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Bio-conjugation and protein engineering are two powerful biochemical methods to alter proteins to have enhanced functions. Bio-conjugation allows for proteins to be attached to other protein and non-protein molecules in ways that would never appear naturally in the biological world, bestowing new properties onto a protein. Protein engineering allows for manipulation of the linear amino-acid sequence of a protein to alter its biochemical properties. These types of substitutions can change the way a protein folds, how it interacts with partner proteins, and its own chemical reactivity. These methods are currently highly utilized in the development of novel therapeutics and research tools. For both methods, location is one of the most important considerations for successful applications. Years of bio-conjugation research has revealed that attachment at a controlled and known site improves the reproducibility and properties of a given conjugate. In protein engineering, not all sites on a protein have the same chemical environment and thus even the same amino acid at a different location may react differently. Thus, even with these powerful biochemical methods, careful research must be done to determine the optimal attachment method, mutation location, and site. However, the optimized conjugate or engineered protein has great potential as a novel therapeutic, diagnostic, or research tool.Here, I describe three approaches using bio-conjugation and protein engineering to develop novel and optimized protein conjugates or engineered protein fusions for various applications. In Chapter 1, I describe the use of a methionine-based conjugation method to attach cytotoxic cancer drugs on antibodies as selective drug delivery carriers. In Chapter 2, I highlight the development of rapid, facile protein biosensors to detect antibodies to COVID-19. In Chapter 3, I describe the development of an improved cell-surface biotinylation method utilizing novel enzyme conjugates.

Therapeutic agents including small molecules, peptides, and proteins comprise most of the pharmaceutics currently on the market and are an invaluable piece of modern medicine. Many of these therapeutics have some level of instability and/or off-target effects when administered in vivo that ultimately limits their effectiveness. Covalent modification of such therapeutics into an inactive prodrug prior to administration is a common modality used within the field of drug delivery to increase the circulation time or limit off-target effects, thus enhancing the therapeutic efficacy compared to administration of the unaltered drug. Due to the large diversity in chemical reactivities across various therapeutics, a wide array of strategies has been developed to covalently modify functional groups of interest. This thesis outlines the development of two such prodrug modalities; the first for the targeted delivery of oxycodone to the small intestine to prevent nonprescribed forms of administration and the second being a platform for the tunable release of peptides and proteins for enhanced circulation time.Biologics including peptides, proteins, and oligonucleotides are an important class of macromolecules that have received an increasing amount of attention over the past few decades for their therapeutic potential. Their widespread adoption has lacked, in part, due to issues with chemical instability and immunogenicity in vivo. One particular avenue towards mitigating these effects is through PEGylation of the biologics, which can lead to enhanced stability and circulation times in vivo. However, PEGylation typically results in decreased binding or activity and in some cases can turn these off altogether. To mitigate these effects, researchers developed small molecule linkages that can be placed in between the protein and polymer, which cleave slowly over time to release native protein with restored binding/activity. Chapter 1 outlines the current strategies used within the field of traceless peptide/protein conjugation, including lonapegsomatropin-tcgp and NKTR-214 the only two traceless protein-polymer conjugates currently approved by the FDA, along with insights into potential future directions for this field.Prescription opioids, although necessary for pain management, are highly addictive and have led to the dramatic increase of opioid involved overdoses over the past 20 years. Abuse-deterrent (AD) opioid formulations are an important avenue towards addressing this national health crisis by increasing the difficulty for abusers to easily obtain large amounts of active opioid. However, there are currently only ten AD formulations on the market, a majority of which are easily circumvented by motivated users. Chapter 2 outlines the design and preparation of a slow-release elastomeric opioid formulation, which requires the presence of two proteases found within the small intestine in order to release the drug. This strategy limits the common abusive routes of administration, including, nasal insufflation and intravenous injection through the protease mediated activation step. In order to do this, the opioid was modified with a dual-enzyme responsive peptide sequence, whereupon proteolytic cleavage by chymotrypsin followed by trypsin triggers a self-immolative based release of the active opioid. The resulting elastomeric formulation was shown to be stable towards mechanical deformation testing following periods of heating and cooling, as well as hydrolytic degradation across a pH range of 2-10.It was later identified that the elastomeric opioid carrier was not entirely necessary for the abuse deterrent properties of the formulation, but rather the same properties could be demonstrated in a small molecule peptide-opioid prodrug. Therein, Chapter 3 highlights the development of a second-generation abuse-deterrent peptide prodrug without the elastomeric carrier. The peptide-opioid prodrug sequence was optimized to mitigate any nonspecific protease cleavage, while also enhancing the rate of trypsin and chymotrypsin cleavage, the two enzymes responsible for activation in the small intestine. Notably, the composition of the amino acid sequence was highly crucial in determining the protease promiscuity which was identified as an issue with the first-generation prodrug. In addition, a third level of protection from abuse was incorporated into the prodrug through an acid mediated activation step to unmask a tyrosine residue. This activation rapidly occurs in simulated gastric fluid producing t-butanol and allows for chymotrypsin to bind and subsequently cleave the tyrosine residue. The development of traceless linkers, outlined in Chapter 1, demonstrates the interest in creating prodrug like protein-polymer conjugates, allowing for the systemic administration of therapeutic peptides and proteins that were previously cleared too rapidly to be clinically useful. To this end, Chapter 4 outlines the development of a new amine reactive benzylamine traceless linker as an alternative to the commonly used carbamate linkages. A small molecule model system was initially used to probe the release kinetics of a primary amine, simulating lysine release from a protein-polymer conjugate. Varying the electronics within the aromatic core of the linker played a vital role in modulating the rate of release, resulting in half-lives ranging from 144 to 20 hours. The most promising linker was then incorporated into as a PEG-end group and used to conjugate to prepare traceless lysozyme-PEG conjugates. These conjugates released more than 95% native lysozyme over the course of 48 hours at a pH of 7.4 with a restoration of lysozyme activity upon release, whereas less than 25% native lysozyme was released within 96 hours at a pH of 4.0.Building upon the results from Chapter 4 and mechanistic insights from density functional theory (DFT) calculations carried out in collaboration with members from the Houk Lab, Chapter 5 discusses the synthetic pursuits, kinetics studies, and protein conjugation studies toward developing a second-generation linker. These linkers incorporate an intramolecular trap to decrease the lifetime of the quinone methide intermediate, which in turn enhanced the rate of release to a 4.5 hour half-life using a small molecule model system. Additionally, these second-generation benzylamine linkers were used to prepare two traceless lysozyme-PEG conjugates with varying electronics and rates of release. The lysozyme-PEG conjugate containing the more electron rich linker demonstrated 98% release of native lysozyme within 12 days while restoring lysozyme activity, whereas the less electron rich linker showed only 50% release within the same time frame. This new class of linkers with tunable release rates expands the traceless linkers toolbox for a variety of bioconjugation applications.

DNA polymerase catalyzes the correct replication and repair of DNA, an essential step in the life cycle of all organisms. The enzyme incorporates incoming deoxynucleotide triphosphates (dNTPs) into a nascent DNA strand that is complementary to a single-stranded DNA (ssDNA) template. This capability makes DNA polymerases workhorses for molecular biology and biotechnology. Single-molecule studies can identify the dynamics of DNA polymerase structural conformations, which are otherwise lost through averaging in ensemble populations. Therefore, the Weiss and Collins labs (UCI) collaboratively developed a single-walled carbon nanotube field-effect transistor (nanocircuit) to translate enzyme motions into electronic signals. These nanocircuits may illuminate hidden conformational transitions of DNA polymerase activity and reveal new mechanisms and dynamics during dNTP incorporation. The goal of this work is to apply this approach to understand the conformational dynamics of the thermostable DNA polymerase from Thermus aquaticus (Taq) and processive DNA polymerase from the Bacillus subtilis bacteriophage phi29 (Φ29). The work here resolves subtle dynamics and transient intermediate states of DNA polymerase that modulate catalytic speed and molecular recognition during DNA synthesis. Additional efforts are focused on evaluating the performance of nanocircuits for direct electrical monitoring of polymerase in future DNA sequencing. Advances in bioconjugation, the ability to link biomolecules to each other, small molecules, surfaces, and more, can spur the development of advanced materials and therapeutics. In work reported here, pyrocinchonimide (Pci) undergoes a surprising transformation with biomolecules. The reaction targets amines and involves an imide transfer, which has not been previously reported for bioconjugation purposes. The Pci motif can reduce combinatorial diversity when many available reactive amines are available, such as in the formation of antibody-drug conjugates. The reaction offers a thermodynamically controlled route to single or multiple modifications of proteins for a wide range of applications. In response to the ongoing Coronavirus disease (COVID-19) pandemic, my laboratory members and I investigated the antibody response of patients infected by SARS-CoV-2 for predicting disease trajectories. Using methods of enzyme-linked immunosorbent assay (ELISA) and coronavirus antigen microarray (COVAM) analysis, antibody epitopes were mapped in the plasma of COVID-19 patients (n = 186) experiencing a wide range of disease states. This work identified antibodies to a 21-residue epitope from nucleocapsid (termed Ep9) associated with severe disease, including admission to the intensive care unit (ICU), requirement for ventilators, or death. By combining a disease risk factor score with a test for anti-Ep9 antibodies, severe COVID-19 outcomes could be predicted with 13.4 likelihood ratio (96.7% specificity). The results lay the groundwork for a new type of COVID-19 prognostic that could guide more effective therapeutic intervention.

Proteins are increasingly applied in commercial manufacturing, medicine, and research. Engineering proteins improves many aspects of such applications (e.g., efficiency, safety, quality) through a variety of genetic and chemical techniques. For instance, directed evolution can improve endogenous protein activities, diminish undesired properties, or introduce new activities. Alternatively, bioconjugation to small molecules extends the functionality of proteins beyond what nature can offer. Additionally, protein immobilization in flow enables the recreation or rearrangement of valuable biosynthetic pathways. In my Ph.D. work, I have sought to implement or improve protein engineering techniques to address distinct problems in biotherapeutics, biosensing, and biocatalysis.Proteases exemplify how protein engineering innovates across sectors. For example, proteases in stain removers are engineered to tolerate alkaline conditions, detergents, and extreme temperatures. In research, engineered proteases enable the proteomic mapping of elusive post-translational modifications. We demonstrate the potential of protein engineering in biotherapeutics by retargeting the hyper-specific metalloprotease domain of the FDA-approved botulinum neurotoxin serotype A (LC/A) via directed evolution. The final LC/A variant displayed specificity for a new therapeutic target, infiltrated neurons, and demonstrated reduced toxicity in mice. Importantly, these results guide further engineering of LC/A and related proteases.Engineered oxidoreductases play an important role in biosensors and basic research. Dihydrofolate reductase (DHFR), for instance, is a model for relating enzymatic conformational motions to biocatalytic events. Many of these studies employed chemically modified DHFR variants, making DHFR attractive for investigating bioconjugation reactions. Indeed, reacting DHFR variants with the maleimide analog pyrocinchonimide revealed higher cysteine-selectivity compared to maleimide. Cystine vs. lysine reactivity is an important factor when constructing protein-drug therapeutics. Moreover, bioconjugating DHFR to boronate enabled the identification of post-translationally modified biomarkers in an electrochemical biosensor. This device sensitively differentiated glycated from non-glycated human serum albumin, an important glycemic control indicator in diabetes.Engineering reversible immobilization tags into proteins enables their biocatalytic application in a vortex fluidic device. Creating stripes of fluorescent proteins offers a proof of concept for the spatial segregation and rearrangement of high-value biosynthetic pathways. Flowing crude lysate through such devices demonstrates the ability to rapidly capture, purify, and use tagged enzymes for continuous-flow biocatalysis.

Pathogenic Gram-positive bacteria cause a range of serious infections in humans and represent a significant threat to global health. The rising emergence of virulent strains which are resistant to our current arsenal of antibiotics escalates these dangers. Many Gram-positive bacteria display an array of proteins on their cell surface that enable them to interact with their environment during infections. Among the most important extracellular virulence factors are bacterial pili, which are adhesive filaments that are constructed by specialized cysteine transpeptidases through isopeptide linkages. This dissertation describes my efforts to elucidate the assembly mechanism of the archetypal SpaA-pilus from Corynebacterium diphtheriae. Further, it describes parallel experiments aimed at repurposing the enzymes used in pilus biogenesis for protein engineering applications. This thesis is divided into two major sections: Pilus Biology (Chapters 2 - 4) and Sortase Bioconjugation (Chapters 5 - 7). The Pilus Biology section describes efforts to elucidate the mechanism of Gram-positive pilus biogenesis using kinetic, structural and cellular experiments. Chapter 2 describes the biochemical reconstitution and characterization of the assembly reaction that builds the C. diphtheriae SpaA-pilus. SpaA-type pili are assembled by a Class C pilin polymerase sortase, CdSrtA. Examination of the structure of this enzyme revealed that CdSrtA is held in an inactive state in vitro by an autoinhibitory “lid” structure. We discovered that amino acid substitutions introduced into the “lid” activate the enzyme and permit biochemical characterization of the polymerization reaction. Chapter 3 characterizes the mechanism and in vitro kinetics of the lysine-isopeptide transpeptidation reaction that builds the SpaA pilus. We identify the rate limiting step in the mechanism and offer a kinetic explanation that explains why “lid” alterations activate CdSrtA. Chapter 4 describes the solution structure of the pilin-pilin linkage which joins successive protomers in the SpaA pilus. Data from NMR dynamics, SAXS and biophysical measurements of protein stability reveal the mechanism by which Gram-positive pili are stabilized at each linkage site throughout the elongated pilus fiber.The Bioconjugation section of this thesis describes our efforts to develop sortase enzymes into useful bioconjugation tools. Chapter 5 describes a versatile sortase-mediated protein nanocage ligation platform that facilitates enzymatic synergy by enhancing pathway flux between enzymes with complementary activities. As a proof of principle, the nanocage scaffold was functionalized with cellulolytic enzymes, demonstrating a marked enhancement in degradative synergy of cellulose substrate compared to unbound cellulases. Chapter 6 builds upon our understanding of the pilus biogenesis reaction to engineer the CdSrtA sortase into a viable bioconjugation tool that can be used to attach peptide fluorophores to proteins via isopeptide bonds. Chapter 7 describes ongoing efforts to further optimize the CdSrtA bioconjugation tool using a directed evolution approach, with the goal of altering its substrate specificity and increasing its thermostability. Together, the research described in this thesis provides new insight into the biogenesis mechanism that is used by Gram-positive bacteria to produce adhesive pili and describes the development of a promising molecular tool for producing novel bioconjugates.

Gelatin sponges are widely employed as hemostatic agents, and are gaining increasing interest as 3D scaffolds for tissue engineering. To broaden their possible application in the field of tissue engineering, a straightforward synthetic protocol able to anchor the disaccharides, maltose and lactose, for specific cell interactions was developed. A high conjugation yield was confirmed by 1H-NMR and FT-IR spectroscopy, and the morphology of the resulting decorated sponges was characterized by SEM. After the crosslinking reaction, the sponges preserve their porous structure as ascertained by SEM. Finally, HepG2 cells cultured on the decorated gelatin sponges show high viability and significant differences in the cellular morphology as a function of the conjugated disaccharide. More spherical morphologies are observed when cultured on maltose-conjugated gelatin sponges, while a more flattened aspect is discerned when cultured onto lactose-conjugated gelatin sponges. Considering the increasing interest in small-sized carbohydrates as signaling cues on biomaterial surfaces, systematic studies on how small carbohydrates might influence cell adhesion and differentiation processes could take advantage of the described protocol.

International audience; A novel Ru(II) cyclometalated photosensitizer (PS), Ru-NH2, for photodynamic therapy (PDT) of formula [Ru(appy)(bphen)2]PF6 (where appy = 4-amino-2-phenylpyridine and bphen = bathophenanthroline) and its cetuximab (CTX) bioconjugates, Ru-Mal-CTX and Ru-BAA-CTX (where Mal = maleimide and BAA = benzoylacrylic acid) were synthesised and characterised. The photophysical properties of Ru-NH2 revealed absorption maxima around 580 nm with an absorption up to 725 nm. The generation of singlet oxygen (1O2) upon light irradiation was confirmed with a 1O2 quantum yield of 0.19 in acetonitrile. Preliminary in vitro experiments revealed the Ru-NH2 was nontoxic in the dark in CT-26 and SQ20B cell lines but showed outstanding phototoxicity when irradiated, reaching interesting phototoxicity indexes (PI) > 370 at 670 nm, and > 150 at 740 nm for CT-26 cells and > 50 with NIR light in SQ20B cells. The antibody CTX was successfully attached to the complexes in view of the selective delivery of the PS to cancer cells. Up to four ruthenium fragments were anchored to the antibody (Ab), as confirmed by MALDI-TOF mass spectrometry. Nonetheless, the bioconjugates were not as photoactive as the Ru-NH2 complex.

Dedicated to Prof. Janine Cossy Recent years have witnessed a considerable progress in research aimed at merging transition metal catalysis with chemical and cell biology. Therefore, a crescent number of metal-catalyzed transformations have been shown compatible with biological media and even with living settings. Of the different transition metals used to build these biocompatible catalysts, ruthenium has demonstrated to be particularly powerful, in part because the resulting complexes exhibit a very good balance between reactivity and biological stability. Indeed, ruthenium complexes have demonstrated utility to promote a great variety of reactions in biologically relevant contexts, from deprotection and redox processes to cycloadditions or photocatalytic transformations. Many of these reactions may enable the development of new type of biological tools and pharmacological strategies PID2020-118579GB−I00; PID2019-108624RB−I00; Consellería de Cultura, Educación e Ordenación Universitaria-Grant Numbers: ED431 C 2021/25, ED431G 2019/03; European Union; Ministerio de Universidades. Grant Number: FPU17/00711 SI

This study reports the development of conjugates based on quantum dots (QD)s and lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL). Cryptococcus neoformans cells were chosen to evaluate the efficiency of the conjugates. Lectins were conjugated to QDs via adsorption, and the optical parameters (emission and absorption) were monitored. Lectin stability in the conjugates towards denaturing agents was investigated via fluorometry. The conjugation was evaluated using fluorescence microplate (FMA) and hemagglutination (HA) assays. The labeling of the C. neoformans cell surface was quantified using flow cytometry and observed via fluorescence microscopy. The QDs-SteLL and QDs-PgTeL conjugates, obtained at pH 7.0 and 8.0, respectively, showed the maintenance of colloidal and optical properties. FMA confirmed the conjugation, and the HA assay indicated that the lectin carbohydrate-binding ability was preserved after conjugation. SteLL and PgTeL showed stability towards high urea concentrations and heating. Conjugates labeled over 90% of C. neoformans cells as observed via flow cytometry and confirmed through fluorescence microscopy. C. neoformans labeling by conjugates was inhibited by glycoproteins, suggesting specific interactions through the lectin carbohydrate-binding site. Thus, an effective protocol for the conjugation of SteLL or PgTeL with QDs was proposed, yielding new nanoprobes useful for glycobiological studies.

Aptamer-Carbon Dot (CD) bioconjugation is an attractive target-tracking strategy in detecting cell surface antigens. This study describes an effective imaging paradigm for CA-125 antigen imaging. Our experience encompasses green CD synthesis and characterization, CD-capture probe conjugation through covalent bonding, the hybridization linkage of CD-probe to aptamer and their coupling confirmation, and fluorescent targeted imaging of ovarian cancer cells. As a result, the synthesized CDs from lemon extract by hydrothermal reaction show average size of 2 nm with maximum fluorescence intensity at excitation/emission 360/450 nm. CD-probe construction was provided by functional group interactions of CD and probe via EDC/NHS chemistry. The linkage of CD-probe to aptamer was conducted by Watson-Crick nucleotide pairing. The assessment of CD-probe and CD-probe-aptamer fabrication was validated by the increase in surface roughness through AFM analysis, the diminish of fluorescence intensity of CD after bioconjugation, and particle size growth of the construct. Conjugates with negligible cytotoxicity, appropriate zeta potential, and good aptamer release were applied in cellular imaging. This targeted diagnosis method was employed the four reported DNA aptamers toward fluorescence intensity. The DOV-3 aptamer showed more qualified detection over other aptamer conjugates during fluorescent microscopy analysis. In conclusion, the CD-probe-aptamer conjugate applications as toxic-free method can open new horizons in fluorescent nano-imaging in the field of targeted cancer cell diagnosis.

The site selectivity for lysine conjugation on a native protein is difficult to control and characterize. Here, we applied mass spectrometry to examine the conjugation kinetics of Trastuzumab-IgG (Her-IgG) and α-lactalbumin under excess linker concentration ([L]0) based on the modified Michaelis–Menten equation, in which the initial rate constant per amine (kNH2 = Vmax/NH2/KM) was determined by the maximum reaction rate (Vmax/NH2) under saturated accessible sites and initial amine–linker affinity (1/KM). Reductive amination (RA) displayed 3–4 times greater Vmax/NH2 and a different panel of conjugation sites than that observed for N-hydroxysuccinimide ester (NHS) chemistry using the same length of polyethylene glycol (PEG) linkers. Moreover, faster conversion power rendered RA site selectivity among accessible amine groups and a greater tunable range of linker/protein ratio for aldehyde-linkers compared to those of the same length of NHS-linkers. Single conjugation with high yield or poly-conjugations with site homogeneity was demonstrated by controlling [L]0 or gradual addition to minimize the [L]0/KM ratio. Formaldehyde, the shortest aldehyde-linker with the greatest 1/KM, exhibited the highest selectivity and was shown to be a suitable probe to predict conjugation profile of aldehyde-linkers. Four linkers on the few probe-predicted hot spots were elucidated by kinetically controlled RA with conserved drug efficacy when conjugated with the payload. This study provides insights into controlling factors for homogenous and predictable amine bioconjugation.

Protein–protein interactions (PPI) are involved in all cellular processes and many represent attractive therapeutic targets. However, the frequently rather flat and large interaction areas render the identification of small molecular PPI inhibitors very challenging. As an alternative, peptide interaction motifs derived from a PPI interface can serve as starting points for the development of inhibitors. However, certain proteins remain challenging targets when applying inhibitors with a competitive mode of action. For that reason, peptide-based ligands with an irreversible binding mode have gained attention in recent years. This review summarizes examples of covalent inhibitors that employ peptidic binders and have been tested in a biological context.

Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi-functionalization of the widely employed carrier protein CRM197 with a carbohydrate-based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM197 followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione. Efficient protein modification required that the reduction and functionalization with a dibromopyridazinedione was performed as a one-step procedure with control over the reaction temperature. Furthermore, ligations were most successful when dibromopyridazinediones were employed having a functional entity such as a TLR7/8 agonist and a cyclooctyne for further modification. Site-specific conjugation avoids modification of T-epitopes of the carrier protein and covalent attachment of an immune potentiator will ensure that cytokines are produced where the vaccine interacts with relevant immune cells resulting in efficient immune potentiation.

Protein bioconjugates are in high demand for applications in biomedicine, diagnostics, chemical biology and bionanotechnology. Proteins are large and sensitive molecules containing multiple different functional groups and in particular nucleophilic groups. In bioconjugation reactions it can therefore be challenging to obtain a homogeneous product in high yield. Numerous strategies for protein conjugation have been developed, of which a vast majority target lysine, cysteine and to a lesser extend tyrosine. Likewise, several methods that involve recombinantly engineered protein tags have been reported. In recent years a number of methods have emerged for chemical bioconjugation to other amino acids and in this review, we present the progress in this area.

We report a strategy for the orthogonal conjugation of the vinyl nucleosides, 5-vinyluridine (5-VU) and 2-vinyladenosine (2-VA), via selective reactivity with maleimide and tris(2-carboxyethyl)phosphine (TCEP), respectively. The orthogonality was investigated using density functional theory (DFT) and confirmed by reactions with vinyl nucleosides. Further, these chemistries were used to modify RNA for fluorescent cell imaging. These reactions allow for the expanded use of RNA metabolic labeling to study nascent RNA expression within different RNA populations.

Biotin is a common functional handle for bioconjugation to proteins and DNA, but its uses are limited to protein-containing conjugation partners such as streptavidin and derivatives thereof. Recently, oxaziridine reagents were developed that selectively conjugate the thioether of methionines on the surface of proteins, a method termed redox-activated chemical tagging (ReACT). These reagents generate sulfimide linkages that range in stability depending on the solvent accessibility and substitutions on the oxaziridine. Here we show that oxaziridine reagents react rapidly with the thioether in biotin to produce sulfimide products that are stable for more than 10 d at 37 °C. This method, which we call biotin redox-activated chemical tagging (BioReACT), expands the utility of biotin labeling and enables a predictable and stable chemical conjugation to biomolecules without the need to screen for a suitable methionine conjugation site. We demonstrate the versatility of this approach by producing a fluorescently labeled antibody, an antibody-drug conjugate, and a small molecule-conjugated oligonucleotide. We anticipate that BioReACT will be useful to rapidly introduce biorthogonal handles into biomolecules using biotin, a functional group that is widespread and straightforward to install.

While a diverse set of design strategies have produced various chemical tools for biomolecule labeling in aqueous media, the development of nonaqueous, biomolecule-compatible media for bioconjugation has significantly lagged behind. In this report, we demonstrate that an aprotic ionic liquid serves as a novel reaction solvent for protein bioconjugation without noticeable loss of the biomolecule functions. The ionic liquid bioconjugation approach led to discovery of a novel triphenylphosphine-mediated amine-azide coupling reaction that forges a stable tetrazene linkage on unprotected peptides and proteins. This strategy of using untraditional media would provide untapped opportunities for expanding the scope of chemical approaches for bioconjugation.

Self-assembly of a monomeric protease to form a multi-subunit protein complex “proteasome” enables targeted protein degradation in living cells. The naturally occurring proteasomes serve as an inspiration and blueprint for the design of artificial protein-based nanoreactors. Here we disclose a general chemical strategy for the design of proteasome-like nanoreactors. Micelle-assisted protein labeling (MAPLab) technology along with the N-terminal bioconjugation strategy is utilized for the synthesis of a well-defined monodisperse self-assembling semi-synthetic protease. The designer protein is programmed to self-assemble into a proteasome-like nanostructure which preserves the functional properties of native protease.

Site-selective chemical modification of protein side chain has probed enormous opportunities in the fundamental understanding of cellular biology and therapeutic applications. Primarily, in the field of biopharmaceuticals, the formulation of bioconjugates has been found to have more potential than an individual constituent. In this regard, Lysine and Cysteine are the most widely used endogenous amino acid for these purposes. Recently, the aromatic side chain residues (Trp, Tyr, and His) that are low abundant in protein have gained more attention in therapeutic applications due to their advantages of chemical reactivity and specificity. This review discusses the site-selective bioconjugation methods for aromatic side chains (Trp, Tyr and His) and highlights the developed strategies in the last three years, along with their applications. Also, the review highlights the prevalent methods published earlier. We have examined that metal-catalyzed and photocatalytic reactions are gaining more attention for bioconjugation, though their practical operation is under development. The review has been summarized with the future perspective of protein and peptide conjugations contemplating therapeutic applications and challenges.

Boronic acids can play diverse roles when applied in biological environments, and employing boronic acid structures in tandem could provide new tools for multifunctional probes. This Letter describes a pair of boronic acid functional groups, 2-nitro-arylboronic acid (NAB) and (E)-alkenylboronic acid (EAB), that enable sequential cross-coupling through stepwise nickel- and copper-catalyzed processes. The selective coupling of NAB groups enables the preparation of stapled peptides, protein-protein conjugates, and other bioconjugates.

In this study, we present a convenient method for the labelling of tyrosine residues on bovine serum albumin (BSA) and human serum albumin (HSA) and report for the first time their subsequent bio-orthogonal conjugation with porphyrins via "click" chemistry. We demonstrate that these serum proteins can be labelled with an alkyne-diazonium heterobifunctional linker and can then undergo chemo-selective bio-orthogonal conjugation with a water-soluble azido metalloporphyrin via "click" chemistry to yield protein-conjugates that retain their photodynamic properties. In our hands, this method was found to be highly reproducible, scalable, and tuneable which allows for the production of bioconjugates where the porphyrin-protein conjugate not only retains an ability to generate singlet oxygen but possess an enhanced relative singlet oxygen quantum yields relative to the porphyrin alone. Furthermore, we have investigated the photochemical properties of these conjugates through photospectrometric techniques and have determined that the porphyrin macrocycles remain appreciably photostable under light irradiation. Our phototoxic protein-photosensitizer-conjugates show excellent photodynamic activity against a human colorectal adenocarcinoma cancer cell line (HT-29) with cell viabilities of 7.7±0.5 % (IC50 8.76±2.14 μM) and 1.7±1.9 % (IC50 8.48±5.11 μM) for BSA and HAS, respectively, when irradiated with 20 J cm-2 of white-light. Importantly, neither of the conjugates was found to possess any significant "dark" toxicity even at concentrations of 100 μM. Furthermore, the natural fluorescent properties of the bioconjugates allowed for the determination of cellular uptake in vitro via fluorescence microscopy thus highlighting the potential theranostic applications of these unique protein-drug-conjugates.

Boron-containing heterocycles are important in a variety of applications from drug discovery to materials science; therefore a clear understanding of their structure and reactivity is desirable to optimize these functions. Although the boranol (B-OH) unit of boronic acids behaves as a Lewis acid to form a tetravalent trihydroxyborate conjugate base, it has been proposed that pseudoaromatic hemiboronic acids may possess sufficient aromatic character to act as Bronsted acids and form a boron oxy conjugate base, thereby avoiding the disruption of ring aromaticity that would occur with a tetravalent boronate anion. Until now no firm evidence existed to ascertain the structure of the conjugate base and the aromatic character of the boron-containing ring of hemiboronic "naphthoid" isosteres. Here, these questions are addressed with a combination of experimental, spectroscopic, X-ray crystallographic, and computational studies of a series of model benzoxazaborine and benzodiazaborine naphthoids. Although these hemiboronic heterocycles are unambiguously shown to behave as Lewis acids in aqueous solutions, boraza derivatives possess partial aromaticity provided their nitrogen lone electron pair is sufficiently available to participate in extended delocalization. As demonstrated by dynamic exchange and crossover experiments, these heterocycles are stable in neutral aqueous medium, and their measured pKa values are consistent with the ability of the endocyclic heteroatom substituent to stabilize a partial negative charge in the conjugate base. Altogether, this study corrects previous inaccuracies and provides conclusions regarding the properties of these compounds that are important toward the methodical application of hemiboronic and other boron heterocycles in catalysis, bioconjugation, and medicinal chemistry.

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells.

Synthetic bioconjugation at cysteine (Cys) residues in peptides and proteins has emerged as a powerful tool in chemistry. Soft nucleophilicity of the sulfur in Cys renders an exquisite chemoselectivity with which various functional groups can be placed onto this residue under benign conditions. While a variety of reactions have been successful at producing Cys-based bioconjugates, the majority of these feature sulfur-carbon bonds. We report Cys-borylation, wherein a benchtop stable Pt(II)-based organometallic reagent can be used to transfer a boron-rich cluster onto a sulfur moiety in unprotected peptides forging a boron-sulfur bond. Cys-borylation proceeds at room temperature and tolerates a variety of functional groups present in complex polypeptides. Further, the bioconjugation strategy can be applied to a model protein modification of Cys-containing DARPin (designed ankyrin repeat protein). The resultant bioconjugates show no additional toxicity compared to their Cys alkyl-based congeners. Finally, we demonstrate how the developed Cys-borylation can enhance the proteolytic stability of the resultant peptide bioconjugates while maintaining the binding affinity to a protein target.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the need for rapid and sensitive protein detection and quantification in simple and robust formats for widespread point-of-care applications. Here, we report on nanobody-functionalized organic electrochemical transistors with a modular architecture for the rapid quantification of single-molecule-to-nanomolar levels of specific antigens in complex bodily fluids. The sensors combine a solution-processable conjugated polymer in the transistor channel and high-density and orientation-controlled bioconjugation of nanobody–SpyCatcher fusion proteins on disposable gate electrodes. The devices provide results after 10 min of exposure to 5 μl of unprocessed samples, maintain high specificity and single-molecule sensitivity in human saliva and serum, and can be reprogrammed to detect any protein antigen if a corresponding specific nanobody is available. We used the sensors to detect green fluorescent protein, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV) spike proteins, and for the COVID-19 screening of unprocessed clinical nasopharyngeal swab and saliva samples with a wide range of viral loads. Organic electrochemical transistors functionalized with antigen-specific nanobodies can rapidly detect attomolar-to-nanomolar levels of the antigens in complex bodily fluids.

Identification of tumors which over-express Epidermal Growth Factor Receptor (EGFR) is important in selecting patients for anti-EGFR therapies. Enzymatic bioconjugation was used to introduce positron-emitting radionuclides (89Zr, 64Cu) into an anti-EGFR antibody fragment for Positron Emission Tomography (PET) imaging the same day as injection. A monovalent antibody fragment with high affinity for EGFR was engineered to include a sequence that is recognized by the transpeptidase sortase A. Two different metal chelators, one for 89ZrIV and one for 64CuII, were modified with a N-terminal glycine to enable them to act as substrates in sortase A mediated bioconjugation to the antibody fragment. Both fragments provided high-quality PET images of EGFR positive tumors in a mouse model at 3 hours post-injection, a significant advantage when compared to radiolabeled full antibodies that require several days between injection of the tracer and imaging. The use of enzymatic bioconjugation gives reproducible homogeneous products with the metal complexes selectively installed on the C-terminus of the antibody potentially simplifying regulatory approval., Enzymatic bioconjugation to introduce positron-emitting radionuclides (89Zr, 64Cu) into an anti-EGFR antibody fragment allows same day imaging with positron emission tomography.

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related to weak substrate binding and ligand relocalization. The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), the most important drug-metabolizing enzyme. Allosteric activation and inhibition of CYP3A4 by pharmaceuticals is an important mechanism of drug interactions. We performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic) or do not show activation while still blocking the allosteric site (antagonistic). This allowed us to better define the position of the allosteric site, the protein structural dynamics associated with allosteric activation, and the presence of coexisting conformers.

Human Interleukin 2 (IL-2) has already achieved impressive results as a therapeutic agent for cancer and autoimmune diseases. However, one of the limitations associated with the clinical application of IL-2 is its short half-life owing to rapid clearance by the kidneys. Modification with fatty acids, as an albumin noncovalent ligand with the advantage of deep penetration into tissues and high activity-to-mass ratio, is a commonly used approach to improve the half-life of native peptides and proteins. In this investigation, we attempted to extend the half-life of IL-2 through conjugation with a fatty acid using sortase A (srtA). We initially designed and optimized three IL-2 analogues with different peptide linkers between the C-terminus of IL-2 and srtA recognition sequence (LPETG). Among these, analogue A3 was validated as the optimal IL-2 analogue for further modification. Next, six fatty acid moieties with the same fatty acid and different hydrophilic spacers were conjugated to A3 through srtA. The six bioconjugates generated were screened for in vitro biological activity, among which bioconjugate B6 was identified as near-optimal to IL-2. Additionally, B6 could effectively bind albumin through the conjugated fatty acid, which contributed to a significant improvement in its pharmacokinetic properties in vivo. In summary, we have developed a novel IL-2 bioconjugate, B6, modified with fatty acids using srtA, which may effectively serve as a new-generation long-acting IL-2 immunotherapeutic agent.

Guanidinoglycosides are a class of non-cytotoxic molecular transporters capable of delivering high molecular weight bioactive cargos into cells at low nanomolar concentrations. Efficient bioconjugation with guanidinoglycosides has been previously demonstrated by utilizing a guanidinoneomycin decorated with a reactive but also unstable N-hydroxysuccinimmide ester-containing linker. Herein we report the synthesis, chemistry, and application of a new, stable guanidinoneomycin derivative armed with a highly specific maleimide moiety which allows for thiol-maleimide click chemistry, a highly popular bioconjugation strategy, widening the field of application of these intriguing and useful delivery vehicles.

Herein we disclose the transformation of maleimides into water-soluble tris(2-carboxyethyl)phosphonium ylides and their subsequent application in the bioconjugation of protein- and peptide-linked aldehydes. The new entry into Wittig bioconjugate chemistry proceeds under mild conditions and relies on highly water soluble reagents, which are likely already part of most biochemists’ inventory. Authors in thesis list of papers: Hartmann, R. W., Pijnappel, M., Nilvebrant, J., Nygren, P.-Å., Lehmann, F., Odell, L. R.

An Ag(i)-catalyzed tandem addition-cyclization of isothiocyanate and propargylamine was successfully applied to the synthesis of 2-amino-4-methylenethiazolines. This route features an unprecedented fast reaction rate with full conversion reached within 10 min at room temperature for aromatic isothiocyanates and excellent chemoselectivity for exocyclic products. The application of this strategy is further highlighted by the accelerated bioconjugation of propargylamine with fluorescein isothiocyanate (FITC) under Ag(i)-catalysis.

Dynamic bonds continually form and dissociate at equilibrium. Carbonyl compounds with proximal boronic acids, including 2-formylphenylboronic acid (2-FPBA), have been reported to form highly dynamic covalent hydrazone and oxime bonds in physiological conditions, but strategies to tune the dynamics have not yet been reported. Here, we characterize the dynamics of 2-FPBA-derived hydrazones and oximes and account for both the rapid rate of formation (∼102-103 M-1 s-1) and the relatively fast rate of hydrolysis (∼10-4 s-1) at physiological pH. We further show that these substrates undergo exchange with α-nucleophiles, which can be reversibly paused and restarted with pH control. Finally, we show that oxidation of the arylboronic acid effectively abolishes the rapid dynamics, which slows the forward reaction by more than 30 000 times and increases the hydrolytic half-life from 50 minutes to 6 months at physiological pH. These results set the stage to explore these linkages in dynamic combinatorial libraries, reversible bioconjugation, and self-healing materials.

Development of conceptually novel and practically useful bioconjugation reactions has been an intense pursuit of chemical biology research. Herein, we report an unprecedented bioconjugation reaction that hinges on a chemical trigger-enabled inverse-electron-demand Diels–Alder (IEDDA) cycloaddition of trans-cycloheptene (TCH) with tetrazine. Unlike the conventional strain-promoted bioconjugation reactions that utilize built-in strained alkenes as reactants, the current one features a “trigger-release-conjugate” reaction model, in which a highly strained TCH species is released from a bench-stable bicyclic N-nitrosourea (BNU) derivative upon treatment with an external stimulus. It is noteworthy that the reactivity–stability balance of BNU derivatives could be tuned by manipulating their N-1 substituents. As a proof-of-concept case, this new chemical trigger-enabled IEDDA reaction has been applied to in vitro protein labeling and pretargeted cell imaging. This work opens a new avenue to utilize BNU derivatives as a new class of chemical reporters in bioconjugate chemistry.

Sulfur electrophiles constitute an important class of covalent small molecules that have found widespread applications in synthetic chemistry and chemical biology. Various electrophilic scaffolds, including sulfonyl fluorides and arylfluorosulfates as recent examples, have been applied for protein bioconjugation to probe ligand sites amenable for chemical proteomics and drug discovery. In this review, we describe the development of sulfonyl-triazoles as a new class of electrophiles for sulfur–triazole exchange (SuTEx) chemistry. SuTEx achieves covalent reaction with protein sites through irreversible modification of a residue with an adduct group (AG) upon departure of a leaving group (LG). A principal differentiator of SuTEx from other chemotypes is the selection of a triazole heterocycle as the LG, which introduces additional capabilities for tuning the sulfur electrophile. We describe the opportunities afforded by modifications to the LG and AG alone or in tandem to facilitate nucleophilic substitution reactions at the SO2 center in cell lysates and live cells. As a result of these features, SuTEx serves as an efficient platform for developing chemical probes with tunable bioactivity to study novel nucleophilic sites on established and poorly annotated protein targets. Here, we highlight a suite of biological applications for the SuTEx electrophile and discuss future goals for this enabling covalent chemistry.

It is imminent to develop a new type of rapid COVID-19 detection method with high sensitivity. Here, we used novel red emission-enhanced carbon dot (CD)-based silica (RCS) spheres as the signals of lateral flow immunochromatography (LFI) to ultrasensitively detect novel severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins (SARS-CoV-2 NPs). The red emission of CDs can be enhanced and enriched in silica spheres by a simple way. The amino ends of the N-β-(aminoethyl)-γ-aminopropyltrimethoxy anchor carboxyl-rich CDs and enhance the red emission, while the other end is embedded in the silica carrier. Then, the composite silica spheres werecoated with 3-(triethylsilyl) propylamine to protect the CDs, promote bioconjugation and obtain RCS spheres. The optimal emission peaks of the aqueous solution and the solid state of RCS spheres were at 634 nm and 638 nm, respectively, with quantum yields (QYs) of 48.5% and 35.7%, respectively. Their red emission has a wide excitation range (from the ultraviolet region to the red region), and the best excitation wavelength is about 580 nm. Two fluorescence detection modes of the RCS-LFI technology for the SARS-CoV-2 NP assay are available: the simple mode of observation under ultraviolet light has a sensitivity of 100 pg mL-1; the advanced mode of detection under a fluorescence microscope has a sensitivity of 10 pg mL-1. This assay also exhibits the advantages of fast detection speed, high specificity, and simple operation. In addition, the feasibility of this method in actual sample detection was verified in human serum by the standard-addition method, and the results show that the method has excellent practicability. We believe that this method will be a valuable supplement for the diagnosis of COVID-19.

Bioconjugation reactions play a central facilitating role in engendering modified peptides and proteins. Early progress in this area was inhibited by challenges such as the limited range of substrates and the relatively poor biocompatibility of bioconjugation reagents. However, the recent developments in visible-light induced photoredox catalysis and electrochemical catalysis reactions have permitted significant novel reactivities to be developed in the field of synthetic and bioconjugation chemistry. This perspective describes recent advances in the use of biocompatible energy input for the modification of peptides and proteins mainly, via the single electron transfer (SET) process, as well as key future developments in this area. Photochemical and electrochemical approaches to protein and peptide modification offer a valuable complement to the use of stoichiometric reagents. Here recent developments in bioconjugation methodology relying on single electron transfer are described.

Here we report the first use of ligand‐directed proximity accelerated bioconjugation chemistry in the tandem delivery and release of a therapeutic payload. To do this, we designed a nitrophenol carbonate for ligand‐directed in situ bioconjugation of a prodrug payload to a protein. The transient nature of our conjugation chemistry renders the protein a depot for time‐dependent release of active drug following hydrolysis and self‐immolation. In our model system, using an immunostimulant prodrug, biotin ligand, and avidin protein, we observe release of bioavailable immunostimulant both spectroscopically and with an immune cell line over 48 h. Avidin co‐crystalized with the nitrophenolate directing group verified the binding pose of the ligand and offered insight into the mechanism of in situ bioconjugation. Overall, this scaffold warrants further investigation for the time‐dependent delivery of therapeutics and use in protein ligand pairs beyond biotin and avidin used for this work., Ligand‐directed proximity accelerated bioconjugation to deliver a therapeutic payload using a novel nitrophenol carbonate derivative. In this work, we demonstrate the utility for the ligand‐directed in situ labeling of a protein avidin with an immunostimulant prodrug. This renders the target protein a depot for time‐dependent release of active drug. The mechanism is probed using crystallography, and time‐dependent drug release is demonstrated in vitro.

A review of the heretofore less explored approach of tyrosine bioconjugation, which is rapidly becoming a constructive alternative/complement to the more well-established strategies, is provided., Protein bioconjugation is an increasingly important field of research, with wide-ranging applications in areas such as therapeutics and biomaterials. Traditional cysteine and lysine bioconjugation strategies are widely used and have been extensively researched, but in some cases they are not appropriate and alternatives are needed or they are not compatible with one another to enable the formation of dually (and distinctly) modified dual-conjugates (an increasingly desired class of bioconjugates). Here we review the heretofore less explored approach of tyrosine bioconjugation, which is rapidly becoming a constructive alternative/complement to the more well-established strategies. Herein we present an overview of the field, and then focus on promising recent methods that can achieve high conversion and chemoselectivity. This suggests that not only can tyrosine bioconjugation be used in conjunction with cysteine and lysine modification to obtain proteins with multiple different modifications, it is also becoming a stand-alone alternative to these more traditional methods.

Copyright © 2020 American Chemical Society, This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods., Financial support for this work was provided by Bristol-Myers Squibb, the NIH (GM-118176), the Marie Skłodowska-CurieGlobal Fellowships (749359-EnanSET to N.M.P) within the European Union’s Research and Innovation Framework Programme (2014−2020), FCT Portugal (IF/00624/2015), and the Royal Society (URF\R\180019). D.F. was supported by the National Center for Advancing Translational Sciences,National Institutes of Health, through Grant UL1 TR002551 and Linked Award TL1 TR002551.

Metal nanoparticles (NP) that exhibit localized surface plasmon resonance play an important role in metal‐enhanced fluorescence (MEF) and surface‐enhanced Raman scattering (SERS). Among the optical biosensors, MEF and SERS stand out to be the most sensitive techniques to detect a wide range of analytes from ions, biomolecules to macromolecules and microorganisms. Particularly, anisotropic metal NPs with strongly enhanced electric field at their sharp corners/edges under a wide range of excitation wavelengths are highly suitable for developing the ultrasensitive plasmon‐enhanced biosensors. In this review, we first highlight the reliable methods for the synthesis of anisotropic gold NPs and silver NPs in high yield, as well as their alloys and composites with good control of size and shape. It is followed by the discussion of different sensing mechanisms and recent advances in the MEF and SERS biosensor designs. This includes the review of surface functionalization, bioconjugation and (directed/self) assembly methods as well as the selection/screening of specific biorecognition elements such as aptamers or antibodies for the highly selective bio‐detection. The right combinations of metal nanoparticles, biorecognition element and assay design will lead to the successful development of MEF and SERS biosensors targeting different analytes both in‐vitro and in‐vivo. Finally, the prospects and challenges of metal‐enhanced biosensors for future nanomedicine in achieving ultrasensitive and fast medical diagnostics, high‐throughput drug discovery as well as effective and reliable theranostic treatment are discussed., Plasmonic nanostructures with anisotropic shapes possess dramatic enhanced electric field when they interact with light. This unique feature has led to the design of metal‐enhanced fluorescence (MEF) biosensors and surface‐enhanced Raman scattering (SERS) biosensors with ultrasensitivity, where metal nanostructure plays an important role as plasmonic enhancer to amplify the optical signals for biological detection.

Conjugate vaccines against encapsulated pathogens like Streptococcus pneumoniae face many challenges, including the existence of multiple serotypes with a diverse global distribution that constantly requires new formulations and higher coverage. Multivalency is usually achieved by combining capsular polysaccharide-protein conjugates from invasive serotypes, and for S. pneumoniae, this has evolved from 7- up to 20-valent vaccines. These glycoconjugate formulations often contain high concentrations of carrier proteins, which may negatively affect glycoconjugate immune response. This work broadens the scope of an efficient multicomponent strategy, leading to multivalent pneumococcal glycoconjugates assembled in a single synthetic operation. The bioconjugation method, based on the Ugi four-component reaction, enables the one-pot incorporation of two different polysaccharide antigens to a tetanus toxoid carrier, thus representing the fastest approach to achieve multivalency. The reported glycoconjugates incorporate three combinations of capsular polysaccharides 1, 6B, 14, and 18C from S. pneumoniae. The glycoconjugates were able to elicit functional specific antibodies against pneumococcal strains comparable to those shown by mixtures of the two monovalent glycoconjugates.

The hydrazine group serves as a great anchor for bioconjugation; however, the application of hydrazone ligation has been limited by poor product stability. We aim to resolve such issues by optimizing the recently established pyrazolone ligation and investigating a new pyrazole ligation. We have identified a new, electron-deficient pyrazolone ligation and a regiospecific pyrazole ligation, both offering aqueous buffer stable and chemically inert products possessing triazole-like structures while not involving any heavy metal catalyst.

A pair of 9-mesityl-10-phenyl acridinium (Mes-Acr+ ) photoredox catalysts were synthesized with an iodoacetamide handle for cysteine bioconjugation. Covalently tethering of the synthetic Mes-Acr+ cofactors with a small panel of thermostable protein scaffolds resulted in 12 new artificial enzymes. The unique chemical and structural environment of the protein hosts had a measurable effect on the photophysical properties and photocatalytic activity of the cofactors. The constructed Mes-Acr+ hybrid enzymes were found to be active photoinduced electron-transfer catalysts, controllably oxidizing a variety of aryl sulfides when irradiated with visible light, and possessed activities that correlated with the photophysical characterization data. Their catalytic performance was found to depend on multiple factors including the Mes-Acr+ cofactor, the protein scaffold, the location of cofactor immobilization, and the substrate. This work provides a framework toward adapting synthetic photoredox catalysts into artificial cofactors and includes important considerations for future bioengineering efforts.

The synthesis of protein–protein and protein–peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras., Enzymatic oxidation of tyrosine residues followed by reaction with cysteine thiols allows the covalent coupling of proteins and peptides using only native amino acid side chains.