Bioconjugation Applications Research Articles

Short DNAzymes for Efficient Catalysis of "Click" Reactions in Solution and on Surface.

We have systematically investigated and found surprising superior catalytic activities of very short DNAzymes for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), both in solution and on surface. As a key reaction of the "click chemistry" class, CuAAC is a highly efficient and specific covalent conjugation tool with demonstrated applications in organic synthesis, bioconjugation, and surface functionalization; however, it requires the presence of the Cu(I) catalyst, which is an unstable species in aqueous solutions. We show here that one ultrashort, 14-nucleotide-truncated fragment of an earlier in vitro selected DNAzyme (CLICK-17) shows a striking and superior catalytic activity toward the in trans CuAAC reaction in solution and on surface in the presence of either Cu(I) or Cu(II), at significantly lowered concentrations. These results obviate the need for long-sequence DNAzymes, selected out of the homogeneous solution phase, for application in complex surface environments.

Interfacing DNA and Aptamers with Gold Nanoparticles: From Fundamental Colloid and Interface Sciences to Biosensors

Comprehensive SummaryInterfacing DNA oligonucleotides and DNA aptamers with gold nanoparticles has generated numerous functional hybrid materials for sensing, self‐assembly and drug delivery applications. Our lab has been working in this area for 15 years. In this article, the current understanding of the adsorption of DNA to gold nanoparticles is summarized, and related applications in bioconjugation of DNA to gold surface is described. In addition, problems of using gold nanoparticles to signaling aptamer binding are discussed. Finally, re‐selection of aptamers for previously reported targets using the library‐immobilization method is reviewed. What is the most favorite and original chemistry developed in your research group?My most favorite and original work is the study of biointerface chemistry between DNA oligonucleotides and gold nanoparticles enabling rapid DNA bioconjugation by lowering the pH and freezing.How do you get into this specific field? Could you please share some experiences with our readers?My PhD training was focused on catalytic DNA for the detection of metal ions, when I used gold nanoparticles to signal the reactions catalyzed by DNA. When I started my independent career in the University of Waterloo in 2009, I realized that there were many fundamental issues regarding gold nanoparticles and DNA to be studied. My first teaching assignment was a course named ‘Surfaces and Interfaces’. By teaching this course, I learned a lot of surface science concepts that were later used in my research. That was the starting point for me to set up my own research program in this area.What is the most important personality for scientific research?Curiosity, careful observation, critical thinking, and keep trying.How do you keep balance between research and family?Research and personal life don’t have to be always in conflict. New ideas may spark when I am in a relaxed family environment.What are your favorite journals?Journal of the American Chemical Society, Angewandte Chemie International Edition, Nucleic Acids Research, Analytical Chemistry, Langmuir.What are your hobbies?Running; traveling; watching movies.

An innovative stepwise activated coupling reaction for fabricating functional fluorescence probes with potential photosensitive applications

Coupling bright organic emitters with biocomponents such as proteins and amino acids to fabricate biocompatible optical functional materials is receiving increasing attention. Previous reports of an efficient, typical reaction between maleic anhydride (MAH) and amino groups indicated that MAH derivatives have great potential for bioconjugation applications. However, the obstacle of complex synthesis methods and weak fluorescence to this kind of derivative structures based on MAH, have greatly limited their applications. Herein, an innovative strategy was developed based on stepwise activated coupling via a multi-step process. First, a series of donor–acceptor emitting frameworks was constructed by introducing different commercially available electron donors into the electron-withdrawing squaric acid core. Then, these frameworks were further activated to obtain different 3,4-diaryl MAH precursors for bioconjugation, which contributed to a novel light-triggered ring-enlargement effect. Importantly, the MAH precursors triggered catalyst-free coupling reactions at ambient temperature with organic small molecules and biological macromolecules containing primary amine groups. Different fluorescent precursors and abundant biological components were effectively combined, and a water-soluble, biocompatible photosensitizer with efficient photodynamic and photothermal therapy effects was obtained. This strategy is expected to have great potential for biological imaging and theragnostic applications.

3-Oxidopyridinium Ions Are Versatile Bioorthogonal Dipoles for Use in Cycloadditions with Cyclooctynes.

3-oxidopyridinium ions are water stable and soluble heteroaromatic betaines that behave as latent dipoles and undergo a wide variety of cycloadditions. Research into the cycloaddition reactions of 3-oxidopyridiniums was spearheaded by Alan R. Katritzky and collaborators from the early 1970s until the late 1980s, but they have yet to be used for bioorthogonal applications. Herein we report that 3-oxidopyridiniums can readily react with 4-dibenzocyclooctynol (DIBO), a common bioorthogonal handle, in a [3+2] cycloaddition. The mechanism was investigated by altering the electronics of the reaction by changing the substituent on the 5 position of the pyridinium. Electron-donating 5-substituents have been shown to significantly increase the rate of the reaction, with bimolecular rate constants ranging from 3.3×10-4 s-1 with 5-trifluoromethyl-N-methyl-3-oxidopyridinium to 1.07 M-1 s-1 with 5-amino-N-methyl-3-oxidopyridinium. 3-oxidopyridiniums' appreciable cycloaddition rates and compatibility with bioorthogonally relevant environments give them the potential to be used in a variety of bioconjugation applications.

Engineering the Propeptide of Microbial Transglutaminase Zymogen: Enabling Substrate-Dependent Activation for Bioconjugation Applications.

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.

Enantioselective sulfur(VI) fluoride exchange reaction of iminosulfur oxydifluorides.

Linkage chemistry and functional molecules derived from the stereogenic sulfur(VI) centre have important applications in organic synthesis, bioconjugation, drug discovery, agrochemicals and polymeric materials. However, existing approaches for the preparation of optically active S(VI)-centred compounds heavily rely on synthetic chiral S(IV) pools, and the reported linkers of S(VI) lack stereocontrol. A modular assembly method, involving sequential ligand exchange at the S(VI) centre with precise control of enantioselectivity, is appealing but remains elusive. Here we report an asymmetric three-dimensional sulfur(VI) fluoride exchange (3D-SuFEx) reaction based on thionyl tetrafluoride gas (SOF4). A key step involves the chiral ligand-induced enantioselective defluorinative substitution of iminosulfur oxydifluorides using organolithium reagents. The resulting optically active sulfonimidoyl fluorides allow for further stereospecific fluoride-exchange by various nucleophiles, thereby establishing a modular platform for the asymmetric SuFEx ligation and the divergent synthesis of optically active S(VI) functional molecules.

Development and Characterization of a Portable Flow Microreactor for Enhanced Bioconjugation Applications

Development and Characterization of a Portable Flow Microreactor for Enhanced Bioconjugation Applications

Bridge Cross-Coupling of Bicyclo[1.1.0]butanes.

Bicyclo[1.1.0]butanes (BCBs) have gained growing popularity in "strain release" chemistry for the synthesis of four-membered-ring systems and para- and meta-disubstituted arene bioisosteres as well as applications in chemoselective bioconjugation. However, functionalization of the bridge position of BCBs can be challenging due to the inherent strain of the ring system and reactivity of the central C-C bond. Here we report the first late-stage bridge cross-coupling of BCBs, mediated by directed metalation/palladium catalysis.

Exploring the Chemical Properties and Medicinal Applications of Tetramethylthiocycloheptyne Sulfoximine Used in Strain-Promoted Azide-Alkyne Cycloaddition Reactions.

The recently developed compound, tetramethylthiocycloheptyne sulfoximine (TMTHSI), has shown to be a promising strained alkyne for strain-promoted azide-alkyne cycloaddition (SPAAC), metal-free click chemistry. This research explores the properties of TMTHSI-based compounds via three aspects: (1) large-scale production, (2) unique stability in acidic conditions and its subsequent use in peptide synthesis, and (3) the functionalization of antibodies. Here, it is shown that (1) scale-up is achieved on a scale of up to 100 g. (2) TMTHSI is remarkably stable against TFA allowing for the site-specific functionalization of peptides on resin. Finally, (3) the functionalization of an antibody with a model payload is very efficient, with antibody conjugation demonstrating more beneficial features such as a high yield and limited hydrophobicity as compared to other alkyne reagent conjugates. These results illustrate the high potential of TMTHSI for diverse bioconjugation applications, with production already being GMP-compatible and a highly efficient conversion resulting in attractive costs of goods.

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine.

The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2-cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2-cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science.

Preparation of 3‐Bromo‐1,2,4,5‐tetrazine

AbstractIn this synthetic procedure, a seven‐step protocol for the preparation of monosubstituted 3‐bromo‐1,2,4,5‐tetrazine is presented. The procedure features efficient transformations and purification methods starting from commercially readily available starting materials and affords the title compound on a gram scale with 13 % overall yield in reliable purity (>97 %). Detailed experimental procedures, supported by images and additional notes, allow the preparation of a valuable advanced building block, enabling further applications in bioconjugation, protein labelling, bio‐orthogonal chemistry, heterocycle syntheses, high energy materials, and drug release, among others.

Facile de Novo Sequencing of Tetrazine-Cyclized Peptides through UV-Induced Ring-Opening and Cleavage from the Solid Phase.

While most FDA-approved peptide drugs are cyclic, the robust cyclization chemistry of peptides and the deconvolution of cyclic peptide sequences by using tandem mass spectrometry render cyclic peptide drug discovery difficult. Here we present the successful design of cyclic peptides on solid phase that addresses both of these problems. We demonstrate that this peptide cyclization method using dichloro-s-tetrazine on solid phase allows successful cyclization of a panel of random peptide sequences with various charges and hydrophobicities. The cyclic peptides can be linearized and cleaved from the solid phase by simple UV light irradiation, and we demonstrate that accurate sequence information can be obtained for the UV-cleaved linearized peptides by using tandem mass spectrometry. The tetrazine linker used in the cyclic peptides can further be explored for inverse electron-demand Diels-Alder (IEDDA) reactions for screening or bioconjugation applications in the future.

A Single Functionalization Agent for Heterotelechelic ROMP Polymers.

Heterotelechelic polymers are an important class of materials finding applications in bioconjugation, imaging, sensing, and synthesis of organic/inorganic hybrid systems with interesting features. However, the synthesis of such polymers is challenging. Here, we report a mechanistically unique and most efficient method based on a single functionalization agent to prepare heterotelechelic polymers by a ring-opening metathesis polymerization. Different functionalization agents can be synthesized in one simple step from inexpensive commercial starting materials. The functionalization agents initially generate a functional initiator from commercial Grubbs' first-generation ruthenium benzylidene catalyst. During this process, a functional dihydrofuran derivative is produced. After functional initiation and propagation of a suitable monomer, the dihydrofuran derivative functionally terminates the polymerization yielding a primary alcohol-terminated heterotelechelic polymer. Molecular weight control is achieved by varying the ratio between monomer and Grubbs' first-generation catalyst. This method may emerge as a popular choice to prepare heterotelechelic polymers due to its simplicity and efficiency.

Deciphering the substrate specificity of housekeeping sortase A and pilus-specific sortase C of probiotic bacterium Lactococcus lactis

Several strains and species of lactic acid bacteria (LAB) are widely used in fermented foods, including dairy products and also as probiotics, because of their contribution to various health benefits in humans. Sortase enzymes decorate the bacterial cell wall with different surface proteins and pili for facilitating the interactions with host and environment for the colonization and beneficial effects. While the sortases and sortase anchored proteins from pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including LAB strains, have attracted attention for their potential applications in vaccine delivery and other clinical interventions. Here, we report the purification and functional characterization of two sortases (housekeeping SrtA and pilus-specific SrtC) from a probiotic Lactococcus lactis. The purified sortases were found to be active against the putative LPXTG motif-based peptide substrates, albeit with differences. The in-silico analysis provides insights into the residues involved in substrate binding and specificity. Overall, this study sheds new light on the aspects of structure, substrate specificity, and function of sortases from non-pathogenic bacteria, which may have physiological ramifications as well as their applications in sortase-mediated protein bioconjugation.

Self-Immolative Hydroxybenzylamine Linkers for Traceless Protein Modification

Traceless self-immolative linkers are widely used for the reversible modification of proteins and peptides. This article describes a new class of traceless linkers based on ortho- or para-hydroxybenzylamines. The introduction of electron-donating substituents on the aromatic core stabilizes the quinone methide intermediate, thus providing a platform for payload release that can be modulated. To determine the extent to which the electronics affect the rate of release, we prepared a small library of hydroxybenzylamine linkers with varied electronics in the aromatic core, resulting in half-lives ranging from 20 to 144 h. Optimization of the linker design was carried out with mechanistic insights from density functional theory (DFT) and the in silico design of an intramolecular trapping agent through the use of DFT and intramolecular distortion energy calculations. This resulted in the development of a faster self-immolative linker with a half-life of 4.6 h. To demonstrate their effectiveness as traceless linkers for bioconjugation, reversible protein-polyethylene glycol conjugates with a model protein lysozyme were prepared, which had reduced protein activity but recovered ≥94% activity upon traceless release of the polymer. This new class of linkers with tunable release rates expands the traceless linkers toolbox for a variety of bioconjugation applications.

Self-Illuminating Triggered Release of Therapeutics from Photocleavable Nanoprodrug for the Targeted Treatment of Breast Cancer.

Photocleavable biomaterials and bioconjugates have been widely researched for tissue engineering, cell culture, and therapeutics delivery. However, most in vivo applications of these materials or conjugates require external irradiation, and some of the light sources used such as ultraviolet (UV) light have poor tissue penetration. To address these key limitations, we synthesized a photocleavable nanoprodrug using luminol (a luminescent donor), chlorambucil (CHL, i.e., an antitumor drug with a photocleavable linker), and polyethylene glycol-folic acid conjugates (a targeted moiety) loaded onto polyamidoamine (PAMAM). The synthesized nanoprodrug can smartly release its payloads through photocleavage of photoresponsive linker by UV light, which was produced in situ by reacting luminol with pathological reactive oxygen species (ROS). The luminescence performance and absorption spectrum of this nanoprodrug was characterized in detail. In vitro cellular assays verified that the nanoprodrugs could be efficiently internalized by 4T1 and MDA-MB-231 cells, and the CHL released from the nanoprodrugs could distinctly decrease cell viability through the damage of DNA in cells. In vivo animal experiments demonstrated that the nanoprodrugs were mainly accumulated at tumor sites, and the antitumor drug CHL could be smartly released from the nanoprodrugs through cleavage of photosensitive linkers at a high level of ROS. The released CHL significantly inhibited the growth of tumors without any obvious adverse effects. Our results provide a practicable strategy to expand the in vivo application of photocleavable biomaterials and bioconjugates.

Preparation and Application of Fluorescent Bioconjugates in Probing the pH and Solvent Relaxation Dynamics in ZIF-8 Cavities

Preparation and Application of Fluorescent Bioconjugates in Probing the pH and Solvent Relaxation Dynamics in ZIF-8 Cavities

Protein Modification via Mild Photochemical Isomerization of Triazenes to Release Aryl Diazonium Ions.

This work describes the development of phenyl diazenyl piperidine triazene derivatives that can be activated to release aryl diazonium ions for labeling of proteins using light. These probes show marked bench stability at room temperature and can be photoisomerized via low-intensity UVA irradiation at physiological pH. Upon isomerization, the triazenes are rendered more basic and readily protonate to release reactive aryl diazonium ions. It was discovered that the intensity and duration of the UV light was essential to the observed diazonium ion reactivity in competition with the traditionally observed photolytic radical pathways. The combination of their synthetic efficiency coupled with their overall stability makes triazenes an attractive candidate for use in bioconjugation applications. Bioorthogonal handles on the triazenes are used to demonstrate the ease by which proteins can be modified.

An Overview of Recent Advances in Biomedical Applications of Click Chemistry.

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is a modular and bio-orthogonal approach that is being adopted for the efficient synthesis of organic and bioorganic compounds. It leads to the selective formation of 1,4-disubstituted 1,2,3-triazole units connecting readily accessible building blocks via a stable and biocompatible linkage. The vast array of the bioconjugation applications of click chemistry has been attributed to its fast reaction kinetics, quantitative yields, minimal byproducts, and high chemospecificity and regioselectivity. These combined advantages make click reactions quite suitable for the lead identification and the development of pharmaceutical agents in the fields of medicinal chemistry and drug discovery. In this review, we have outlined the key aspects, the mechanistic details and merits and demerits of the click reaction. In addition, we have also discussed the recent pharmaceutical applications of click chemistry, ranging from the development of anticancer, antibacterial, and antiviral agents to that of biomedical imaging agents and clinical therapeutics.

Pentafulvene-Maleimide Cycloaddition for Bioorthogonal Ligation.

The applications of bioconjugation chemistry are rapidly expanding, and the addition of new strategies to the bioconjugation and ligation toolbox will further advance progress in this field. Herein, we present a detailed study of the Diels-Alder cycloaddition (DAC) reaction between pentafulvenes and maleimides in aqueous solutions and investigate the reaction as an emerging bioconjugation strategy. The DAC reactions were found to proceed efficiently, quantitatively yielding cycloadducts with reaction rates ranging up to ∼0.7 M-1 s-1 for a series of maleimides, including maleimide-derivatized peptides and proteins. The absence of cross-reactivity of the pentafulvene with a large panel of functional (bio)molecules and biological media further demonstrated the bioorthogonality of this approach. The utility of the DAC reaction for bioorthogonal bioconjugation applications was further demonstrated in the presence of biological media and proteins, as well as through protein derivatization and labeling, which was comparable to the widely employed sulfhydryl-maleimide coupling chemistry.