ABSTRACT
Real-time tracking of intracellular carbohydrates remains challenging. While click chemistry allows bio-orthogonal tagging with fluorescent probes, the reaction permanently alters the target molecule and only allows a single snapshot. Here, we demonstrate click-free mid-infrared photothermal (MIP) imaging of azide-tagged carbohydrates in live cells. Leveraging the micromolar detection sensitivity for 6-azido-trehalose (TreAz) and the 300-nm spatial resolution of MIP imaging, the trehalose recycling pathway in single mycobacteria, from cytoplasmic uptake to membrane localization, is directly visualized. A peak shift of azide in MIP spectrum further uncovers interactions between TreAz and intracellular protein. MIP mapping of unreacted azide after click reaction reveals click chemistry heterogeneity within a bacterium. Broader applications of azido photothermal probes to visualize the initial steps of the Leloir pathway in yeasts and the newly synthesized glycans in mammalian cells are demonstrated.
Subject(s)
Azides , Click Chemistry , Azides/chemistry , Click Chemistry/methods , Humans , Trehalose/metabolism , Trehalose/chemistry , Carbohydrates/chemistry , Fluorescent Dyes/chemistry , Biological TransportABSTRACT
To effectively solve the problem of significant loss of transplanted cells caused by thrombosis during cell transplantation, this study simulates the human fibrinolytic system and combines metabolic oligosaccharide engineering with strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry to construct a cell surface with fibrinolytic activity. First, a copolymer (POL) of oligoethylene glycol methacrylate (OEGMA) and 6-amino-2-(2-methylamido)hexanoic acid (Lys) was synthesized by reversible addition-fragmentation chain transfer (RAFT) copolymerization, and the dibenzocyclooctyne (DBCO) functional group was introduced into the side chain of the copolymer through an active ester reaction, resulting in a functionalized copolymer DBCO-PEG4-POL with ε-lysine ligands. Then, azide functional groups were introduced onto the surface of HeLa model cells through metabolic oligosaccharide engineering, and DBCO-PEG4-POL was further specifically modified onto the surface of HeLa cells via the SPAAC "click" reaction. In vitro investigations revealed that compared with unmodified HeLa cells, modified cells not only resist the adsorption of nonspecific proteins such as fibrinogen and human serum albumin but also selectively bind to plasminogen in plasma while maintaining good cell viability and proliferative activity. More importantly, upon the activation of adsorbed plasminogen into plasmin, the modified cells exhibited remarkable fibrinolytic activity and were capable of promptly dissolving the primary thrombus formed on their surfaces. This research not only provides a novel approach for constructing transplantable cells with fibrinolytic activity but also offers a new perspective for effectively addressing the significant loss of transplanted cells caused by thrombosis.
Subject(s)
Click Chemistry , Cycloaddition Reaction , Fibrinolysis , Oligosaccharides , Humans , HeLa Cells , Oligosaccharides/chemistry , Fibrinolysis/drug effects , Metabolic Engineering , Azides/chemistry , Polyethylene Glycols/chemistry , Methacrylates/chemistry , Alkynes/chemistry , Animals , Cell Survival/drug effects , Plasminogen/chemistry , Plasminogen/metabolism , Surface PropertiesABSTRACT
Spotted fever group rickettsiae (SFGR) are obligate intracellular bacteria that cause spotted fever. The limitations of gene manipulation pose great challenges to studying the infection mechanisms of Rickettsia. By combining bioorthogonal metabolism and click chemistry, we developed a method to label R. heilongjiangensis via azide moieties and achieved rapid pathogen localization without complex procedures. Moreover, we constructed a C57BL/6 mice infection model by simulating tick bites and discovered that the stomach is the target organ of R. heilongjiangensis infection through in vivo imaging systems, which explained the occurrence of gastrointestinal symptoms following R. heilongjiangensis infection in some cases. This study offers a unique perspective for subsequent investigations into the pathogenic mechanisms of SFGR and identifies a potential target organ for R. heilongjiangensis.
Subject(s)
Click Chemistry , Mice, Inbred C57BL , Rickettsia , Animals , Rickettsia/genetics , Rickettsia/physiology , Mice , Click Chemistry/methods , Stomach/microbiology , Disease Models, Animal , Spotted Fever Group Rickettsiosis/microbiology , Female , Rickettsia Infections/microbiology , Azides/chemistryABSTRACT
Phospholipase D (PLD) is an enzyme with many functions, one of which is the synthesis of phosphatidic acid (PA), a molecule with a myriad of effects on various organ systems and processes. These numerous roles make it hard to understand the true action of PA in cellular and bodily processes. Imaging PLD activity is one way to better understand the synthesis of PA and start to elucidate its function. However, many of the current imaging techniques for PLD come with limitations. This chapter presents a thorough methodology of a new imaging technique for PLD activity with clickable alcohols via transphosphatidylation (IMPACT) and Real-Time IMPACT (RT-IMPACT) that takes advantage of clickable chemistry to overcome current limitations. Using strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron-demand Diels-Alder (IEDDA), and the synthesis of various organic compounds, this chapter will explain a step-by-step procedure of how to perform the IMPACT and RT-IMPACT method(s).
Subject(s)
Alcohols , Click Chemistry , Phospholipase D , Phospholipase D/metabolism , Phospholipase D/chemistry , Click Chemistry/methods , Alcohols/chemistry , Alcohols/metabolism , Cycloaddition Reaction , Humans , Phosphatidic Acids/metabolism , Phosphatidic Acids/chemistry , Azides/chemistry , Molecular Imaging/methods , Alkynes/chemistryABSTRACT
Extracellular vesicles (EVs) are nanoparticles secreted by various organisms. Methods for modifying EVs functionally have garnered attention for developing EV-based therapeutic systems. However, most technologies used to integrate these functions are limited to mammalian-derived EVs and a promising modification method for bacteria-derived EVs has not yet been developed. In this study, we propose a novel method for the versatile functionalization of immunostimulatory probiotic Bifidobacteria-derived EVs (B-EVs) using amino acid metabolic labeling and azide-alkyne click reaction. Azide D-alanine (ADA), a similar molecule to D-alanine in bacteria cell-wall peptidoglycan, was selected as an azide group-functionalized amino acid. Azide-modified B-EVs were isolated from Bifidobacteria incubated with ADA. The physicochemical and compositional characteristics, as well as adjuvanticity of B-EVs against immune cells were not affected by azide loading, demonstrating that this functionalization approach can retain the endogenous usefulness of B-EVs. By using the fluorescent B-EVs obtained by this method, the intracellular trafficking of B-EVs after uptake by immune cells was successfully observed. Furthermore, this method enabled the formulation of B-EVs for hydrogelation and enhanced adjuvanticity in the host. Our findings will be helpful for further development of EV-based immunotherapy.
Subject(s)
Azides , Bifidobacterium , Click Chemistry , Extracellular Vesicles , Immunotherapy , Extracellular Vesicles/metabolism , Bifidobacterium/metabolism , Azides/chemistry , Animals , Immunotherapy/methods , Alanine/chemistry , Probiotics/administration & dosage , Mice , Amino Acids/chemistry , Amino Acids/metabolism , Humans , RAW 264.7 CellsABSTRACT
Lipids have demonstrated tremendous promise for mRNA delivery, as evidenced by the success of Covid-19 mRNA vaccines. However, existing lipids are mostly used as delivery vehicles and lack the ability to monitor and further modulate the target cells. Here, for the first time, we report a class of unnatural lipids (azido-DOTAP) that can efficiently deliver mRNAs into cells and meanwhile metabolically label cells with unique chemical tags (e.g., azido groups). The azido tags expressed on the cell membrane enable the monitoring of transfected cells, and can mediate subsequent conjugation of cargos via efficient click chemistry for further modulation of transfected cells. We further demonstrate that the dual-functional unnatural lipid is applicable to different types of cells including dendritic cells, the prominent type of antigen presenting cells, potentially opening a new avenue to developing enhanced mRNA vaccines.
Subject(s)
Azides , Click Chemistry , RNA, Messenger , RNA, Messenger/administration & dosage , Humans , Azides/chemistry , Dendritic Cells/metabolism , Lipids/chemistry , Fatty Acids, Monounsaturated/chemistry , Transfection/methods , COVID-19 , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Animals , Quaternary Ammonium CompoundsABSTRACT
In this study, a low field nuclear magnetic resonance (LF-NMR) homogeneous sensor was constructed for detection of Escherichia coli (E. coli) based on the copper metabolism of E. coli triggered click reaction. When live E. coli was present, a large amount of Cu2+ ions were transformed into Cu+ via copper metabolism, which then catalyzed a Cu+-catalyzed azide-alkyne cycloaddition (CuAAC) reaction between two materials, azide group modified gadolinium oxide nanorods (Gd2O3-Az) and PA-GO@Fe3O4 i.e., graphene oxide (GO) loaded with large amounts of alkynyl (PA) groups and Fe3O4 nanoparticles simultaneously. After magnetic separation, unbound Gd2O3-Az was dissolved by added hydrochloric acid (HCl) to generate homogeneous Gd3+ solution, enabling homogeneous detection of E. coli. Triple signal amplification was achieved through the CuAAC reaction induced by E. coli copper metabolism, functional nanomaterials, and HCl assisted homogeneous detection. Under the optimal experimental conditions, the linear range and limit of detection (LOD) for E. coli were 10-1.0 × 107 CFU/mL and 3.5 CFU/mL, respectively, and the relative standard deviations (RSDs) were all less than 2.8 %. In addition, the sensor has satisfactory selectivity, stability and practical sample application capability, providing a new approach for the LF-NMR detection of food-borne pathogenic bacteria.
Subject(s)
Alkynes , Azides , Click Chemistry , Copper , Escherichia coli , Graphite , Escherichia coli/isolation & purification , Copper/chemistry , Azides/chemistry , Graphite/chemistry , Alkynes/chemistry , Magnetic Resonance Spectroscopy , Cycloaddition Reaction , Gadolinium/chemistry , Limit of Detection , Nanotubes/chemistryABSTRACT
The hyphenation of HPLC with its high separation ability and ICP-MS with its excellent sensitivity, allows the analysis of Pt drugs in biological samples at the low nanomolar concentration levels. On the other hand, LC-MS provides molecular structural confirmation for each species. Using a combination of these methods, we have investigated the speciation of the photoactive anticancer complex diazido Pt(IV) complex trans, trans, trans-[Pt(N3)2(OH)2(py)2] (FM-190) in aqueous solution and biofluids at single-digit nanomolar concentrations before and after irradiation. FM-190 displays high stability in human blood plasma in the dark at 37 °C. Interestingly, the polyhydroxido species [{PtIV(py)2(OH)4} + Na]+ and [{PtIV(py)2(N3)(OH)3} + Na]+ resulting from the replacement of azido ligands, as determined by LC-MS, were the major products after photoirradiation of FM-190 with blue light (463 nm). This finding suggests that such photosubstituted Pt(IV) tri- and tetra-hydroxido species could play important roles in the biological activity of this anticancer complex. Density Functional Theory (DFT) and Time-Dependent DFT (TDDFT) calculations show that these Pt(IV) species arising from FM-190 in aqueous media can be formed directly from a singlet excited state. The results highlight how speciation analysis (metallomics) can shed light on photoactivation pathways for FM-190 and formation of potential excited-state pharmacophores. The ability to detect and identify photoproducts at physiologically-relevant concentrations in cells and tissues will be important for preclinical development studies of this class of photoactivatable platinum drugs.
Subject(s)
Antineoplastic Agents , Oxidation-Reduction , Photochemical Processes , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Humans , Organoplatinum Compounds/chemistry , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/chemical synthesis , Light , Azides/chemistry , Platinum/chemistry , Molecular Structure , Coordination Complexes/chemistry , Coordination Complexes/chemical synthesisABSTRACT
Biotinylation is probably the most frequent and practically useful modification of molecules to facilitate selective and highly affine binding to (strept)avidin for immobilization, enrichment, and purification for further (bio)chemical or (bio)physical investigations. We present a protecting-group-free synthesis of a branched biotin bis-azide that enables dual-payload late-stage functionalization with arbitrary alkynes via click chemistry. Utility of the chassis is briefly showcased on the example of a valuable Pittsburgh B analogue, which binds pathological protein aggregates, commonly found in neurodegenerative diseases.
Subject(s)
Alkynes , Biotin , Biotinylation , Click Chemistry , Molecular Structure , Biotin/chemistry , Alkynes/chemistry , Thiazoles/chemistry , Thiazoles/chemical synthesis , Azides/chemistryABSTRACT
Macromolecule branching upon polyhedral oligomeric silsesquioxanes (POSS) via "click" chemistry has previously been reported for promoting natural biological responses in vitro, particularly when regarding their demonstrated biocompatibility and structural robustness as potential macromolecule anchoring points. However, "clicking" of large molecules around POSS structures uncovers two main challenges: (1) a synthetic challenge encompassing multi-covalent attachment of macromolecules to a single nanoscale-central position, and (2) purification and separation of fully adorned nanocages from those that are incomplete due to their similar physical characteristics. Here we present peptide decoration to a T8POSS nanocage through the attachment of azido-modified trimers. Triglycine- and trialanine-methyl esters "clicked" to 97% and 92% completion, respectively, resulting in 84% and 68% yields of the fully-adorned octamers. The "clicks" halt within 27-h of the reaction time, and efforts to further increase the octamer yield were of negligible benefit. Exploration of reaction conditions reveals multiple factors preventing full octa-arm modification to all available POSS nanocages, and offers insights into macromolecule attachment between both peptides and small inorganic-organic structures, all of which require consideration for future work of this nature.
Subject(s)
Click Chemistry , Organosilicon Compounds , Peptides , Peptides/chemistry , Organosilicon Compounds/chemistry , Nanostructures/chemistry , Azides/chemistryABSTRACT
Surface functionalization of nano drug carriers allows for precise delivery of therapeutic molecules to the target site. This technique involves attaching targeting molecules to the nanoparticle surface, facilitating selective interaction. In this study, we engineered virus-like particles (VLPs) to enhance their targeting capabilities. Azide groups incorporated on the lipid membranes of VLPs enabled bioorthogonal click reactions for conjugation with cycloalkyne-bearing molecules, providing efficient conjugation with high specificity. HIV-1 Gag VLPs were chosen due to their envelope, which allows host membrane component incorporation, and the Gag protein, which serves as a recognition motif for human T cells. This combination, along with antibody-mediated targeting, addresses the limitations of intracellular delivery to T cells, which typically exhibit low uptake of exogenous materials. The selective uptake of azide VLPs by CD3-positive T cells was evaluated in a co-culture system. Even without antibody conjugation, VLP uptake was enhanced in T cells, indicating their intrinsic targeting potential. Antibody conjugation further amplified this effect, demonstrating the synergistic benefits of the combined targeting approach. Our study shows that recombinant production of azide functionalized VLPs results in engineered nanoparticles that can be easily modified using bioorthogonal click reactions, providing high specificity and versatility for conjugation with various molecules, making it applicable to a wide range of biological products.
Subject(s)
Azides , Click Chemistry , T-Lymphocytes , Humans , Azides/chemistry , T-Lymphocytes/immunology , Nanoparticles/chemistry , gag Gene Products, Human Immunodeficiency Virus , HIV-1 , Coculture Techniques , Drug Delivery Systems , Surface PropertiesABSTRACT
Nucleic acids play a pivotal role in the diagnosis of diseases. However, rapid, cost-efficient, and ultrasensitive identification of nucleic acid targets still represents a significant challenge. Herein, we describe an enzyme-free DNA amplification method capable of achieving accurate and ultrasensitive nucleic acid detection via DNA-templated click ligation chain reaction (DT-CLCR) catalyzed by a heterogeneous nanocatalyst made of Cu2O (hnCu2O). This hnCu2O-DT-CLCR method is built on two cross-amplifying hnCu2O-catalyzed DNA-templated azide-alkyne cycloaddition-driven DNA ligation reactions that boast a fast reaction rate and a high DNA ligation yield in minutes, enabling rapid exponential amplification of specific DNA targets. This newly developed hnCu2O-DT-CLCR-enabled DNA amplification strategy is further integrated with two signal reporting mechanisms to achieve low-cost and easy-to-use biosensors: an electrochemical sensor through the conjugation of a methylene blue redox reporter to a DNA probe used in hnCu2O-DT-CLCR and a colorimetric sensor through the incorporation of the split-to-intact G-quadruplex DNAzyme encoded into hnCu2O-DT-CLCR. Both sensors are able to achieve specific detection of the intended DNA target with a limit of detection at aM ranges, even when challenged in complex biological matrices. The combined hnCu2O-DT-CLCR and sensing strategies offer attractive universal platforms for enzyme-free and yet efficient detection of specific nucleic acid targets.
Subject(s)
Click Chemistry , Copper , DNA , Nucleic Acid Amplification Techniques , Copper/chemistry , DNA/chemistry , Catalysis , Humans , Biosensing Techniques/methods , Limit of Detection , DNA, Catalytic/chemistry , DNA, Catalytic/metabolism , Azides/chemistry , Colorimetry/methods , Electrochemical Techniques/methods , Cycloaddition ReactionABSTRACT
Aim: The goal of this study is to synthesize new metal complexes containing N-methyl-1-(pyridin-2-yl)methanimine and azide ligands as α-glucosidase inhibitors for Type 2 diabetes. Materials & methods: The target complexes (12-16) were synthesized by reacting N-methyl-1-(pyridin-2-yl)methanimine (L1) with sodium azide in the presence of corresponding metal salts. The investigation of target protein interactions, vibrational, electronic and nonlinear optical properties for these complexes was performed by molecular docking and density functional theory studies. Results: Among these complexes, complex 13 (IC50 = 0.2802 ± 0.62 µM) containing Hg ion showed the highest α-glucosidase inhibitory property. On the other hand, significant results were detected for complexes containing Cu and Ag ions. Conclusion: Complex 13 may be an alternate anti-diabetic inhibitor according to in vitro/docking results.
[Box: see text].
Subject(s)
Azides , Coordination Complexes , Density Functional Theory , Glycoside Hydrolase Inhibitors , Molecular Docking Simulation , alpha-Glucosidases , Glycoside Hydrolase Inhibitors/chemistry , Glycoside Hydrolase Inhibitors/pharmacology , Glycoside Hydrolase Inhibitors/chemical synthesis , alpha-Glucosidases/metabolism , alpha-Glucosidases/chemistry , Coordination Complexes/chemistry , Coordination Complexes/pharmacology , Coordination Complexes/chemical synthesis , Azides/chemistry , Humans , Molecular Structure , Hypoglycemic Agents/chemistry , Hypoglycemic Agents/pharmacology , Hypoglycemic Agents/chemical synthesis , Diabetes Mellitus, Type 2/drug therapy , Structure-Activity RelationshipABSTRACT
Bacterial nonulosonic acids (NulOs), which feature a nine-carbon backbone, are associated with the biological functions of bacterial glycans. Here, an orthogonally protected 5-amino-7-azido-3,5,7,9-tetradeoxy-d-glycero-l-gluco-2-nonulosonic acid related to Fusobacterium nucleatum ATCC 23726 NulO was synthesized from N-acetylneuraminic acid with sequential performance of C5,7 azidation, C9 deoxygenation, C4 epimerization, and N5,7 differentiation. The C5 azido group in the obtained 5,7-diazido-NulO can be regioselectively reduced to differentiate the two amino groups.
Subject(s)
N-Acetylneuraminic Acid , Sugar Acids , N-Acetylneuraminic Acid/chemistry , N-Acetylneuraminic Acid/chemical synthesis , Molecular Structure , Sugar Acids/chemistry , Sugar Acids/chemical synthesis , Fusobacterium nucleatum/chemistry , Azides/chemistryABSTRACT
SARS-CoV-2 is a highly infectious virus responsible for the COVID-19 pandemic. Therefore, it is important to assess the risk of SARS-CoV-2 infection, especially in persistently positive patients. Rapid discrimination between infectious and non-infectious viruses aids in determining whether prevention, control, and treatment measures are necessary. For this purpose, a method was developed and utilized involving a pre-treatment with 50 µM of propidium monoazide (PMAxx, a DNA intercalant) combined with a digital droplet PCR (ddPCR). The ddPCR method was performed on 40 nasopharyngeal swabs (NPSs) both before and after treatment with PMAxx, revealing a reduction in the viral load at a mean of 0.9 Log copies/mL (SD ± 0.6 Log copies/mL). Furthermore, six samples were stratified based on the Ct values of SARS-CoV-2 RNA (Ct < 20, 20 < Ct < 30, Ct > 30) and analyzed to compare the results obtained via a ddPCR with viral isolation and a negative-chain PCR. Of the five samples found positive via a ddPCR after the PMAxx treatment, two of the samples showed the highest post-treatment SARS-CoV-2 loads. The virus was isolated in vitro from both samples and the negative strand chains were detected. In three NPS samples, SARS CoV-2 was present post-treatment at a low level; it was not isolated in vitro, and, when detected, the strand was negative. Our results indicate that the established method is useful for determining whether the SARS-CoV-2 within positive NPS samples is intact and capable of causing infection.
Subject(s)
Azides , COVID-19 , Nasopharynx , Propidium , SARS-CoV-2 , Viral Load , Humans , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Azides/chemistry , Propidium/analogs & derivatives , Propidium/chemistry , COVID-19/virology , Viral Load/methods , Nasopharynx/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , COVID-19 Nucleic Acid Testing/methods , Polymerase Chain Reaction/methodsABSTRACT
Nowadays, medical polyurethanes with favorable and durable antibacterial properties received more attention, because of avoiding repeated replacement of interventional materials and reducing patients' pain. In this thesis, non-soluble antibacterial polyurethane (NAPU) based on cation antibacterial mechanism was prepared by photo-grafting chitosan azide and heparin azide into polyurethane (PU). -NH3+of chitosan azide absorbed bacteria, inhibiting and breaking their mobility and structures. Heparin azide prevented cations from penetrating bacteria's membranes and inhibited their growth. The results showed that chitosan azide and heparin azide were successfully grafted into PU. The highest antibacterial rate was 92.07%, cytotoxicity grade ranging from 0-1 (RGR standard) and water contact angle exhibiting 60°, attributing to cation antibacterial effect and -OH existing. Tensile strength was up to 23.91 MPa and was suitable for using as medical materials. NAPU with long-lasting coating both possessed antibacterial properties and persistence, which can solve the problem of medical catheters' long-term using.
Subject(s)
Anti-Bacterial Agents , Azides , Cations , Chitosan , Heparin , Polyurethanes , Polyurethanes/chemistry , Chitosan/chemistry , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Heparin/chemistry , Azides/chemistry , Materials Testing , Tensile Strength , Escherichia coli/drug effects , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Animals , Humans , Microbial Sensitivity Tests , Staphylococcus aureus/drug effects , Mice , SolubilityABSTRACT
Nanotechnology has revolutionized the fabrication of hybrid species with tailored functionalities. A milestone in this field is the deoxyribonucleic acid (DNA) conjugation of nanoparticles, introduced almost 30 years ago, which typically exploits the affinity between thiol groups and metallic surfaces. Over the last decades, developments in colloidal research have enabled the synthesis of an assortment of nonmetallic structures, such as high-index dielectric nanoparticles, with unique properties not previously accessible with traditional metallic nanoparticles. However, to stabilize, integrate, and provide further functionality to nonmetallic nanoparticles, reliable techniques for their functionalization with DNA will be crucial. Here, we combine well-established dibenzylcyclooctyne-azide click-chemistry with a simple freeze-thaw method to achieve the functionalization of silica and silicon nanoparticles, which form exceptionally stable colloids with a high DNA surface density of â¼0.2 molecules/nm2. Furthermore, we demonstrate that these functionalized colloids can be self-assembled into high-index dielectric dimers with a yield of over 50% via the use of DNA origami. Finally, we extend this method to functionalize other important nanomaterials, including oxides, polymers, core-shell, and metal nanostructures. Our results indicate that the method presented herein serves as a crucial complement to conventional thiol functionalization chemistry and thus greatly expands the toolbox of DNA-functionalized nanoparticles currently available.
Subject(s)
Click Chemistry , DNA , Nanoparticles , Silicon Dioxide , DNA/chemistry , Nanoparticles/chemistry , Silicon Dioxide/chemistry , Silicon/chemistry , Azides/chemistry , Surface PropertiesABSTRACT
A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each ß-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier.
Subject(s)
Alkynes , Azides , Cycloaddition Reaction , Hemoglobins , Serum Albumin , Alkynes/chemistry , Azides/chemistry , Humans , Hemoglobins/chemistry , Serum Albumin/chemistry , Oxygen/chemistry , Maleimides/chemistryABSTRACT
Protein phosphorylation is catalyzed by kinases to regulate cellular events and disease states. Identifying kinase-substrate relationships represents a powerful strategy to understand cell biology and disease yet remains challenging due to the rapid dynamics of phosphorylation. Over the last decade, several γ-phosphoryl modified ATP analogs containing crosslinkers were developed to covalently conjugate kinases, their substrates, and their associated proteins for subsequent characterization. Here, kinetics and crosslinking experiments demonstrated that the UV-activated analogs, ATP-aryl azide and ATP-benzophenone, offered the most robust crosslinking, whereas electrophilic ATP-aryl fluorosulfate promoted the most effective proximity-enabled crosslinking. The data will guide future applications of kinase-catalyzed crosslinking to study normal and disease biology.
Subject(s)
Adenosine Triphosphate , Cross-Linking Reagents , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/chemistry , Cross-Linking Reagents/chemistry , Cross-Linking Reagents/chemical synthesis , Benzophenones/chemistry , Benzophenones/chemical synthesis , Molecular Structure , Azides/chemistry , Humans , Kinetics , PhosphorylationABSTRACT
The limitations of commonly used sodium ascorbate-based catalyst system for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction include excess production of reactive oxygen species and rapid catalyst deactivation. In this study instead of using a highly active reducing agent, such as, sodium ascorbate, we chose reducing sugar as a mild reducing agent to build up the catalyst system for CuAAC reaction. Interestingly, the bicinchoninic acid (BCA) assay system containing reducing sugar satisfies the essential elements of the catalyst system for CuAAC reaction. We found that CuSO4/BCA/Reducing sugar system can catalyze the CuAAC reaction but with low yield. Rational analyses of various parameters in CuSO4/BCA/Glucose catalyst system suggested storage at room temperature might enhance the catalytic activity, which was proven to be the case. Importantly, the system remains stable at room temperature and minimal H2O2 was detected. Notably, our study showed that the coordination between the slow reduction of Cu(I) by reducing sugar and the selective chelation of Cu(I) by BCA is key to developing this system. The CuSO4/BCA/Reducing sugar catalyst system was successfully applied to various CuAAC reaction based bioanalyses, and it is suitable for the CuAAC reaction based bioanalyses that are sensitive to ROS or request long reaction time.