Facile Labeling Research Articles

Assembling a new generation of radiopharmaceuticals with supramolecular theranostics.

Supramolecular chemistry has been used to tackle some of the major challenges in modern science, including cancer therapy and diagnosis. Supramolecular platforms provide synthetic flexibility, rapid generation through self-assembly, facile labelling, unique topologies, tunable reversibility of the enabling noncovalent interactions, and opportunities for host-guest chemistry and mechanical bonding. In this Review, we summarize recent advances in the design and radiopharmaceutical application of discrete self-assembled coordination complexes and mechanically interlocked molecules - namely, metallacages and rotaxanes, respectively - as well as in situ-forming supramolecular aggregates, specifically pinpointing their potential as next-generation radiotheranostic agents. The outlook of suchsupramolecular constructs for potential applications in the clinic is discussed.

Absolute Quantification of Dynamic Cellular Uptake of Small Extracellular Vesicles via Lanthanide Element Labeling and ICP-MS.

Small extracellular vesicles (sEVs) are increasingly reported to play important roles in numerous physiological and pathological processes. Cellular uptake of sEVs is of great significance for functional regulation in recipient cells. Although various sEV quantification, labeling, and tracking methods have been reported, it is still highly challenging to quantify the absolute amount of cellular uptake of sEVs and correlate this information with phenotypic variations in the recipient cell. Therefore, we developed a novel strategy using lanthanide element labeling and inductively coupled plasma-mass spectrometry (ICP-MS) for the absolute and sensitive quantification of sEVs. This strategy utilizes the chelation interaction between Eu3+ and the phosphate groups on the sEV membrane for specific labeling. sEVs internalized by cells can then be quantified by ICP-MS using a previously established linear relationship between the europium content and the particle numbers. High Eu labeling efficiency and stability were demonstrated by various evaluations, and no structural or functional alterations in the sEVs were discovered after Eu labeling. Application of this method revealed that 4020 ± 171 sEV particles/cell were internalized by HeLa cells at 37 °C and 61% uptake inhibition at 4 °C. Further investigation led to the quantitative differential analysis of sEV cellular uptake under the treatment of several chemical endocytosis inhibitors. A 23% strong inhibition indicated that HeLa cells uptake sEVs mainly through the macropinocytosis pathway. This facile labeling and absolute quantification strategy of sEVs with ppb-level high sensitivity is expected to become a potential tool for studying the functions of sEVs in intracellular communication and cargo transportation.

Exploration of 68Ga-DOTA-MAL as a Versatile Vehicle for Facile Labeling of a Variety of Thiol-Containing Bioactive Molecules.

Efficient and site-specific radiolabeling reactions are essential in molecular probe synthesis. Thus, selecting an effective method for radiolabeling that does not affect bioactivity of the molecule is critical. Varieties of bifunctional chelating agents provide a solution in this matter. As a chemo-specific chelator, maleimido-mono-amide-DOTA (DOTA-Mal) holds significant potential for 68Ga labeling of bioactive molecules; it can react specifically with free sulfhydryl groups under mild conditions. Compared with amino and carboxylic acid groups, free sulfhydryl groups are relatively less common in most biomolecules and can serve as site-specific radiolabeling targets. Labeling of 68Ga usually employs a two-step labeling strategy; first, chelators are conjugated to the biomolecules, which is followed by radiolabeling. However, the bioactivity of biomolecules may be affected by harsh labeling conditions. In this study, three 68Ga-labeled bioactive molecules, namely, 68Ga-DOTA-RGD, 68Ga-DOTA-FA, and 68Ga-DOTA-BSA, were prepared using a novel strategy under mild conditions (pH of 8.0 at room temperature). Using this strategy, DOTA-Mal was labeled by 68Ga before it reacted with the sulfhydryl group-containing biomolecules, which avoided damage to said biomolecules caused by the harsh reaction conditions required in 68Ga-labeling procedures. The biological and chemical properties of these three radiotracers synthesized using this strategy are well manifested. Through a series of experiments, the effectiveness of this strategy is demonstrated, and we believe that this site-specific bioactivity-friendly reaction strategy will facilitate developments and translation applications of varieties of 68Ga-labeled positron emission tomography probes.

P-239 - A trifluoroborate (BF3) radioprosthetic group bearing FAP-targeting ligand for facile labeling with 18F via the 18F-19F isotope exchange reaction: synthesis, radiolabeling and in vivo evaluation

P-239 - A trifluoroborate (BF3) radioprosthetic group bearing FAP-targeting ligand for facile labeling with 18F via the 18F-19F isotope exchange reaction: synthesis, radiolabeling and in vivo evaluation

P-038 - [11C]Carboxylated tetrazines for facile labeling of trans-cyclooctene-functionalized macromolecules

P-038 - [11C]Carboxylated tetrazines for facile labeling of trans-cyclooctene-functionalized macromolecules

11 C]Carboxylated Tetrazines for Facile Labeling of Trans-Cyclooctene-Functionalized PeptoBrushes.

Functionalization of macromolecules (antibodies, polymers, nanoparticles) with click-reactive groups greatly enhances the versatility of their potential applications. Click chemistry based on tetrazine - trans-cyclooctene (TCO) ligation is especially promising and is already widely applied for pretargeted imaging and therapy. Indirect radiolabeling of TCO-functionalized macromolecules with substoichiometric amounts of radioactive tetrazines is a convenient way to monitor the fate of those macromolecules by means of positron emission tomography (PET) imaging after their administration into the test subject. In this work, the preparation is reported of TCO-containing graft copolymers, namely PeptoBrushes (polyglutamic acid-graft-polysarcosine), novel [11 C]carboxylated tetrazines, and their combined use in radiolabeling the polymer by inverse electron demand Diels Alder reaction, to investigate it is potential for an application in pretarget imaging or injectable brachytherapy. The procedure for [11 C]tetrazine production is easy and scalable, while indirect TCO-PeptoBrushes labeling with these [11 C]tetrazines is mild, fast, and quantitative. This strategy allows facile 11 C-labeling of diverse TCO-functionalized macromolecules, so that their localization and distribution shortly after injection can be assessed by PET.

Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales.

Superresolution Imaging of Live Cells with Genetically Encoded Silicon Rhodamine-Binding RNA Aptamers

The key role of fluorescence microscopy in the molecular life sciences rests to a large extent on the availability of genetically encoded fluorescence marker proteins, allowing facile labeling of specific proteins within living cells. Although there is an urgent need to establish a comparable, easy-to-use technology for specific, genetically encoded fluorescence labeling of ribonucleic acid (RNA) molecules, such strategies are less advanced. Here, we present studies exploring the suitability of SiRA, a novel, silicon rhodamine (SiR)-binding RNA aptamer for super-resolution microscopy in live cells. Selective binding of SiRA to the fluorescent, zwitterionic form of SiR dyes, rather than to the non-fluorescent, spiro-cyclized form, results in a significant fluorescence turn-on, generating an extremely photostable and, in fact, the brightest far-red light-up aptamer system reported to date. We have found that SiRA can be utilized as an excellent marker for stimulated emission depletion (STED) nanoscopy; further research toward applications in single-molecule based localization microscopy is presently ongoing.

Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

We introduce UnaG as a green-to-dark photoswitching fluorescent protein capable of high-quality super-resolution imaging with photon numbers equivalent to the brightest photoswitchable red protein. UnaG only fluoresces upon binding of a fluorogenic metabolite, bilirubin, enabling UV-free reversible photoswitching with easily controllable kinetics and low background under Epi illumination. The on- and off-switching rates are controlled by the concentration of the ligand and the excitation light intensity, respectively, where the dissolved oxygen also promotes the off-switching. The photo-oxidation reaction mechanism of bilirubin in UnaG suggests that the lack of ligand-protein covalent bond allows the oxidized ligand to detach from the protein, emptying the binding cavity for rebinding to a fresh ligand molecule. We demonstrate super-resolution single-molecule localization imaging of various subcellular structures genetically encoded with UnaG, which enables facile labeling and simultaneous multicolor imaging of live cells. UnaG has the promise of becoming a default protein for high-performance super-resolution imaging.

Efficient Labeling Of Mesenchymal Stem Cells For High Sensitivity Long-Term MRI Monitoring In Live Mice Brains.

BackgroundRegenerative medicine field is still lagging due to the lack of adequate knowledge regarding the homing of therapeutic cells towards disease sites, tracking of cells during treatment, and monitoring the biodistribution and fate of cells. Such necessities require labeling of cells with imaging agents that do not alter their biological characteristics, and development of suitable non-invasive imaging modalities.PurposeWe aimed to develop, characterize, and standardize a facile labeling strategy for engineered mesenchymal stem cells without altering their viability, secretion of FGF21 protein (neuroprotective), and differentiation capabilities for non-invasive longitudinal MRI monitoring in live mice brains with high sensitivity.MethodsWe compared the labeling efficiency of different commercial iron oxide nanoparticles towards our stem cells and determined the optimum labeling conditions using prussian blue staining, confocal microscopy, transmission electron microscopy, and flow cytometry. To investigate any change in biological characteristics of labeled cells, we tested their viability by WST-1 assay, expression of FGF21 by Western blot, and adipogenic and osteogenic differentiation capabilities. MRI contrast-enhancing properties of labeled cells were investigated in vitro using cell-agarose phantoms and in mice brains transplanted with the therapeutic stem cells.ResultsWe determined the nanoparticles that showed best labeling efficiency and least extracellular aggregation. We further optimized their labeling conditions (nanoparticles concentration and media supplementation) to achieve high cellular uptake and minimal extracellular aggregation of nanoparticles. Cell viability, expression of FGF21 protein, and differentiation capabilities were not impeded by nanoparticles labeling. Low number of labeled cells produced strong MRI signal decay in phantoms and in live mice brains which were visible for 4 weeks post transplantation.ConclusionWe established a standardized magnetic nanoparticle labeling platform for stem cells that were monitored longitudinally with high sensitivity in mice brains using MRI for regenerative medicine applications.

Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry.

The lanthanides (Ln3+), or rare earth elements, have proven to be useful tools for biomolecular NMR, X-ray crystallographic, and fluorescence analyses due to their unique 4f orbitals. However, their utility in biological applications has been limited because site-specific incorporation of a chelating element is required to ensure efficient binding of the free Ln3+ ion. Additionally, current Ln3+ chelator syntheses complicate efforts to directly incorporate Ln3+ chelators into proteins as the multi-step processes and a reliance on organic solvents promote protein denaturation and aggregation which are generally incompatible with direct incorporation into the protein of interest. To overcome these limitations, herein we describe a two-step aqueous synthesis of a small molecule lanthanide chelating agent amenable to site-specific incorporation into a protein using copper-free click chemistry with unnatural amino acids. The bioconjugate combines a diethylenetriaminepentaacetic acid (DTPA) chelating moiety with a clickable dibenzylcyclooctyne-amine (DBCO-amine) to facilitate the reaction with an azide containing unnatural amino acid. Incorporating the DBCO-amine avoids the use of the cytotoxic Cu2+ ion as a catalyst. The clickable lanthanide chelator (CLC) reagent reacted readily with p-azidophenylalanine (paF) without the need of a copper catalyst, thereby demonstrating proof-of-concept. Implementation of the orthogonal click chemistry reaction has the added advantage that the chelator can be used directly in a protein labeling reaction, without the need of extensive purification. Given the inherent advantages of Cu2+-free click chemistry, aqueous synthesis, and facile labeling, we believe that the CLC will find abundant use in both structural and biophysical studies of proteins and their complexes.

One-Step 18F-Labeling and Preclinical Evaluation of Prostate-Specific Membrane Antigen Trifluoroborate Probes for Cancer Imaging.

After the identification of the high-affinity glutamate-ureido scaffold, the design of several potent 18F- and 68Ga-labeled tracers has allowed spectacular progress in imaging recurrent prostate cancer by targeting the prostate-specific membrane antigen (PSMA). We evaluated a series of PSMA-targeting probes that are 18F-labeled in a single step for PET imaging of prostate cancer. Methods: We prepared 8 trifluoroborate constructs for prostate cancer imaging, to study the influence of the linker and the trifluoroborate prosthetic on pharmacokinetics and image quality. After 1-step labeling by 19F–18F isotopic exchange, the radiotracers were injected in mice bearing LNCaP xenografts, with or without blocking controls, to assess specific uptake. PET/CT images and biodistribution data were acquired at 1 h after injection and compared with 18F-DCFPyL on the same mouse strain and tumor model. Results: All tracers exhibited nanomolar affinities, were labeled in good radiochemical yields at high molar activities, and exhibited high tumor uptake in LNCaP xenografts with clearance from nontarget organs. Most derivatives with a naphthylalanine linker showed significant gastrointestinal excretion. A radiotracer incorporating this linker with a dual trifluoroborate-glutamate labeling moiety showed high tumor uptake, low background activity, and no liver or gastrointestinal track accumulation. Conclusion: PSMA-targeting probes with trifluoroborate prosthetic groups represent promising candidates for prostate cancer imaging because of facile labeling while affording high tumor uptake values and contrast ratios that are similar to those obtained with 18F-DCFPyL.

PH-Responsive diblock copolymers with two different fluorescent labels for simultaneous monitoring of micellar self-assembly and degree of protonation

Facile labelling of both blocks of a pH-responsive diblock copolymer with different fluorophores allows monitoring of polymer aggregation and deprotonation.

Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

Fast Diazaborine Formation of Semicarbazide Enables Facile Labeling of Bacterial Pathogens.

Bioorthogonal conjugation chemistry has enabled the development of tools for the interrogation of complex biological systems. Although a number of bioorthogonal reactions have been documented in literature, they are less ideal for one or several reasons including slow kinetics, low stability of the conjugated product, requirement of toxic catalysts, and side reactions with unintended biomolecules. Herein we report a fast (>103 M-1 s-1) and bioorthogonal conjugation reaction that joins semicarbazide to an aryl ketone or aldehyde with an ortho-boronic acid substituent. The boronic acid moiety greatly accelerates the initial formation of a semicarbazone conjugate, which rearranges into a stable diazaborine. The diazaborine formation can be performed in blood serum or cell lysates with minimal interference from biomolecules. We further demonstrate that application of this conjugation chemistry enables facile labeling of bacteria. A synthetic amino acid D-AB3, which presents a 2-acetylphenylboronic acid moiety as its side chain, was found to incorporate into several bacterial species through cell wall remodeling, with particularly high efficiency for Escherichia coli. Subsequent D-AB3 conjugation to a fluorophore-labeled semicarbazide allows robust detection of this bacterial pathogen in blood serum.

A platform with connections in many directions - further remarkable facets to the multifaceted methylbiquinoxen dication.

The N,N'-dimethyl-3,3'-biquinoxalinium "methylbiquinoxen" dicationic platform is revealed to have even more fascinating possibilities than we originally thought in terms of its chemical versatility. In addition to its rich redox chemistry and coordination abilities, we have now unveiled an unexpected Lewis acid/base chemistry linked with a tuneable switching of its luminescence properties. This, amongst other things, allows for the facile fluorescent covalent labelling of hydroxyl-terminated materials. This platform provides intriguing chemical prospects realised in molecular systems such as porphyrins as well as an easy alternative functionalisation methodology to that provided by click-chemistry.

Preparation, Characterization and Intracellular Imaging of 2,4-Dichlorophenoxyacetic Acid Conjugated Gold Nanorods.

Visualizing the biodistribution of pesticides inside living cells is great importance for enhancing targeting of pesticides. Here we reported for the first time that gold nanorods (Au NRs) with size of 39.4 nm x 11.3 nm could be used as a fluorescent tracer to examine the distribution of a typical herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), in tobacco bright yellow 2 (BY-2) cells. The nanostructures of hybrid materials were analyzed by using Raman spectra and X-ray photoelectron spectroscopy (XPS), including spectra assignments and electronic property. These data revealed 2,4-D has successfully conjugated MP-Au NRs according to Raman and XPS. The biodistribution of the conjugates inside BY-2 cells was directly examined at 12 and 24 h by the two-photon microscopy. The intensity of two-photon luminescence (TPL) inside cells demonstrated that the conjugates could be localized and excluded by BY-2 cells. Thus, this labeling approach opens up new avenues to the facile and efficient labeling of pesticides.

Fast and selective labeling of N-terminal cysteines at neutral pH via thiazolidino boronate formation

Facile labeling of proteins of interest is highly desirable in proteomic research as well as in the development of protein therapeutics. Herein we report a novel method that allows for fast and selective labeling of proteins with an N-terminal cysteine. Although N-terminal cysteines are well known to conjugate with aldehydes to give thiazolidines, the reaction requires acidic conditions and suffers from slow kinetics. We show that benzaldehyde with an ortho-boronic acid substituent readily reacts with N-terminal cysteines at neutral pH, giving rate constants on the order of 103 M-1 s-1. The product features a thiazolidono boronate (TzB) structure and exhibits improved stability due to formation of the B-N dative bond. While stable at neutral pH, the TzB complex dissociates upon mild acidification. These characteristics make the TzB conjugation chemistry potentially useful for the development of drug-protein conjugates that release the small molecule drug in acidic endosomes.

Facile labelling of graphene oxide for superior capacitive energy storage and fluorescence applications.

The majority of supercapacitor research studies on graphene materials today have been based upon developing electrochemical double-layer capacitors (EDLCs) using reduced graphenes. In contrast, graphene oxide (GO) is often neglected as a supercapacitor candidate due to its low electrical conductivity and surface area. Nonetheless, we present herein a fast (1 h) labelling of GO with o-phenylenediamine (PD) to produce PD-GO, exploiting inherent oxygen groups in creating new functionalities that exhibit capacitive enhancement from pseudo-capacitance. A high specific capacitance of 191 F g(-1) was obtained (at 0.2 A g(-1)), comparable to recent binder-free graphene supercapacitors. The large surface-normalized capacitance of up to 628 μF cm(-2) is also many times greater than the intrinsic capacitance of single-layer graphene (21 μF cm(-2)) as a result of additional pseudo-capacitance. A high capacity retention of ∼85% with each 10-fold increase in current density further indicates excellent rate performance. Hence, this approach in enhancing GO pseudo-capacitance may be similarly feasible as graphene EDLCs. Additionally, PD-GO was also found to exhibit a bright green fluorescence with a 540 nm maximum. The strongest fluorescence intensities arose from the smallest PD-GO fragments, and we attribute the origin to localised sp(2) domains and newly formed phenazine edge groups. The dual enhancement of dissimilar properties such as capacitance and fluorescence emphasizes the continued significance of covalent functionalisation towards tuning of properties in graphene-type materials.

Carbon-11 radiolabelling of organosulfur compounds: (11) C synthesis of the progesterone receptor agonist tanaproget.

Herein a new (11) C radiolabelling strategy for the fast and efficient synthesis of thioureas and related derivatives using the novel synthon, (11) CS2 , is reported. This approach has enabled the facile labelling of a potent progesterone receptor (PR) agonist, [(11) C]Tanaproget, by the intramolecular reaction of the acyclic aminohydroxyl precursor with (11) CS2 , which has potential applications as a positron emission tomography radioligand for cancer imaging.