Development of Multivalent Conjugates with a Single Non-Canonical Amino Acid.

Proteins represent powerful biomacromolecules due to their unique functionality and broad utility both in the cell and in non-biological applications. The genetic encoding of non-canonical amino acids (ncAAs) facilitates functional diversification of these already powerful proteins. Specifically, ncAAs have been demonstrated to provide unique functional handles to bioorthogonally introduce novel functionality via conjugation reactions. Herein we examine the ability of a single ncAA to serve as a handle to generate multivalent bioconjugates to introduce two or more additional components to a protein, yielding a multivalent conjugate. To accomplish this aim, p-bromopropargyloxyphenyalanine (pBrPrF) was genetically encoded into both superfolder green fluorescent protein (sfGFP) and ubiquitin model proteins to serve as a conjugation handle. A sequential bioconjugation sequence involving a copper-assisted cycloaddition reaction coupled with a subsequent Sonogashira cross-coupling was then optimized. The linkage of two additional molecules to the model protein via these reactions yielded the desired multivalent bioconjugate. This domino approach using a single ncAA has a plethora of applications in both therapeutics and diagnostics as multiple unique moieties can be introduced into proteins in a highly controlled fashion.

Employing non-canonical amino acids towards the immobilization of a hyperthermophilic enzyme to increase protein stability.

A carboxylesterase derived from Sulfolobus solfataricus P1 was immobilized onto an epoxy-activated Sepharose resin via non-canonical amino acids. The immobilized enzyme exhibited heightened performance in organic solvents, recyclability, and stability at room temperature for over two years. The incorporation of a non-canonical amino acid afforded a high degree of control over the bioorthogonal immobilization reaction. These results indicate that the specificity conferred by genetic code expansion produces advantages in protein immobilization and broadens the utility of such proteins to non-biological settings.

Protein Modification Employing Non-Canonical Amino Acids to Prepare SUMOylation Detecting Bioconjugates.

Protein modification with non-canonical amino acids (ncAAs) represents a useful technology to afford homogenous samples of bioconjugates with site-specific modification. This technique can be directly applied to the detection of aberrant SUMOylation patterns, which are often indicative of disease states. Modified SUMO-trapping proteins, consisting of a catalytically inactive ULP1 fragment (UTAG) fused to the maltose-binding protein MBP, are useful reagents for the binding and labeling of SUMOylated proteins. Mutation of this UTAG fusion protein to facilitate amber suppression technologies for the genetic incorporation of ncAAs was assessed to provide a functional handle for modification. Ultimately, two sites in the maltose-binding protein (MBP) fusion were identified as ideal for incorporation and bioconjugation without perturbation to the SUMO-trapping ability of the UTAG protein. This functionality was then employed to label SUMOylated proteins in HeLa cells and demonstrate their enrichment in the nucleus. This modified UTAG-MBP-ncAA protein has far-reaching applications for both diagnostics and therapeutics.

Effects of Structural Variations on Antibacterial Properties for Conjugated Diynes Generated through Glaser Hay Couplings.

Antibiotic resistance is a growing problem facing global societies today. Many new antibiotics are derivatized versions of already existing antibiotics, which allows for antibiotic resistance to arise. To combat this issue, new antibiotics with different core structures need to be elucidated. Asymmetrical polyacetylenes have been isolated from natural products and they have previously been demonstrated to exhibit antimicrobial and antibacterial activity; however, their synthetic preparation has not made them easily amenable to rapid derivatization for SAR studies. Using a combination of solution and solid-supported chemistries, an array of diynes inspired by a known natural product were prepared and assessed for antibacterial activity. Ultimately, several compounds were identified with improved activity in bacterial viability assays. Moreover, some compounds were discovered that displayed a degree of specificity for E. coli over P. fluorescens and vice versa. These new compounds show promise, and further investigation is needed to pinpoint the specific structural components that elicit biological activity.

Photoregulation of PRMT-1 Using a Photolabile Non-Canonical Amino Acid.

Protein methyltransferases are vital to the epigenetic modification of gene expression. Thus, obtaining a better understanding of and control over the regulation of these crucial proteins has significant implications for the study and treatment of numerous diseases. One ideal mechanism of protein regulation is the specific installation of a photolabile-protecting group through the use of photocaged non-canonical amino acids. Consequently, PRMT1 was caged at a key tyrosine residue with a nitrobenzyl-protected Schultz amino acid to modulate protein function. Subsequent irradiation with UV light removes the caging group and restores normal methyltransferase activity, facilitating the spatial and temporal control of PRMT1 activity. Ultimately, this caged PRMT1 affords the ability to better understand the protein's mechanism of action and potentially regulate the epigenetic impacts of this vital protein.

Genetic Encoding of a Bioconjugation Handle for [2+2+2] Cycloaddition Reactions.

Protein bioconjugates have many critical applications, especially in the development of therapeutics. Consequently, the design of novel methodologies to prepare protein bioconjugates is of great importance. Herein we present the development and optimization of a novel strategy to prepare bioconjugates through a genetically encoded [2+2+2] cycloaddition reaction. To do this, a novel unnatural amino acid (UAA) containing a dipropargyl amine functionality was synthesized and incorporated site specifically. This UAA-containing protein was reacted with an alkyne-containing fluorophore to afford a covalently linked, well-defined protein bioconjugate. This reaction is convenient with an optimized reaction time of just two hours at room temperature and yields a stable, polysubstituted benzene ring. Overall, this work contributes a new bioconjugation strategy to the growing toolbox of reactions to develop protein bioconjugates, which have a myriad of applications.

Mechanistic investigation and further optimization of the aqueous Glaser-Hay bioconjugation.

The Glaser-Hay bioconjugation has recently emerged as an efficient and attractive method to generate stable, useful bioconjugates with numerous applications, specifically in the field of therapeutics. Herein, we investigate the mechanism of the aqueous Glaser-Hay coupling to better understand optimization strategies. In doing so, it was identified that catalase is able to minimize protein oxidation and improve coupling efficiency, suggesting that hydrogen peroxide is produced during the aqueous Glaser-Hay bioconjugation. Further, several new ligands were investigated to minimize protein oxidation and maximize coupling efficiency. Finally, two novel strategies to streamline the Glaser-Hay bioconjugation and eliminate the need for secondary purification have been developed.

Playing with the Molecules of Life.

Our understanding of the complex molecular processes of living organisms at the molecular level is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of macromolecules, is allowing chemists to to harness and reprogram the cellular machinery in ways previously unimaged. Here we review one example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins with unprecedented precision.

Investigation of copper-free alkyne/azide 1,3-dipolar cycloadditions using microwave irradiation.

The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and chemistry highlights the utility of these reactions. However, the use of a copper catalyst can be prohibitive to some applications. Consequently, we have optimized a copper-free microwave-assisted reaction to alleviate the necessity for the copper catalyst. A small array of triazoles was prepared to examine the scope of this approach, and the methodology was translated to a protein context through the use of unnatural amino acids to demonstrate one of the first microwave-mediated bioconjugations involving a full length protein.

Development of optimized conditions for Glaser-Hay bioconjugations.

The efficient preparation of protein bioconjugates represents a route to novel materials, diagnostics, and therapeutics. We previously reported a novel bioorthogonal Glaser-Hay reaction for the preparation of covalent linkages between proteins and a reaction partner; however, deleterious protein degradation was observed under extended reaction conditions. Herein, we describe the systematic optimization of the reaction to increase coupling efficiency and decrease protein degradation. Two optimized conditions were identified varying either the pH of the reaction or the bidentate ligand employed, allowing for more rapid conjugations and/or less protein oxidation.

Fluorescence Modulation of Green Fluorescent Protein Using Fluorinated Unnatural Amino Acids.

The ability to modulate protein function through minimal perturbations to amino acid structure represents an ideal mechanism to engineer optimized proteins. Due to the novel spectroscopic properties of green fluorescent protein, it has found widespread application as a reporter protein throughout the fields of biology and chemistry. Using site-specific amino acid mutagenesis, we have incorporated various fluorotyrosine residues directly into the fluorophore of the protein, altering the fluorescence and shifting the pKa of the phenolic proton associated with the fluorophore. Relative to wild type GFP, the fluorescence spectrum of the protein is altered with each additional fluorine atom, and the mutant GFPs have the potential to be employed as pH sensors due to the altered electronic properties of the fluorine atoms.

Utilization of alkyne bioconjugations to modulate protein function.

The ability to introduce or modify protein function has widespread application to multiple scientific disciplines. The introduction of unique unnatural amino acids represents an excellent mechanism to incorporate new functionality; however, this approach is limited by ability of the translational machinery to recognize and incorporate the chemical moiety. To overcome this potential limitation, we aimed to exploit the functionality of existing unnatural amino acids to perform bioorthogonal reactions to introduce the desired protein modification, altering its function. Specifically, via the introduction of a terminal alkyne containing unnatural amino acid, we demonstrated chemically programmable protein modification through the Glaser-Hay coupling to other terminal alkynes, altering the function of a protein. In a proof-of-concept experiment, this approach has been utilized to modify the fluorescence spectrum of green fluorescent protein.

Application of the Solid-Supported Glaser-Hay Reaction to Natural Product Synthesis.

The Glaser-Hay coupling of terminal alkynes is a useful synthetic reaction for the preparation of polyynes; however, chemoselectivity issues have precluded its widespread utilization. Conducting the reaction on a solid-support provides a mechanism to alleviate the chemoselectivity issues and provide products in high purities and yields. Moreover, the polyyne core is a key component to several natural products. Herein, we describe the application of a solid-supported Glaser-Hay reaction in the preparation of several natural products. These compounds were then screened for antibacterial activity, illustrating the utility of the methodology.

Synthesis and incorporation of a caged tyrosine amino acid possessing a bioorthogonal handle.

Reversing a bioconjugation in a spatial and temporal fashion has widespread applications, especially toward targeted drug delivery. We report the synthesis and incorporation of an unnatural amino acid with an alkyne modified dimethoxy-ortho-nitrobenzyl caging group. This unnatural amino acid can be utilized in a Glaser-Hay conjugation to generate a bioconjugate, but also is able to disrupt the bioconjugate when irradiated with light. These combined features allow for the preparation of bioconjugates with a high degree of site-specificity and allow for the separation of the two components if necessary.

Expanding the scope of alkyne-mediated bioconjugations utilizing unnatural amino acids.

The importance of bioconjugates within the field of chemistry drives the need for novel methodologies for their preparation. Well-defined and stable bioconjugates are easily accessible via the utilization of unnatural amino acids (UAAs). As such, we have synthesized and incorporated two new UAAs into green fluorescent protein, and optimized a novel Cadiot-Chodkiewicz bioconjugation, effectively expanding the toolbox of chemical reactions that can be employed in the preparation of bioconjugates.

Site-specific incorporation of a fluorescent terphenyl unnatural amino acid.

The site-specific incorporation of unnatural amino acids into proteins has a wide range of biological implications. Of particular interest is the incorporation of fluorescent probes as a mechanism to track protein function, transport, and folding. Thus, the development of a novel system for the incorporation of new fluorescent unnatural amino acids has significant utility. Specifically, we have elucidated an aminoacyl-tRNA synthetase capable of recognizing a terphenyl UAA derivative, and charging a cognate tRNA with this amino acid for protein incorporation. Moreover, we have successfully incorporated this fluorescent UAA into GFP at several key residues, demonstrating a novel means to modulate fluorescence within the protein.

Synthesis and Incorporation of Unnatural Amino Acids To Probe and Optimize Protein Bioconjugations.

The utilization of unnatural amino acids (UAAs) in bioconjugations is ideal due to their ability to confer a degree of bioorthogonality and specificity. In order to elucidate optimal conditions for the preparation of bioconjugates with UAAs, we synthesized 9 UAAs with variable methylene tethers (2-4) and either an azide, alkyne, or halide functional group. All 9 UAAs were then incorporated into green fluorescent protein (GFP) using a promiscuous aminoacyl-tRNA synthetase. The different bioconjugations were then analyzed for optimal tether length via reaction with either a fluorophore or a derivatized resin. Interestingly, the optimal tether length was found to be dependent on the type of reaction. Overall, these findings provide a better understanding of various parameters that can be optimized for the efficient preparation of bioconjugates.

Development and Optimization of Glaser-Hay Bioconjugations.

The prevalence of bioconjugates in the biomedical sciences necessitates the development of novel mechanisms to facilitate their preparation. Towards this end, the translation of the Glaser-Hay coupling to an aqueous environment is examined, and its potential as a bioorthogonal conjugation reaction is demonstrated. This optimized, novel, and aqueous Glaser-Hay reaction is applied towards the development of bioconjugates utilizing protein expressed with an alkynyl unnatural amino acid. Unnatural amino acid technology provides a degree of bioorthognality and specificity not feasible with other methods. Moreover, the scope of the reaction is demonstrated through protein-small molecule couplings, small-molecule-solid-support couplings, and protein-solid-support immobilizations.

Optically Triggered Immune Response through Photocaged Oligonucleotides.

Bacterial and viral CpG oligonculeotides are unmethylated cytosine-phosphate-guanosine dinucleotide sequences and trigger an innate immune response through activation of the toll-like receptor 9 (TLR9). We have developed synthetic photocaged CpGs via site-specific incorporation of nitropiperonyloxymethyl (NPOM)-caged thymidine residues. These oligonucleotides enable the optical control of TLR9 function and thereby provide light-activation of an immune response. We provide a proof-of-concept model by applying a reporter assay in live cells and by quantification of endogenous production of interleukin 6.

Optimization of solid-supported Glaser-Hay reactions in the microwave.

The translation of organometallic reactions into a microwave reactor has numerous advantages. Herein, we describe the application of a previously developed solid-supported Glaser-Hay reaction to microwave conditions. Overall, an array of diynes has been prepared demonstrating the ability to conduct chemoselective reactions in the microwave within 20 min compared to the 16 h thermal conditions. Moreover, non-microwave transparent alkynes have been found to react more quickly, preventing catalyst quenching, and resulting in higher yields.