A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full‐length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε‐amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site‐specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site‐specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.     

Sugar–Protein Connectivity Impacts on the Immunogenicity of Site‐Selective Salmonella O‐Antigen Glycoconjugate Vaccines

A series of glycoconjugates with defined connectivity were synthesized to investigate the impact of coupling Salmonella typhimurium O‐antigen to different amino acids of CRM₁₉₇ protein carrier. In particular, two novel methods for site‐selective glycan conjugation were developed to obtain conjugates with single attachment site on the protein, based on chemical modification of a disulfide bond and pH‐controlled transglutaminase‐catalyzed modification of lysine, respectively. Importantly, conjugation at the C186‐201 bond resulted in significantly higher anti O‐antigen bactericidal antibody titers than coupling to K37/39, and in comparable titers to conjugates bearing a larger number of saccharides. This study demonstrates that the conjugation site plays a role in determining the immunogenicity in mice and one single attachment point may be sufficient to induce high levels of bactericidal antibodies.     

Affinity-guided protein conjugation: the trilogy of covalent protein labeling,assembly and inhibition

Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an “affinity-guided covalent conjugation” strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative (which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made “reactive peptides” for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of “affinity-guided covalent conjugation” in various applications.     

Retaining the structural integrity of disulfide bonds in diphtheria toxoid carrier protein is crucial for the effectiveness of glycoconjugate vaccine candidates

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases. The covalent conjugation of relevant polysaccharide antigens to immunogenic carrier proteins enables the induction of a long-lasting and robust IgG antibody response, which is not observed for pure polysaccharide vaccines. Although there has been remarkable progress in the development of glycoconjugate vaccines, many crucial parameters remain poorly understood. In particular, the influence of the conjugation site and strategy on the immunogenic properties of the final glycoconjugate vaccine is the focus of intense research. Here, we present a comparison of two cysteine selective conjugation strategies, elucidating the impact of both modifications on the structural integrity of the carrier protein, as well as on the immunogenic properties of the resulting glycoconjugate vaccine candidates. Our work suggests that conjugation chemistries impairing structurally relevant elements of the protein carrier, such as disulfide bonds, can have a dramatic effect on protein immunogenicity.

The introduction of glycoconjugate vaccines marks an important point in the fight against various infectious diseases.     

Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.     

Site-specific PEGylation of Human Growth Hormone by Mutated Sortase A

Human growth hormone(hGH), a classic therapeutic protein, which promotes growth and wound healing, is released from the pituitary gland. As a protein drug, its short half-life is its main barrier to therapeutic efficacy. Various strategies have been designed to prolong its serum half-life, the most common of which is the conjugation with polyethylene glycol(PEG), as this has been shown to significantly extend protein's serum half-life. However, PEGylation often results in random conjugation, which can lead to impaired protein function and hinder purification, characterization and evaluation of the PEGylated protein. Therefore, site specific PEGylation is a promising direction for PEG-protein conjugation. Here we took advantages of the mutated sortase A(7M) enzyme, which can enzymatically ligate the universal α-amino acids to a C-terminal tagged protein. This then allows specific modification of the C-terminal of hGH with PEG. This site-specific bound PEG-hGH has similar efficacy, receptor binding and cell proliferation as wild-type hGH; however, pharmacokinetic analysis demonstrates that its serum half-life is almost 24 times that of wild-type hGH. Herein, we provided a promising advancement in the development of site specific PEGylated therapeutic proteins.     

Biological–synthetic hybrid block copolymers: Combining the best from two worlds

Although biopolymers and synthetic polymers share many common features, each of these two classes of materials is also characterized by a distinct and very specific set of advantages and disadvantages. Combining biopolymer elements with synthetic polymers into a single macromolecular conjugate is an interesting strategy for synergetically merging the properties of the individual components and overcoming some of their limitations. This article focuses on a special class of biological–synthetic hybrids that are obtained by site‐selective conjugation of a protein or peptide and a synthetic polymer. The first part of the article gives an overview of the different liquid‐phase and solid‐phase techniques that have been developed for the synthesis of well‐defined, that is, site‐selectively conjugated, synthetic polymer–protein hybrids. In the second part, the properties and potential applications of these materials are discussed. The conjugation of biological and synthetic macromolecules allows the modulation of protein binding and recognition properties and is a powerful strategy for mediating the self‐assembly of synthetic polymers. Synthetic polymer–protein hybrids are already used as medicines and show significant promise for bioanalytical applications and bioseparations. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1–17, 2005     

Site‐Selective Labeling of a Lysine Residue in Human Serum Albumin

Conjugation to human serum albumin (HSA) has emerged as a powerful approach for extending the in vivo half‐life of many small molecule and peptide/protein drugs. Current HSA conjugation strategies, however, can often yield heterogeneous mixtures with inadequate pharmacokinetics, low efficacies, and variable safety profiles. Here, we designed and synthesized analogues of TAK‐242, a small molecule inhibitor of Toll‐like receptor 4, that primarily reacted with a single lysine residue of HSA. These TAK‐242‐based cyclohexene compounds demonstrated robust reactivity, and Lys64 was identified as the primary conjugation site. A bivalent HSA conjugate was also prepared in a site‐specific manner. Additionally, HSA‐cyclohexene conjugates maintained higher levels of stability both in human plasma and in mice than the corresponding maleimide conjugates. This new conjugation strategy promises to broadly enhance the performance of HSA conjugates for numerous applications.     

A chemoenzymatic approach enables the site‐specific conjugation of recombinant proteins

Many biotechniques including protein microarray, drug screening, biosensors rely on the immobilization of recombinant proteins on the solid supports. It is well known that random orientation of the immobilized proteins could impair their biologic functions. Thus, it is very important to develop new site‐specific immobilization approach. In this study, we presented a chemoenzymatic approach for site‐specific conjugation of recombinant proteins onto solid support. In this strategy, the affinity tag on recombinant protein was enzymatically cleaved to expose the N‐terminal serine, which was oxidized to carry an aldehyde group and was then covalently coupled to hydrazide resin through hydrazone ligation. As this approach takes advantage of the most frequently used TEV protease, it requires no further sequence design on recombinant protein. This method was validated by site specific coupling of a synthetic peptide and a recombinant protein onto solid supports. It was found that the site specific immobilized SH2 domain is functional and could be used to enrich tyrosine phosphorylated peptides.     

Enzyme-Polymer-Conjugate-Based Pickering Emulsions for Cell-Free Expression and Cascade Biotransformation

In this study, we addressed the limitations of conventional enzyme-polymer-conjugate-based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from nonspecific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non-canonical amino acid (ncAA) at a specific site on target enzymes, we enabled precise polymer-enzyme conjugation. These engineered conjugates then acted as biocatalytically active emulsifiers to stabilize Pickering emulsions, while encapsulating a cell-free protein synthesis (CFPS) system in the aqueous phase for targeted enzyme expression. The resulting cascade reaction system leveraged enzymes expressed in the aqueous phase and on the emulsion interface for optimized chemical biosynthesis. The use of the cell-free system eliminated the need for intact whole cells or purified enzymes, representing a significant advancement in biocatalysis. Remarkably, the integration of Pickering emulsion, precise enzyme-polymer conjugation, and CFPS resulted in a fivefold enhancement in catalytic performance as compared to traditional single-phase reactions. Therefore, our approach harnesses the combined strengths of advanced biochemical engineering techniques, offering an efficient and practical solution for the synthesis of value-added chemicals in various biocatalysis and biotransformation applications.     

A Cyclopropene Electrophile that Targets Glutathione S‐Transferase Omega‐1 in Cells

Cyclopropenes are an important new addition to the portfolio of functional groups that can be used for bioorthogonal couplings. The inert nature of these highly strained compounds in complex biological systems is almost counterintuitive given their established electrophilic properties in organic synthesis. Here we provide the first demonstration of a cyclopropene that is capable of direct conjugation to protein targets in cells and show that this compound preferentially alkylates the active site cysteine of glutathione S‐transferase omega‐1 (GSTO1).     

Separation efficiency of free‐solution conjugated electrophoresis with drag‐tags incorporating a synthetic amino acid

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge‐to‐friction ratio in free solution, DNA fragments cannot be separated by size. However, size‐based separation of DNA is possible in free‐solution conjugate electrophoresis (FSCE) if a “drag‐tag” is attached to DNA fragments because the tag breaks the linear charge‐to‐friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free‐solution conjugated electrophoresis, generation of a monodisperse drag‐tag and identification of a strong, site‐specific conjugation method between a DNA fragment and a drag‐tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin‐like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site‐specific conjugation, a methionine residue in drag‐tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site.     

Semi-synthesis of a protease-activatable collagen targeting probe

A protease-activatable collagen targeting probe (proCNA35) is synthesized by conjugation of a synthetic collagen fragment to the collagen binding protein CNA35 via a protease-cleavable linker. Cleavage of the linker by MMP1 releases the intramolecular inhibition of the collagen binding site and restores its collagen binding capacity.     

Monitoring Endocytic Trafficking of Anthrax Lethal Factor by Precise and Quantitative Protein Labeling

Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site‐specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF’s membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild‐type LF. This allowed time‐lapse imaging of LF’s natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies.     

Synthesis of fluorescent oligonucleotide--EYFP conjugate: towards supramolecular construction of semisynthetic biomolecular antennae

A novel species of DNA--protein conjugate was synthesized by chemically linking DNA oligonucleotides to Aequorea victoria green fluorescent protein mutant EYFP. An additional cysteine was added to the C-terminus of the EYFP by genetic engineering and used to covalently attach amino-modified oligonucleotide with the aid of the heterobifunctional crosslinker sSMCC. EYFP maintained its fluorescence upon conjugation. The oligonucleotide provides an additional binding site to the fluorescent protein, and hence, the EYFP conjugate could be specifically hybridized with both complementary DNA-protein conjugates in-solution as well as immobilized at capture oligonucleotides attached to a solid substrate. These studies are paving the way for future applications in the self-assembly of photoactive supramolecular complexes, such as artificial light-harvesting systems.     

Insights Into the Allosteric Inhibition of the SUMO E2 Enzyme Ubc9

Conjugation of the small ubiquitin‐like modifier (SUMO) to protein substrates is an important disease‐associated posttranslational modification, although few inhibitors of this process are known. Herein, we report the discovery of an allosteric small‐molecule binding site on Ubc9, the sole SUMO E2 enzyme. An X‐ray crystallographic screen was used to identify two distinct small‐molecule fragments that bind to Ubc9 at a site distal to its catalytic cysteine. These fragments and related compounds inhibit SUMO conjugation in biochemical assays with potencies of 1.9–5.8 mm . Mechanistic and biophysical analyses, coupled with molecular dynamics simulations, point toward ligand‐induced rigidification of Ubc9 as a mechanism of inhibition.     

Biocompatible functionalization of polymersome surfaces: a new approach to surface immobilization and cell targeting using polymersomes

Vesicles assembled from amphiphilic block copolymers represent promising nanomaterials for applications that include drug delivery and surface functionalization. One essential requirement to guide such polymersomes to a desired site in vivo is conjugation of active, targeting ligands to the surface of preformed self-assemblies. Such conjugation chemistry must fulfill criteria of efficiency and selectivity, stability of the resulting bond, and biocompatibility. We have here developed a new system that achieves these criteria by simple conjugation of 4-formylbenzoate (4FB) functionalized polymersomes with 6-hydrazinonicotinate acetone hydrazone (HyNic) functionalized antibodies in aqueous buffer. The number of available amino groups on the surface of polymersomes composed of poly(dimethylsiloxane)-block-poly(2-methyloxazoline) diblock copolymers was investigated by reacting hydrophilic succinimidyl-activated fluorescent dye with polymersomes and evaluating the resulting emission intensity. To prove attachment of biomolecules to polymersomes, HyNic functionalized enhanced yellow fluorescent protein (eYFP) was attached to 4FB functionalized polymersomes, resulting in an average number of 5 eYFP molecules per polymersome. Two different polymersome-antibody conjugates were produced using either antibiotin IgG or trastuzumab. They showed specific targeting toward biotin-patterned surfaces and breast cancer cells. Overall, the polymersome-ligand platform appears promising for therapeutic and diagnostic use.     

Orthogonal Cysteine Protection Enables Homogeneous Multi‐Drug Antibody–Drug Conjugates

A strategy for the preparation of homogeneous antibody–drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site‐specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug‐linkers that have differing physiochemical properties and exert complementary anti‐cancer activities. Dual‐auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual‐drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site‐specific protein modification.     

Site‐Selective Cysteine–Cyclooctyne Conjugation

We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water.     

Determination of ornithine conjugates of some carboxylic acids in birds by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the determination of ornithine conjugation of some carboxylic acids in vitro has been developed. The ornithine conjugates of benzoic acid, p-nitrobenzoic acid, furancarboxylic acid and phenylacetic acid in an incubation mixture with kidney mitochondria were well separated on a reversed-phase C18 column using a mixture of 10 mM ammonium acetate buffer and methanol as the mobile phase. In addition, by varying the pH of the mobile phase and utilizing the absorption wavelengths (nm) of the conjugates it was possible to resolve and specifically detect each conjugate. The calibration curves were linear in the range of 0.2-16 micrograms/ml for all compounds and the detection limits were about 50 ng/ml except for the ornithine conjugate of phenyl acetic acid (S/N = 2). The ornithine conjugation of some carboxylic acids with chicken kidney mitochondria were determined by this assay method. The activity of ornithine conjugation of benzoic acid, furancarboxylic acid, p-nitrobenzoic acid and phenylacetic acid were 14.5, 5.5, 0.5 and 6.9 nmol/mg of protein, respectively. Moreover, the ornithine conjugation and the glycine conjugation of benzoic acid were examined in birds and rodents. The ornithine conjugation was observed only in chicken (14.5 nmol/mg of protein) and mallard (0.99 nmol/mg of protein).