutic carriers because they can increase the signal-to-background ratio of imaging agents, improve the efficacy of drugs, and reduce adverse effects by concentrating the therapeutic molecule in the region of interest. The genome-free capsid of bacteriophage MS2 has several features that make it well-suited for use in delivery applications, such as facile production and modification, the ability to display multiple copies of targeting ligands, and the capacity to deliver large payloads. Anti-EGFR antibodies were conjugated to MS2 capsids to construct nanoparticles targeted toward receptors overexpressed on breast cancer cells. The MS2 agents showed good stability in physiological conditions up to 2 days and specific binding to the targeted receptors in <i>in vitro</i> experiments. Capsids radiolabeled with ⁶⁴Cu isotopes were injected into mice possessing tumor xenografts, and both positron emission tomography–computed tomography (PET/CT) and scintillation counting of the organs <i>ex vivo</i> were used to determine the localization of the agents. The capsids exhibit surprisingly long circulation times (10–15% ID/g in blood at 24 h) and moderate tumor uptake (2–5% ID/g). However, the targeting antibodies did not lead to increased uptake <i>in vivo</i> despite <i>in vitro</i> enhancements, suggesting that extravasation is a limiting factor for delivery to tumors by these particles