Allie C. Obermeyer

Oxidative Modification of Native Protein Residues Using Cerium(IV) Ammonium Nitrate

A new protein modification strategy has been developed that is based on an oxidative coupling reaction that targets electron-rich amino acids. This strategy relies on cerium(IV) ammonium nitrate (CAN) as an oxidation reagent and results in the coupling of tyrosine and tryptophan residues to phenylene diamine and anisidine derivatives. The methodology was first identified and characterized on peptides and small molecules, and was subsequently adapted for protein modification by determining appropriate buffer conditions. Using the optimized procedure, native and introduced solvent-accessible residues on proteins were selectively modified with polyethylene glycol (PEG) and small peptides. This unprecedented bioconjugation strategy targets these under-utilized amino acids with excellent chemoselectivity and affords good-to-high yields using low concentrations of the oxidant and coupling partners, short reaction times, and mild conditions.

Rapid Chemoselective Bioconjugation through Oxidative Coupling of Anilines and Aminophenols

A highly efficient protein bioconjugation method is described involving addition of anilines to o-aminophenols in the presence of sodium periodate. The reaction takes place in aqueous buffer at pH 6.5 and can reach high conversion in 2-5 min. The major product was characterized using X-ray crystallography, which revealed that an unprecedented oxidative ring contraction occurs after the coupling step. The compatibility of the reaction with protein substrates has been demonstrated through attachment of small molecules, polymer chains, and peptides to p-aminophenylalanine residues introduced into viral capsids through amber stop codon suppression. Coupling of anilines to o-aminophenol groups derived from tyrosine residues is also described. The compatibility of this method with thiol modification chemistry is shown through attachment of a near-IR fluorescent chromophore to cysteine residues inside the viral capsid shells, followed by attachment of integrin-targeting RGD peptides to anilines on the exterior surface.

N-terminal modification of proteins with o-aminophenols.

The synthetic modification of proteins plays an important role in chemical biology and biomaterials science. These fields provide a constant need for chemical tools that can introduce new functionality in specific locations on protein surfaces. In this work, an oxidative strategy is demonstrated for the efficient modification of N-terminal residues on peptides and N-terminal proline residues on proteins. The strategy uses o-aminophenols or o-catechols that are oxidized to active coupling species in situ using potassium ferricyanide. Peptide screening results have revealed that many N-terminal amino acids can participate in this reaction, and that proline residues are particularly reactive. When applied to protein substrates, the reaction shows a stronger requirement for the proline group. Key advantages of the reaction include its fast second-order kinetics and ability to achieve site-selective modification in a single step using low concentrations of reagent. Although free cysteines are also modified by the coupling reaction, they can be protected through disulfide formation and then liberated after N-terminal coupling is complete. This allows access to doubly functionalized bioconjugates that can be difficult to access using other methods.

Mild bioconjugation through the oxidative coupling of ortho-aminophenols and anilines with ferricyanide.

Using a small-molecule-based screen, ferricyanide was identified as a mild and efficient oxidant for the coupling of anilines and o-aminophenols on protein substrates. This reaction is compatible with thiols and 1,2-diols, allowing its use in the creation of complex bioconjugates for use in biotechnology and materials applications.

Multivalent viral capsids with internal cargo for fibrin imaging.

Thrombosis is the cause of many cardiovascular syndromes and is a significant contributor to life-threatening diseases, such as myocardial infarction and stroke. Thrombus targeted imaging agents have the capability to provide molecular information about pathological clots, potentially improving detection, risk stratification, and therapy of thrombosis-related diseases. Nanocarriers are a promising platform for the development of molecular imaging agents as they can be modified to have external targeting ligands and internal functional cargo. In this work, we report the synthesis and use of chemically functionalized bacteriophage MS2 capsids as biomolecule-based nanoparticles for fibrin imaging. The capsids were modified using an oxidative coupling reaction, conjugating ∼90 copies of a fibrin targeting peptide to the exterior of each protein shell. The ability of the multivalent, targeted capsids to bind fibrin was first demonstrated by determining the impact on thrombin-mediated clot formation. The modified capsids out-performed the free peptides and were shown to inhibit clot formation at effective concentrations over ten-fold lower than the monomeric peptide alone. The installation of near-infrared fluorophores on the interior surface of the capsids enabled optical detection of binding to fibrin clots. The targeted capsids bound to fibrin, exhibiting higher signal-to-background than control, non-targeted MS2-based nanoagents. The in vitro assessment of the capsids suggests that fibrin-targeted MS2 capsids could be used as delivery agents to thrombi for diagnostic or therapeutic applications.

Photoactivated Bioconjugation Between ortho-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.

Bioconjugation of gold nanoparticles through the oxidative coupling of ortho-aminophenols and anilines.

While there are a number of methods for attaching gold nanoparticles (AuNPs) to biomolecules, the existing strategies suffer from nonspecific AuNP adsorption, reagents that are unstable in aqueous solutions, and/or long reaction times. To improve upon existing AuNP bioconjugation strategies, we have adapted a recently reported potassium ferricyanide-mediated oxidative coupling reaction for the attachment of aniline-functionalized AuNPs to o-aminophenol-containing oligonucleotides, peptides, and proteins. The aniline-AuNPs are stable in aqueous solutions, show little-to-no nonspecific adsorption with biomolecules, and react rapidly (30 min) with o-aminophenols under mild conditions (pH 6.5, 1 mM oxidant).

Three‐Dimensional Ordered Antibody Arrays Through Self‐Assembly of Antibody–Polymer Conjugates

Three-dimensional (3D) ordered arrays of human immunoglobulin G (IgG) were fabricated using well-defined full-length antibody-polymer conjugates (APCs). The conjugates were prepared through a two-step sequential click approach with a combination of oxime ligation and strain promoted alkyne-azide cycloaddition. They were able to self-assemble into lamellar nanostructures with alternating IgG and poly(N-isopropylacrylamide) (PNIPAM) nanodomains. As a proof-of-concept, these materials were fabricated into thin films and their specific binding ability was tested. The nanostructure not only improves the packing density and the proper orientation of the IgG, but also provides nanochannels to facilitate substrate transport.

Phase Separation Behavior of Supercharged Proteins and Polyelectrolytes

Membraneless organelles, like membrane-bound organelles, are essential to cell homeostasis and provide discrete cellular subcompartments. Unlike classical organelles, membraneless organelles possess no physical barrier but rather arise by phase separation of the organelle components from the surrounding cytoplasm or nucleoplasm. Complex coacervation, the liquid-liquid phase separation of oppositely charged polyelectrolytes, is one of several phenomena that are hypothesized to drive the formation and regulation of some membraneless organelles. Studies of the molecular properties of globular proteins that drive complex coacervation are limited as many proteins do not form complexes with oppositely charged macromolecules at neutral pH and moderate ionic strengths. Protein supercharging overcomes this problem and drives complexation with oppositely charged macromolecules. In this work, several distinct cationic supercharged green fluorescent protein (GFP) variants were designed to examine the phase behavior with oppositely charged polyanionic macromolecules. Cationic GFP variants phase separated with oppositely charged macromolecules at various mixing ratios, salt concentrations, and pH values. Efficient protein incorporation in the macromolecule rich phase occurred over a range of protein and polymer mass fractions, but the protein encapsulation efficiency was highest at the midpoint of the phase separation regime. More positively charged proteins phase separated over broader pH and salt ranges than those of proteins with a lower charge density. Interestingly, each GFP variant phase separated at higher salt concentrations with anionic synthetic macromolecules compared to anionic biological macromolecules. Optical microscopy revealed that most variants, depending on solution conditions, formed liquid-liquid phase separations, except for GFP/DNA pairs that formed solid aggregates under all tested conditions.