Copper-free Click Chemistry Research Articles

Abstract LB-071: A facile strategy for exosome labeling and tracking

Abstract Exosomes are extracellular vesicles of endocytic origin with a size range of 40-150 nm and a lipid bilayer membrane. Though exosomes are known as dynamic mediators of intercellular communication, its characteristics and function have not been fully studied. In this report, we used a metabolic labeling method to prepare fluorescent exosomes to investigate the characteristics and the movement of exosomes derived from various breast cancer cells. MCF-7 and MDA-MB-231 cells were treated with three types of azido sugars, Ac4ManNAz, Ac4GalNAz, or Ac4GlcNAz (50 μM), for 3 days to produce the azide (-N3) containing exosomes through metabolic glycosylation. It is confirmed that the azido sugar decorated exosomes were able to maintain their original characteristics such as size, lipid bilayer morphology, and protein profile. The collected exosomes were labeled with Adibo-cy3 and Adibo-cy5.5 fluorescence dyes using copper-free click chemistry. The exosomes prepared with Ac4ManNAz have shown the highest labeling efficiency, in MCF-7 and MDA-MB-231 cells at 17.2% and 14.8%, respectively. To study their homing capability, the labeled exosomes from different origins were incubated with a panel of breast cancer cells, including MCF-7, MDA-MB-231, MDA-MB-468 and BT-549, and normal NIH/3T3 fibroblast cells for 24 hours. The cells were then evaluated by fluorescence microscopic imaging and flow cytometry. It was observed that exosomes from different origins have a different homing efficiency, suggesting that each exosome may have its unique navigation systems. Furthermore, the cells which have aggressive metastatic potential, such as MDA-MB-231, showed a higher uptake in all exosomes. Our development has demonstrated an effective labeling method that facilitates exosome research by providing a new way of quantification and tracking in vitro and potentially in vivo studies. Citation Format: Young Kim, Tae-Sup Lee, Ching H. Tung. A facile strategy for exosome labeling and tracking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-071. doi:10.1158/1538-7445.AM2017-LB-071

Abstract 871: [18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expression in vivo

Abstract Objectives Inhibition of the Programmed Death Ligand-1 (PD-L1)/PD-1 interaction allows for potent anti-tumor activity and antibodies that disrupt this interaction have been approved for the treatment of multiple cancer types. PD-L1 expression has been investigated clinically as a potential biomarker to predict response to anti-PD-1/PD-L1 therapy. BMS-986192, an Adnectin with high affinity and specificity for human PD-L1, was selected in vitro from a complex library. Here we report the discovery and first preclinical evaluation of [18F]BMS-986192 as a PET imaging agent to detect PD-L1 expression in vivo. Methods [18F]BMS-986192 was radiolabeled via copper-free click chemistry and assessed for its ability to detect PD-L1 expression. Tracer binding to human L2987 (PD-L1+) and HT-29 (PD-L1-) xenografts as well as human non-small cell lung cancer (NSCLC) tissue samples was assessed by autoradiography (ARG). Tracer binding was compared to PD-L1 expression assessed independently with anti-PD-L1 immunohistochemistry (IHC). In vivo performance of the tracer was also assessed by PET imaging in mice bearing bilateral L2987 and HT-29 xenografts, and tracer biodistribution was further assayed in these animals ex vivo by gamma counter. Finally, initial in vivo biodistribution and radiation dosimetry was measured by PET in cynomolgus monkey. Results ARG studies showed increased [18F]BMS-986192 total binding to PD-L1(+) L2987 xenograft compared to PD-L1(-) HT-29 xenograft tissue. Radiotracer binding was higher in all tested human NSCLC tissue samples compared to xenografts. Dose-dependent blockade was seen in all PD-L1(+) tissues co-incubated with cold BMS-986192, and binding was unaffected by co-incubation with cold non-PD-L1 binding control. Visual comparison of tracer binding aligns closely with PD-L1 IHC both spatially as well as in intensity. Preferential accumulation of [18F]BMS-986192 was noted in PD-L1(+) L2987 compared to PD-L1(-) HT-29 xenografts in tumor-bearing mice. PET studies in cynomolgus monkeys confirmed binding to PD-L1(+) tissue (e.g. spleen) with minimal nonspecific background signal exclusive of primary clearance organs. Radiation dosimetry of [18F]BMS-986192 indicates an estimated single administration dose limit of 6.2 mCi for an average human subject. Conclusions ARG, PET studies, and ex vivo measurements in rodent and cynomolgus monkey demonstrated sensitive and specific [18F]BMS-986192 binding to PD-L1. Low background signal in cynomolgus monkey in the context of endogenous PD-L1 expression further supports the potential of this tracer for sensitive detection of PD-L1(+) lesions in vivo. Radiation dosimetry suggests that [18F]BMS-986192 can be safely administered in human trials, with estimated absorbed radiation doses well within safe parameters for human administration. [18F]BMS-986192 has potential as a sensitive PD-L1 imaging agent for same-day imaging in patients. Citation Format: Ralph A. Smith, David Donnelly, Paul E. Morin, Dasa Lipovsek, Jochem Gokemeijer, Daniel Cohen, Joonyoung Kim, Adrienne Pena, Olufemi Adelakun, Xi-Tao Wang, Patrick Chow, Samuel J. Bonacorsi, Wendy Hayes. [18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expression in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 871. doi:10.1158/1538-7445.AM2017-871

A Modular Dual-Labeling Scaffold That Retains Agonistic Properties for Somatostatin Receptor Targeting.

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and 64Cu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC50]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC50: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in 64Cu-MMC(IR800)-TOC uptake whereas 64Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for 64Cu-MMC(IR800)-TOC than 64Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of 64Cu-MMC(IR800)-TOC by 66%. Excretion of 64Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas 64Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

Invivo stem cell tracking with imageable nanoparticles that bind bioorthogonal chemical receptors on the stem cell surface.

It is urgently necessary to develop reliable non-invasive stem cell imaging technology for tracking the invivo fate of transplanted stem cells in living subjects. Herein, we developed a simple and well controlled stem cell imaging method through a combination of metabolic glycoengineering and bioorthogonal copper-free click chemistry. Firstly, the exogenous chemical receptors containing azide (-N3) groups were generated on the surfaces of stem cells through metabolic glycoengineering using metabolic precursor, tetra-acetylated N-azidoacetyl-d-mannosamine(Ac4ManNAz). Next, bicyclo[6.1.0]nonyne-modified glycol chitosan nanoparticles (BCN-CNPs) were prepared as imageable nanoparticles to deliver different imaging agents. Cy5.5, iron oxide nanoparticles and gold nanoparticles were conjugated or encapsulated to BCN-CNPs for optical, MR and CT imaging, respectively. These imageable nanoparticles bound chemical receptors on the Ac4ManNAz-treated stem cell surface specifically via bioorthogonal copper-free click chemistry. Then they were rapidly taken up by the cell membrane turn-over mechanism resulting in higher endocytic capacity compared non-specific uptake of nanoparticles. During invivo animal test, BCN-CNP-Cy5.5-labeled stem cells could be continuously tracked by non-invasive optical imaging over 15 days. Furthermore, BCN-CNP-IRON- and BCN-CNP-GOLD-labeled stem cells could be efficiently visualized using invivo MR and CT imaging demonstrating utility of our stem cell labeling method using chemical receptors. These results conclude that our method based on metabolic glycoengineering and bioorthogonal copper-free click chemistry can stably label stem cells with diverse imageable nanoparticles representing great potential as new stem cell imaging technology.

Zwitterionic Silane Copolymer for Ultra-Stable and Bright Biomolecular Probes Based on Fluorescent Quantum Dot Nanoclusters.

Fluorescent semiconductor quantum dots (QDs) exhibit several unique properties that make them suitable candidates for biomolecular sensing, including high brightness, photostability, broad excitation, and narrow emission spectra. Assembling these QDs into robust and functionalizable nanosized clusters (QD-NSCs) can provide fluorescent probes that are several orders of magnitude brighter than individual QDs, thus allowing an even greater sensitivity of detection with simplified instrumentation. However, the formation of compact, antifouling, functionalizable, and stable QD-NSCs remains a challenging task, especially for a use at ultralow concentrations for single-molecule detection. Here, we describe the development of fluorescent QD-NSCs envisioned as a tool for fast and sensitive biomolecular recognition. First, QDs were assembled into very compact 100-150 nm diameter spherical aggregates; the final QD-NSCs were obtained by growing a cross-linked silica shell around these aggregates. Hydrolytic stability in several concentration and pH conditions is a key requirement for a potential and efficient single-molecule detection tool. However, the hydrolysis of Si-O-Si bonds leads to desorption of monosilane-based surface groups at very low silica concentrations or in a slightly basic medium. Thus, we designed a novel multidentate copolymer composed of multiple silane as well as zwitterionic monomers. Coating silica beads with this multidentate copolymer provided a robust surface chemistry that was demonstrated to be stable against hydrolysis, even at low concentrations. Copolymer-coated silica beads also showed low fouling properties and high colloidal stability in saline solutions. Furthermore, incorporation of additional azido-monomers enabled easy functionalization of QD-NSCs using copper-free bio-orthogonal cyclooctyne-azide click chemistry, as demonstrated by a biotin-streptavidin affinity test.

Ligand Characteristics Important to Avidity Interactions of Multivalent Nanoparticles.

Multivalent interactions involve the engagement of multiple ligand-receptor pairs and are important in synthetic biology as design paradigms for targeted nanoparticles (NPs). However, little is known about the specific ligand parameters important to multivalent interactions. We employed a series of oligonucleotides as ligands conjugated to dendrimers as nanoparticles, and used complementary oligonucleotides on a functionalized SPR surface to measure binding. We compared the effect of ligand affinity to ligand number on the avidity characteristics of functionalized NPs. Changing the ligand affinity, either by changing the temperature of the system or by substitution noncomplementary base pairs into the oligonucleotides, had little effect on multivalent interaction; the overall avidity, number of ligands required for avidity per particle, and the number of particles showing avidity did not significantly change. We then made NP conjugates with the same oligonucleotide using an efficient copper-free click chemistry that resulted in essentially all of the NPs in the population exceeding the threshold ligand value. The particles exceeding the threshold ligand number again demonstrated high avidity interactions. This work validates the concept of a threshold ligand valence and suggests that the number of ligands per nanoparticle is the defining factor in achieving high avidity interactions.

Abstract 238: Sub-Endothelial Matrix Targeted Liposomal Nanoparticles For Vascular Therapeutics

Objectives: Vascular intervention results in intimal denudation, exposure of the sub-endothelial matrix, and subsequent intimal hyperplasia (IH), under the control of numerous remodeling mechanisms. Reduction of IH-induced restenosis may be achieved by manipulation of these remodeling pathways through targeted molecular inhibition. Spatially-controlled nanoparticles designed to co-localize to exposed sub-endothelial matrices could provide an optimal delivery system for targeted vascular therapeutics. To his end, we aimed to develop the framework for a surface-modified liposomal drug delivery platform designed to preferentially bind collagen type IV. Methods: Non-targeted control liposomes (NTL) were formed with bulk DOPC-PEG, 30% cholesterol, and 0.1 mol% Rhodamine-DOPE. DSPE-PEG-DBCO lipids were conjugated to peptides previously shown to bind collagen IV via copper-free click chemistry and inserted at 5 mol% to form collagen-targeting liposomes (CTL), either at hydration (PreCTL) or by post-insertion via micellar transfer (PostCTL). Peptide conjugation was confirmed by MALDI-TOF, and liposomes were characterized by DLS and electrophoretic mobility. Liposome binding was assayed on collagen IV matrices dried at 3ug/cm 2 and quantified by fluorescence at 0-24hr static 37°C incubation. Results: All liposome formulations exhibited a narrow size distribution (~100nm) and neutral-low positive charge. CTLs demonstrated a significant increase in binding vs. NTLs (Fig1). Conclusions: CTLs demonstrated significant affinity for collagen IV binding in a static environment compared with NTLs. Future studies aim to optimize the binding capacity of CTLs via further lipid modifications and under flow conditions mimicking vessel wall hemodynamics. Considering the efficacy demonstrated here, CTLs show promise as the framework for a spatially-controlled drug delivery platform for future application in targeted vascular therapeutics.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Molecular imaging of activated platelets via antibody-targeted ultra-small iron oxide nanoparticles displaying unique dual MRI contrast.

Magnetic resonance imaging (MRI) is a powerful and indispensable tool in medical research, clinical diagnosis, and patient care due to its high spatial resolution and non-limited penetration depth. The simultaneous use of positive and negative MRI imaging that employs the same contrast agents will significantly improve detection accuracy. Here we report the development of functional multimodal iron oxide nanoparticles for targeted MRI of atherothrombosis using a combination of chemical and biological conjugation techniques. Monodisperse, water-soluble and biocompatible ultra-small magnetic dual contrast iron oxide nanoparticles (DCIONs) were generated using a high-temperature co-precipitation route and appeared to be efficient positive and negative dual contrast agents for magnetic resonance imaging. Using a unique chemo-enzymatic approach involving copper-free click chemistry and Staphylococcus aureus sortase A enzyme conjugation, DCIONs were functionalized with single-chain antibodies (scFv) directed against activated platelets for targeting purposes. The DCIONs were also labelled with fluorescent molecules to allow for optical imaging. The antigen binding activity of the scFv was retained and resulted in the successful targeting of contrast agents to thrombosis as demonstrated in a range of invitro and invivo experiments. T1- and T2-weighted MRI of thrombi was recorded and demonstrated the great potential of dual T1/T2 contrast iron oxide particles in imaging of cardiovascular disease.

Precision Templated Bottom-Up Multiprotein Nanoassembly through Defined Click Chemistry Linkage to DNA.

We demonstrate an approach that allows attachment of single-stranded DNA (ssDNA) to a defined residue in a protein of interest (POI) so as to provide optimal and well-defined multicomponent assemblies. Using an expanded genetic code system, azido-phenylalanine (azF) was incorporated at defined residue positions in each POI; copper-free click chemistry was used to attach exactly one ssDNA at precisely defined residues. By choosing an appropriate residue, ssDNA conjugation had minimal impact on protein function, even when attached close to active sites. The protein-ssDNA conjugates were used to (i) assemble double-stranded DNA systems with optimal communication (energy transfer) between normally separate groups and (ii) generate multicomponent systems on DNA origami tiles, including those with enhanced enzyme activity when bound to the tile. Our approach allows any potential protein to be simply engineered to attach ssDNA or related biomolecules, creating conjugates for designed and highly precise multiprotein nanoscale assembly with tailored functionality.

Bifunctional Diaminoterephthalate Fluorescent Dye as Probe for Cross-Linking Proteins.

Diaminoterephthalates are fluorescent dyes and define scaffolds, which can be orthogonally functionalized at their two carboxylate residues with functional residues bearing task specific reactive groups. The synthesis of monofunctionalized dyes with thiol groups for surface binding, an azide for click chemistry, and a biotinoylated congener for streptavidin binding is reported. Two bifunctionalized dyes were prepared: One with an azide for click chemistry and a biotin for streptavidin binding, the other with a maleimide for reaction with thiol and a cyclooctyne moiety for ligation with copper-free click chemistry. In general, the compounds are red to orange, fluorescent materials with an absorption at about 450 nm and an emission at 560 nm with quantum yields between 2-41 %. Of particular interest is the maleimide-functionalized compound, which shows low fluorescence quantum yield (2 %) by itself. After addition of a thiol, the fluorescence is "turned on"; quantum yield 41 %.

Patterning Three-Dimensional Hydrogel Microenvironments Using Hyperbranched Polyglycerols for Independent Control of Mesh Size and Stiffness.

The extracellular matrix is an environment rich with structural, mechanical, and molecular signals that can impact cell biology. Traditional approaches in hydrogel biomaterial design often rely on modifying the concentration of cross-linking groups to adjust mechanical properties. However, this strategy provides limited capacity to control additional important parameters in 3D cell culture such as microstructure and molecular diffusivity. Here we describe the use of multifunctional hyperbranched polyglycerols (HPGs) to manipulate the mechanical properties of polyethylene glycol (PEG) hydrogels while not altering biomolecule diffusion. This strategy also provides the ability to separately regulate spatial and temporal distribution of biomolecules tethered within the hydrogel. The functionalized HPGs used here can also react through a copper-free click chemistry, allowing for the encapsulation of cells and covalently tethered biomolecules within the hydrogel. Because of the hyperbranched architecture and unique properties of HPGs, their addition into PEG hydrogels affords opportunities to locally alter hydrogel cross-linking density with minimal effects on global network architecture. Additionally, photocoupling chemistry allows micropatterning of bioactive cues within the three-dimensional gel structure. This approach therefore enables us to tailor mechanical and diffusive properties independently while further allowing for local modulation of biomolecular cues to create increasingly complex cell culture microenvironments.

Highly robust and optimized conjugation of antibodies to nanoparticles using quantitatively validated protocols.

Antibody-conjugated nanoparticles (NPs) have attracted great attention in diagnostic and therapeutic applications due to their high sensitivity and specificity for biotargets, as well as their wide applicability. Unfortunately, these features are significantly affected by antibody conjugation methods in terms of conjugation efficiency, orientation of the target binding site in the antibody, and denaturation during chemical conjugation reactions. Furthermore, the number of conjugated antibodies on each NP and the overall targeting efficacy are critical factors for a quantitative bioassay with antibody-conjugated NPs. Herein, we report a versatile and oriented antibody conjugation method using copper-free click chemistry. Moreover, the number of conjugated antibodies and their binding capacity were quantitatively and experimentally evaluated using fluorescently-labeled antibodies and antigens. The strong binding capability of antibody-conjugated NPs prepared using the copper-free click chemistry-based conjugation strategy was 8 times superior to the binding capability seen following the use of the EDC/NHS-coupling method. Additionally, the versatility of the developed antibody conjugation method was also demonstrated by conjugation of the antibody to three kinds of silica-encapsulated NPs.

Overcoming multidrug resistance using folate receptor-targeted and pH-responsive polymeric nanogels containing covalently entrapped doxorubicin.

Multidrug resistance (MDR) contributes to failure of chemotherapy. We here show that biodegradable polymeric nanogels are able to overcome MDR via folic acid targeting. The nanogels are based on hydroxyethyl methacrylamide-oligoglycolates-derivatized poly(hydroxyethyl methacrylamide-co-N-(2-azidoethyl)methacrylamide) (p(HEMAm-co-AzEMAm)-Gly-HEMAm), covalently loaded with the chemotherapeutic drug doxorubicin (DOX) and subsequently decorated with a folic acid-PEG conjugate via copper-free click chemistry. pH-Responsive drug release is achieved via the acid-labile hydrazone bond between DOX and the methacrylamide polymeric network. Cellular uptake and cytotoxicity analyses in folate receptor-positive B16F10 melanoma versus folate receptor-negative A549 lung carcinoma cells confirmed specific uptake of the targeted nanogels. Confocal microscopy demonstrated efficient internalization, lysosomal trafficking, drug release and nuclear localization of DOX. We also show that DOX resistance in 4T1 breast cancer cells results in upregulation of the folate receptor, and that folic acid targeted nanogels can be employed to bypass drug efflux pumps, resulting in highly efficient killing of resistant cancer cells. In conclusion, folic acid functionalized nanogels with pH-controlled drug release seem to hold significant potential for treating multidrug resistant malignancies.

Toward Artificial Immunotoxins: Traceless Reversible Conjugation of RNase A with Receptor Targeting and Endosomal Escape Domains.

The specific transport of bioactive proteins into designated target cells is an interesting and challenging perspective for the generation of innovative biopharmaceuticals. Natural protein cytotoxins perform this task with outstanding efficacy. They enter cells with receptor-targeted specificity, respond to changing intracellular microenvironments, and by various mechanisms translocate their cytotoxic protein subunit into the cytosol. Here we imitate this toxin-based delivery strategy in an artificial setting, by bioreversible conjugation of a cytotoxic cargo protein (RNase A) with receptor-targeting PEG-folate and the pH-specific endosomolytic peptide INF7 as synthetic delivery domains. Covalent modification of the cargo protein was achieved using the pH-labile AzMMMan linker and copper-free click chemistry with DBCO-modified delivery modules. This linkage is supposed to enable traceless intracellular release of the RNase A after exposure to the endosomal weakly acidic environment. Delivery of RNase A via this polycation-free delivery strategy resulted in high cytotoxicity against receptor-positive KB tumor cells only when both PEG-folate and INF7 were attached.

Direct imaging of glycans in Arabidopsis roots via click labeling of metabolically incorporated azido-monosaccharides.

BackgroundCarbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques.ResultsArabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, l-fucose, and l-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions.ConclusionsOur results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands the molecular toolbox for direct glycan imaging in plants, from three to eight glycan analogs, which enables more extensive future studies of spatiotemporal glycan dynamics in a wide variety of plant tissues and species. We also show, for the first time in metabolic labeling and imaging of plant glycans, the potential of two copper-free click chemistry methods that are bio-orthogonal and lead to more uniform labeling. These improved labeling methods can be generalized and extended to already existing and future click chemistry-enabled monosaccharide analogs in Arabidopsis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0907-0) contains supplementary material, which is available to authorized users.

Non-invasive stem cell tracking in hindlimb ischemia animal model using bio-orthogonal copper-free click chemistry

Labeling of stem cells aims to distinguish transplanted cells from host cells, understand in vivo fate of transplanted cells, particularly important in stem cell therapy. Adipose-derived mesenchymal stem cells (ASCs) are considered as an emerging therapeutic option for tissue regeneration, but much remains to be understood regarding the in vivo evidence. In this study, a simple and efficient cell labeling method for labeling and tracking of stem cells was developed based on bio-orthogonal copper-free click chemistry, and it was applied in a mouse hindlimb ischemia model. The human ASCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate glycoprotein with unnatural azide groups on the cell surface, and the generated azide groups were fluorescently labeled by specific binding of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5). The safe and long-term labeling of the hASCs by this method was first investigated in vitro. Then the DBCO-Cy5-hASCs were transplanted into the hindlimb ischemia mice model, and we could monitor and track in vivo fate of the cells using optical imaging system. We could clearly observe the migration potent of the hASCs toward the ischemic lesion. This approach to design and tailor new method for labeling of stem cells may be useful to provide better understanding on the therapeutic effects of transplanted stem cells into the target diseases.

ChemInform Abstract: A Practical Guide on the Synthesis of Metal Chelates for Molecular Imaging and Therapy by Means of Click Chemistry

AbstractReview: [67 refs.

Multifunctional and High Affinity Polymer Ligand that Provides Bio-Orthogonal Coating of Quantum Dots.

We detail the design of hydrophilic metal-coordinating ligands and their use for the effective coating of luminescent quantum dots (QDs). The ligand design exploits the specific, reagent-free nucleophilic addition reaction of amine-modified molecules toward maleic anhydride to introduce several lipoic acid metal anchors, hydrophilic zwitterion moieties, and specific reactive groups along a poly(isobutylene-alt-maleic anhydride) (PIMA) chain. Tunable reactive groups tested in this study include azide, biotin, carboxyl, and amine. Cap exchange with these multilipoic acid ligands via a photochemical ligation strategy yields homogeneous QD dispersions that are colloidally stable over several biologically relevant conditions and for extended periods of time. The zwitterionic coating yields compact nanoparticle size and imparts nonsticky surface properties onto the QDs, preventing protein absorption. The introduction of a controllable number of reactive groups allows conjugation of the QDs to biomolecules via bio-orthogonal coupling chemistries including (1) attachment of the neurotransmitter dopamine to QDs via amine-isothiocyanate reaction to produce a platform capable of probing interactions with cysteine in proteins, based on charge transfer interactions; (2) self-assembly of biotinylated QDs with streptavidin-dye; and (3) ligation of azide-functionalized QDs to cyclooctyne-modified transferrin via copper-free click chemistry, used for intracellular delivery. This ligand design strategy can be used to prepare an array of metal-coordinating ligands adapted for coating other inorganic nanoparticles, including magnetic and plasmonic nanomaterials.

Efficient Formation of Site-Specific Protein-DNA Hybrids Using Copper-Free Click Chemistry.

Protein-DNA hybrids have become increasingly popular molecular building blocks in bionanotechnology and single-molecule studies to synergistically combine the programmability of DNA with the chemical diversity of proteins. The growing demand for protein-DNA hybrids requires powerful strategies for their conjugation. Here, we present an efficient two-step method for protein-DNA assembly based on copper-free click chemistry. The method allows site-specificity and high coupling efficiency, while maintaining the conservation of protein activity. We compare our method to a commonly used protocol of direct linkage of maleimide-modified oligos. We demonstrate the significantly higher yield with a protein-DNA conjugate, which is analyzed using single-molecule force spectroscopy.