Strain-promoted Azide-alkyne Cycloaddition Research Articles

Addition of Lauryldimethylamine N-Oxide (LDAO) to a Copper-Free Click Chemistry Reaction Improves the Conjugation Efficiency of a Cell-Free Generated CRM197 Variant to Clinically Important Streptococcus pneumoniae Serotypes.

Strain-promoted azide–alkyne cycloaddition (SPAAC) reactions like click chemistry have the potential to be highly scalable, robust, and cost-effective methods for generating small- and large-molecule conjugates for a variety of applications. However, despite method improvements, the rates of copper-based click chemistry reactions continue to be much faster than the rates of copper-free click chemistry reactions, which makes broader deployment of click chemistry challenging from a safety and compatibility standpoint. In this study, we used a zwitterionic detergent, namely, lauryldimethylamine N-oxide (LDAO), in a copper-free click chemistry reaction to investigate its impact on the generation of conjugate vaccines (CVs). For this, we utilized an Xpress cell-free protein synthesis (CFPS) platform to generate a proprietary variant of CRM197 (eCRM) containing non-native amino acids (nnAA) with azide-containing side chains as a carrier protein for conjugation to several clinically relevant dibenzocyclooctyne (DBCO)-derivatized S. pneumoniae serotypes (types 3, 5, 18C, and 19A). For conjugation, we performed copper-free click chemistry in the presence and absence of LDAO. Our results show that the addition of LDAO significantly enhanced the reaction kinetics to generate larger conjugates, which were similarly immunogenic and equally stable to conjugates generated without LDAO. Most importantly, the addition of LDAO substantially improved the efficiency of the conjugation process. Thus, our results for the first time show that the addition of a zwitterionic surfactant to a copper-free click chemistry reaction can significantly accelerate the reaction kinetics along with improving the efficiency of the conjugation process.

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins.

Post-translational glycosylation of proteins results in complex mixtures of heterogeneous protein glycoforms. Glycoproteins have many potential applications from fundamental studies of glycobiology to potential therapeutics, but generating homogeneous recombinant glycoproteins using chemical or chemoenzymatic reactions to mimic natural glycoproteins or creating homogeneous synthetic neoglycoproteins is a challenging synthetic task. In this work, we use a site-specific bioorthogonal approach to produce synthetic homogeneous glycoproteins. We develop a bifunctional, bioorthogonal linker that combines oxime ligation and strain-promoted azide–alkyne cycloaddition chemistry to functionalize reducing sugars and glycan derivatives for attachment to proteins. We demonstrate the utility of this minimal length linker by producing neoglycoprotein inhibitors of cholera toxin in which derivatives of the disaccharide lactose and GM1os pentasaccharide are attached to a nonbinding variant of the cholera toxin B-subunit that acts as a size- and valency-matched multivalent scaffold. The resulting neoglycoproteins decorated with GM1 ligands inhibit cholera toxin B-subunit adhesion with a picomolar IC50.

Lysine-Directed Site-Selective Bioconjugation for the Creation of Radioimmunoconjugates.

The synthesis of radioimmunoconjugates via the stochastic attachment of bifunctional chelators to lysines can yield heterogeneous products with suboptimal in vitro and in vivo behavior. In response to this, several site-selective approaches to bioconjugation have been developed, yet each has intrinsic drawbacks, such as the need for expensive reagents or the complexity of incorporating unnatural amino acids into IgGs. Herein, we describe the use of a simple and facile approach to lysine-directed site-selective bioconjugation for the generation of radioimmunoconjugates. This strategy relies upon on the selective modification of single lysine residues within each light chain of the monoclonal antibody (mAb) with a branched azide-bearing perfluorophenyl ester (PFP-bisN3) followed by the ligation of dibenzocyclooctyne (DBCO)-bearing payloads to these bioorthogonal handles via the strain-promoted azide-alkyne cycloaddition. This methodology was used to create [89Zr]Zr-SSKDFO-pertuzumab, a radioimmunoconjugate of the HER2-targeting mAb pertuzumab labeled with desferrioxamine (DFO) and the positron-emitting radiometal zirconium-89 (89Zr). [89Zr]Zr-SSKDFO-pertuzumab was compared to a pair of analogous probes: one synthesized via random lysine modification ([89Zr]Zr-DFO-pertuzumab) and another via thiol-maleimide chemistry ([89Zr]Zr-malDFO-pertuzumab). The bioconjugation strategy was assessed using ESI mass spectrometry, SDS-PAGE, and autoradiography. All three immunoconjugates demonstrated comparable binding to HER2 via flow cytometry and surface plasmon resonance (SPR), and 89Zr-labeled variants of each were synthesized in >99% radiochemical yield and molar activities of up to ∼55.5 GBq/μmol (10 mCi/mg). Subsequently, the in vivo behavior of this trio of 89Zr-immunoPET probes was interrogated in athymic nude mice bearing subcutaneous HER2-expressing BT-474 human breast cancer xenografts. [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab produced positron emission tomography (PET) images with high tumoral uptake and high tumor-to-healthy organ activity concentration ratios. A terminal biodistribution study complemented the PET results, revealing tumoral activity concentrations of 126.9 ± 50.3%ID/g, 86.9 ± 53.2%ID/g, and 92.5 ± 27.2%ID/g at 144 h post-injection for [89Zr]Zr-SSKDFO-pertuzumab, [89Zr]Zr-malDFO-pertuzumab, and [89Zr]Zr-DFO-pertuzumab, respectively. Taken together, the data clearly illustrate that this highly modular and facile approach to site-selective bioconjugation produces radioimmunoconjugates that are better-defined and more homogeneous than stochastically modified constructs and also exhibit excellent in vitro and in vivo performance. Furthermore, we contend that this lysine-directed strategy holds several key advantages over extant approaches to site-selective bioconjugation, especially in the context of production for the clinic.

Polymer selection impacts the pharmaceutical profile of site-specifically conjugated Interferon-α2a.

Conjugation of poly(ethylene glycol) (PEG) to biologics is a successful strategy to favorably impact the pharmacokinetics and efficacy of the resulting bioconjugate. We compare bioconjugates synthesized by strain-promoted azide-alkyne cycloaddition (SPAAC) using PEG and linear polyglycerol (LPG) of about 20kDa or 40kDa, respectively, with an azido functionalized human Interferon-α2a (IFN-α2a) mutant. Site-specific PEGylation and LPGylation resulted in IFN-α2a bioconjugates with improved in vitro potency compared to commercial Pegasys. LPGylated bioconjugates had faster disposition kinetics despite comparable hydrodynamic radii to their PEGylated analogues. Overall exposure of the PEGylated IFN-α2a with a 40kDa polymer exceeded Pegasys, which, in return, was similar to the 40kDa LPGylated conjugates. The study points to an expanded polymer design space through which the selected polymer class may result in a different distribution of the studied bioconjugates.

PEtOxylated Interferon-α2a Bioconjugates Addressing H1N1 Influenza A Virus Infection.

Influenza A viruses (IAV), including the pandemic 2009 (pdm09) H1N1 or avian influenza H5N1 virus, may advance into more pathogenic, potentially antiviral drug-resistant strains (including loss of susceptibility against oseltamivir). Such IAV strains fuel the risk of future global outbreaks, to which this study responds by re-engineering Interferon-α2a (IFN-α2a) bioconjugates into influenza therapeutics. Type-I interferons such as IFN-α2a play an essential role in influenza infection and may prevent serious disease courses. We site-specifically conjugated a genetically engineered IFN-α2a mutant to poly(2-ethyl-2-oxazoline)s (PEtOx) of different molecular weights by strain-promoted azide-alkyne cyclo-addition. The promising pharmacokinetic profile of the 25 kDa PEtOx bioconjugate in mice echoed an efficacy in IAV-infected ferrets. One intraperitoneal administration of this bioconjugate, but not the marketed IFN-α2a bioconjugate, changed the disease course similar to oseltamivir, given orally twice every study day. PEtOxylated IFN-α2a bioconjugates may expand our therapeutic arsenal against future influenza pandemics, particularly in light of rising first-line antiviral drug resistance to IAV.

Intracellular Crowding by Bio-Orthogonal Hydrogel Formation Induces Reversible Molecular Stasis.

To survive extreme conditions, certain animals enter a reversible protective stasis through vitrification of the cytosol by polymeric molecules such as proteins and polysaccharides. In this work, synthetic gelation of the cytosol in living cells is used to induce reversible molecular stasis. Through the sequential lipofectamine-mediated transfection of complementary poly(ethylene glycol) macromers into mammalian cells, intracellular crosslinking occurs through bio-orthogonal strain-promoted azide-alkyne cycloaddition click reactions. This achieves efficient polymer uptake with minimal cell death (99% viable). Intracellular crosslinking decreases DNA replication and protein synthesis, and increases the quiescent population by 2.5-fold. Real-time tracking of single cells containing intracellular crosslinked polymers identifies increases in intermitotic time (15 hvs 19 h) and decreases in motility (30 µm h-1 vs 15µm h-1 ). The cytosol viscosity increases threefold after intracellular crosslinking and results in disordered cytoskeletal structure in addition to the disruption of cellular coordination in a scratch assay. By incorporating photodegradable nitrobenzyl moieties into the polymer backbone, the effects of intracellular crosslinking are reversed upon exposure to light, thereby restoring proliferation (80% phospho-Rb+ cells), protein translation, and migration. Reversible intracellular crosslinking provides a novel method for dynamic manipulation of intracellular mechanics, altering essential processes that determine cellular function.

Synthetic Heparan Sulfate Hydrogels Regulate Neurotrophic Factor Signaling and Neuronal Network Activity.

Three-dimensional (3D) synthetic heparan sulfate (HS) constructs possess promising attributes for neural tissue engineering applications. However, their sulfation-dependent ability to facilitate molecular recognition and cell signaling has not yet been investigated. We hypothesized that fully sulfated synthetic HS constructs (bearing compound 1) that are functionalized with neural adhesion peptides will enhance fibroblast growth factor-2 (FGF2) binding and complexation with FGF receptor-1 (FGFR1) to promote the proliferation and neuronal differentiation of human neural stem cells (hNSCs) when compared to constructs with unsulfated controls (bearing compound 2). We tested this hypothesis in vitro using 2D and 3D substrates consisting of different combinations of HS tetrasaccharides (compounds 3 and 4) and an engineered integrin-binding chimeric peptide (CP), which were assembled using strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry. Results indicated that the adhesion of hNSCs increased significantly when cultured on 2D glass substrates functionalized with chimeric peptide. hNSCs encapsulated in 1-CP hydrogels and cultured in media containing the mitogen FGF2 exhibited significantly higher neuronal differentiation when compared to hNSCs in 2-CP hydrogels. These observations were corroborated by Western blot analysis, which indicated the enhanced binding and retention of both FGF2 and FGFR1 by 1 as well as downstream phosphorylation of extracellular signal-regulated kinases (ERK1/2) and enhanced proliferation of hNSCs. Lastly, calcium activity imaging revealed that both 1 and 2 hydrogels supported the neuronal growth and activity of pre-differentiated human prefrontal cortex neurons. Collectively, these results demonstrate that synthetic HS hydrogels can be tailored to regulate growth factor signaling and neuronal fate and activity.

Efficient Strategy for the Preparation of Chemical Probes of Biologically Active Glycosides Using a Boron-Mediated Aglycon Delivery (BMAD) Method

Abstract Development of an efficient method for the analysis and identification of the target proteins with which biologically active glycosides directly interact is highly desirable in many research fields. In this article, we report an efficient strategy for the preparation of chemical probes of biologically active glycosides using a reaction sequence of i) a boron-mediated aglycon delivery (BMAD) with an N3-functionalized 1,2-anhydroglucose donor, ii) deprotection, and iii) strain-promoted azide-alkyne cycloaddition. Using the synthesized chemical probes, we successfully demonstrated that the target proteins of a cardiac glycoside, lanatoside C (1), can be visualized and identified in human colon cancer HCT116 cells.

Controlling the Structure, Properties and Surface Reactivity of Clickable Azide-Functionalized Au25 (SR)18 Nanocluster Platforms Through Regioisomeric Ligand Modifications.

To fine-tune structure-property correlations of thiolate-protected gold nanoclusters through post-assembly surface modifications, we report the synthesis of the o, m, and p regioisomeric forms of the anionic azide-functionalized [Au25 (SCH2 CH2 -C6 H4 -N3 )18 ]1- platform. They can undergo cluster-surface strain-promoted alkyne-azide cycloaddition (CS-SPAAC) chemistry with complementary strained-alkynes. Although their optical properties are similar, the electrochemical properties appear to correlate with the position of the azido group. The ability to conduct CS-SPAAC chemistry without altering the parent nanocluster structure is different as the isomeric form of the surface ligand is changed, with the [Au25 (SCH2 CH2 -p-C6 H4 -N3 )18 ]1- isomer having the highest reaction rates, while the [Au25 (SCH2 CH2 -o-C6 H4 -N3 )18 ]1- isomer is not stable following CS-SPAAC. Single-crystal X-ray diffraction provide the molecular structure of the neutral forms of the three regioisomeric clusters, [Au25 (SCH2 CH2 -o/m/p-C6 H4 -N3 ]0 , which illustrates correlated structural features of the central core as the position of the azido moiety is changed.

Lipofuscin labeling through biorthogonal strain-promoted azide-alkyne cycloaddition for the detection of senescent cells.

A new method for senescent cell detection is described, which is based on lipofuscin labeling with a fluorescent reporter through a biorthogonal strain-promoted azide-alkyne cycloaddition. The sensing protocol involves a first step where the interaction of lipofuscin with a Sudan Black B derivative containing an azide moiety (SBB-N3 ) is carried out. In the final step, the azide moiety reacts with a fluorophore containing a cyclooctene ring (BODIPY). The efficacy of this two-step protocol is assessed in senescent melanoma SK-MEL-103 cells, senescent triple-negative breast cancer MDA-MB-231 cells and senescent WI-38 fibroblasts. In all cases, a clear fluorescence pattern was observed in senescent cells, compared to proliferative cells, only when the SBB-N3 -BODIPY probe was formed. Our results provide an alternative tool for the detection of senescent cells, based on an in situ bio-orthogonal reaction for lipofuscin labeling.

A robust post-insertion method for the preparation of targeted siRNA LNPs

Targeted delivery of nucleic acids is gaining momentum due to improved efficacy, selectivity, increased circulation time and enhanced tissue retention in target cells. Using nucleic acid-based therapies previously undruggable targets have proven now to be amenable for treatment. Currently, several methods for preparing targeted or labelled delivery vehicles for nucleic acids are based on liposomal formulations. Lipid nanoparticles (LNPs) are structurally different from liposomes and these methods should therefore be evaluated before being translated to siRNA LNPs preparation protocols. Here, we describe a robust and facile method for the preparation of targeted or fluorescently labelled siRNA LNPs. Using a copper free strain-promoted azide-alkyne cycloaddition (SPAAC) we demonstrate that post-insertion of ligand-lipid conjugates into preformed LNPs is superior to direct-surface modification because it preserves the physicochemical parameters of the LNPs. We found that the time point of solvent removal by dialysis is critical and affects the hydrodynamic diameter of the LNPs; post-insertion after dialysis shows the smallest increase in hydrodynamic diameter and polydispersity index (PDI). The post-insertion of ligand-lipid conjugates also proceeded with rapid kinetics and high efficacy over a wide temperature range. Using this optimised protocol, we generated siRNA LNPs containing both targeting and fluorescent tracking ligands allowing us to monitor siRNA LNP uptake kinetics in dependence of the targeting ligand. In aggregate, we describe a robust approach for the generation of targeted and labelled siRNA LNPs that allows their controlled and facile decoration with ligand combinations.

Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase.

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo–enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo–enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels–Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar KD values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.

Bivalent EGFR-Targeting DARPin-MMAE Conjugates.

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria.

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Multiscale imaging of therapeutic anti-PD-L1 antibody localization using molecularly defined imaging agents

BackgroundWhile immune checkpoint inhibitors such as anti-PD-L1 antibodies have revolutionized cancer treatment, only subgroups of patients show durable responses. Insight in the relation between clinical response, PD-L1 expression and intratumoral localization of PD-L1 therapeutics could improve patient stratification. Therefore, we present the modular synthesis of multimodal antibody-based imaging tools for multiscale imaging of PD-L1 to study intratumoral distribution of PD-L1 therapeutics.ResultsTo introduce imaging modalities, a peptide containing a near-infrared dye (sulfo-Cy5), a chelator (DTPA), an azide, and a sortase-recognition motif was synthesized. This peptide and a non-fluorescent intermediate were used for site-specific functionalization of c-terminally sortaggable mouse IgG1 (mIgG1) and Fab anti-PD-L1. To increase the half-life of the Fab fragment, a 20 kDa PEG chain was attached via strain-promoted azide-alkyne cycloaddition (SPAAC). Biodistribution and imaging studies were performed with 111In-labeled constructs in 4T1 tumor-bearing mice. Comparing our site-specific antibody-conjugates with randomly conjugated antibodies, we found that antibody clone, isotype and method of DTPA conjugation did not change tumor uptake. Furthermore, addition of sulfo-Cy5 did not affect the biodistribution. PEGylated Fab fragment displayed a significantly longer half-life compared to unPEGylated Fab and demonstrated the highest overall tumor uptake of all constructs. PD-L1 in tumors was clearly visualized by SPECT/CT, as well as whole body fluorescence imaging. Immunohistochemistry staining of tumor sections demonstrated that PD-L1 co-localized with the fluorescent and autoradiographic signal. Intratumoral localization of the imaging agent could be determined with cellular resolution using fluorescent microscopy.ConclusionsA set of molecularly defined multimodal antibody-based PD-L1 imaging agents were synthesized and validated for multiscale monitoring of PD-L1 expression and localization. Our modular approach for site-specific functionalization could easily be adapted to other targets.Graphical

A low-fouling, self-assembled, graft co-polymer and covalent surface coating for controlled immobilization of biologically active moieties

Current biointerfaces aiming to steer specific biological responses frequently lack either stability due to purely electrostatic interactions, bioactivity due to unspecific conjugation chemistries, specificity due to uncontrolled biological interactions such as fouling, or cytocompatibility due to harsh and toxic coating procedures. Here, we report a versatile surface modification platform for covalent tethering of selected biomolecules. New in this approach is the particular combination of modular binding blocks as graft co-polymer. Grafted to the backbone of PAcrAmTM multiple functionalities are strategically combined: covalent (silane) and non-covalent (lysine) surface binding groups for stability and self-assembly in mild buffered solution, PEG-azide chains for low fouling properties, and specific, controlled, covalent, linking of biologically active molecules. This modular strategy overcomes the previously mentioned limitations, for instance regarding bioactivity of the biological moiety due to highly specific strain-promoted azide-alkyne cycloaddition. The successful grafting of the copolymer was confirmed by 1H NMR. The immobilization of RGD peptides was characterized by combining surface analytical techniques, such as ToF-SIMS and ellipsometry, allowing quantification of immobilized molecules over an extensive range of concentrations (0.008–1.95 pmol·cm−2). The bioactivity over this range of concentrations was confirmed by in vitro cell studies, presenting a differential endothelial cell attachment and spreading. The modified substrates enabled the formation of an interconnected monolayer of endothelial cells. Furthermore, the modular platform allowed the co-immobilization of two bioactive functional groups, RGD and biotin, on the same surface, which could be exploited for the further development of controlled multi-functional biointerfaces for diverse biological applications in the future.

Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea.

The study of protein production and degradation in a quantitative and time-dependent manner is a major challenge to better understand cellular physiological response. Among available technologies bioorthogonal noncanonical amino acid tagging (BONCAT) is an efficient approach allowing for time-dependent labeling of proteins through the incorporation of chemically reactive noncanonical amino acids like L-azidohomoalanine (L-AHA). The azide-containing amino-acid derivative enables a highly efficient and specific reaction termed click chemistry, whereby the azide group of the L-AHA reacts with a reactive alkyne derivate, like dibenzocyclooctyne (DBCO) derivatives, using strain-promoted alkyne-azide cycloaddition (SPAAC). Moreover, available DBCO containing reagents are versatile and can be coupled to fluorophore (e.g., Cy7) or affinity tag (e.g., biotin) derivatives, for easy visualization and affinity purification, respectively.Here, we describe a step-by-step BONCAT protocol optimized for the model archaeon Haloferax volcanii , but which is also suitable to harness other biological systems. Finally, we also describe examples of downstream visualization, affinity purification of L-AHA-labeled proteins and differential expression analysis.In conclusion, the following BONCAT protocol expands the available toolkit to explore proteostasis using time-resolved semiquantitative proteomic analysis in archaea .

The influence of degree of labelling upon cellular internalisation of antibody-cell penetrating peptide conjugates.

Antibody-based agents are increasingly used as therapeutics and imaging agents, yet are generally restricted to cell surface targets due to inefficient cellular internalisation and endosomal entrapment. Enhanced cell membrane translocation of antibodies can be achieved by the covalent attachment of cell-penetrating peptides, including the HIV-1-derived transactivator of transcription (TAT) peptide. This study evaluated the cellular internalisation properties of five anti-HER2 Herceptin-TAT conjugates with degrees of TAT labelling (DOLTAT) ranging from one to five. Herceptin-TAT conjugates were synthesised via a strain-promoted alkyne-azide cycloaddition reaction, characterised by UV-vis spectroscopy, MALDI-TOF, and gel electrophoresis, then radiolabelled with zirconium-89 to permit measurement of cellular internalisation by gamma counting. [89Zr]Zr-DFO-Her-TAT(0-5) conjugates were isolated in high radiochemical purity (>99%) and exhibited high stability in murine and human serum over 7 days at 37 °C. Significant increases in cellular internalisation were observed for [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT values of 2 and above in SKBR3 (high HER2) cells over 48 h, in contrast to low-level non-specific uptake in MDA-MB-468 (low HER2) cells that did not increase over time. Notably, [89Zr]Zr-DFO-Her-TAT conjugates with DOLTAT of 3, 4, and 5 reached uptake values in SKBR3 cells of 5, 6, and 8% of the applied dose at 48 h respectively, representing 9, 10, 14-fold increases relative to the TAT-free control conjugate, [89Zr]Zr-DFO-Her-TAT(0).

Strain-Promoted Azide-Alkyne Cycloaddition-Based PSMA-Targeting Ligands for Multimodal Intraoperative Tumor Detection of Prostate Cancer.

Strain-promoted azide–alkyne cycloaddition (SPAAC) is a straightforward and multipurpose conjugation strategy. The use of SPAAC to link different functional elements to prostate-specific membrane antigen (PSMA) ligands would facilitate the development of a modular platform for PSMA-targeted imaging and therapy of prostate cancer (PCa). As a first proof of concept for the SPAAC chemistry platform, we synthesized and characterized four dual-labeled PSMA ligands for intraoperative radiodetection and fluorescence imaging of PCa. Ligands were synthesized using solid-phase chemistry and contained a chelator for 111In or 99mTc labeling. The fluorophore IRDye800CW was conjugated using SPAAC chemistry or conventional N-hydroxysuccinimide (NHS)–ester coupling. Log D values were measured and PSMA specificity of these ligands was determined in LS174T-PSMA cells. Tumor targeting was evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wild-type tumors using μSPECT/CT imaging, fluorescence imaging, and biodistribution studies. SPAAC chemistry increased the lipophilicity of the ligands (log D range: −2.4 to −4.4). In vivo, SPAAC chemistry ligands showed high and specific accumulation in s.c. LS174T-PSMA tumors up to 24 h after injection, enabling clear visualization using μSPECT/CT and fluorescence imaging. Overall, no significant differences between the SPAAC chemistry ligands and their NHS-based counterparts were found (2 h p.i., p > 0.05), while 111In-labeled ligands outperformed the 99mTc ligands. Here, we demonstrate that our newly developed SPAAC-based PSMA ligands show high PSMA-specific tumor targeting. The use of click chemistry in PSMA ligand development opens up the opportunity for fast, efficient, and versatile conjugations of multiple imaging moieties and/or drugs.

Ferritin Conjugates With Multiple Clickable Amino Acids Encoded by C-Terminal Engineered Pyrrolysyl-tRNA Synthetase.

This study reports the application of expanding genetic codes in developing protein cage-based delivery systems. The evolved Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS)•tRNAPyl pairs derived from directed evolution are examined to probe their recognition for para-substituted phenylalanine analogs. The evolved MmPylRS, AzFRS, harboring a wide range of substrates, is further engineered at the C-terminal region into another variant, AzFRS-MS. AzFRS-MS shows suppression of the elevated sfGFP protein amount up to 10 TAG stop codons when charging p-azido-l-phenylalanine (AzF, 4), which allows the occurrence of click chemistry. Since protein nanocages used as drug delivery systems that encompass multiple drugs through a site-specific loading approach remain largely unexplored, as a proof of concept, the application of AzFRS-MS for the site-specific incorporation of AzF on human heavy chain ferritin (Ftn) is developed. The Ftn-4 conjugate is shown to be able to load multiple fluorescence dyes or a therapeutic agent, doxorubicin (Dox), through the strain-promoted azide-alkyne cycloaddition (SPAAC) click reaction. Aiming to selectively target Her2+ breast cancer cells, Ftn-4-DOX conjugates fused with a HER2 receptor recognition peptide, anti-Her2/neu peptide (AHNP), is developed and demonstrated to be able to deliver Dox into the cell and to prolong the drug release. This work presents another application of evolved MmPylRS systems, whose potential in developing a variety of protein conjugates is noteworthy.