Chemical Conjugation Research Articles

Chemical–biological approaches for the direct regulation of cell–cell aggregation

AbstractCell–cell aggregation is one of the most well‐known modes of intercellular communication. The aggregation also plays a vital role in the formation of multicellularity, thus manipulating the growth and development of organisms. In the past decades, cell–cell aggregation‐related bioprocesses and molecular mechanisms have attracted enormous interest from scientists in biology, and bioengineering. People have developed a series of strategies to artificially regulate cell–cell aggregation through chemical–biological approaches. To date, not only the chemical reagents such as coordination compounds and polymers but also the biomacromolecules such as proteins and nucleic acids, are employed as the “cell glue” to achieve the control of the cell aggregation. So it is meaningful to review the recent advances of the chemical–biological approaches in cell–cell aggregation manipulation. In this review, we discuss the mechanisms and features of recently developed strategies to control cell–cell aggregation. We introduce molecules and designs relying on chemical reactions and biological conjugations respectively, and talk about their advantages and suitable applications. A perspective on the challenges in future applications in cell manipulation and cell‐based therapy is also proposed. We expect this review could inspire innovative work on manipulating cell–cell aggregation and further modulate cell–cell interactions in the research of bio/chemical fields.

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.

Evaluation of novel conjugated resveratrol polymeric nanoparticles in reduction of plasma degradation, hepatic metabolism and its augmentation of anticancer activity in vitro and in vivo

Resveratrol (RSV) is a natural product with multiple biological benefits including anticancer properties. Unfortunately, its biological benefits are limited by its low bioavailability and rapid hepatic metabolism and degradation in the body. The aim of this study was to develop an effective delivery system for RSV that would enhance the plasmatic stability and decrease the metabolism rate of RSV through a dual strategy of chemical modification and nanoparticle formulation. The effectiveness of this strategy was tested for the application of RSV anticancer treatment in a mouse cancer model. Chemical modification of RSV was achieved by conjugating RSV to a low molecular weight co-polymer mPEG-PLA. This conjugated RSV together with free RSV were formulated into mPEG-PLA nanoparticles (conjugated RSV NPs). These NPs showed a stable plasma stability profile and decreased liver metabolism rate compared to nanoparticles encapsulating free RSV in mPEG-PLA (encapsulated RSV NPs) and free RSV alone. However, in vitro cell studies using B16-F10 cancer cells showed that conjugated RSV NPs were less effective compared to encapsulated RSV NPs, possibly due to the lack of biotransformation of conjugated RSV to the active form RSV in the simple cell studies. To study the actual effect of our strategy, an in vivo C57BL/6J mouse model with subcutaneous B16-F10 melanoma using intraperitoneal administration was used to reveal the relationship between the improved plasma stability and reduced liver metabolism rate of RSV in conjugated RSV NPs, and suppression of the tumour growth in mice. In vivo, a better tumour suppression trend with conjugated RSV NPs was noted. Our study suggests that the use of chemical conjugation with NP formulation is an effective strategy to reduce the degradation and metabolism rate of RSV and consequently increase the antitumour activity of RSV in vivo. This strategy has potential to be further developed for the suppression of early growth of tumours with no side effects.

Tumor-Associated Immune-Cell-Mediated Tumor-Targeting Mechanism with NIR-II Fluorescence Imaging.

The strategy of structure-inherent tumor targeting (SITT) with cyanine-based fluorophores is receiving more attention because no chemical conjugation of targeting moieties is required. However, the targeting mechanism behind SITT has not yet been well explained. Here, it is demonstrated that heptamethine-cyanine-based fluorophores possess not only targetability of tumor microenvironments without the need for additional targeting ligands but also second near-infrared spectral window (NIR-II) imaging capabilities, i.e., minimum scattering and ultralow autofluorescence. The new SITT mechanism suggests that bone-marrow-derived and/or tissue-resident/tumor-associated immune cells can be a principal target for cancer detection due to their abundance in tumoral tissues. Among the tested, SH1 provides ubiquitous tumor targetability and a high tumor-to-background ratio (TBR) ranging from 9.5 to 47 in pancreatic, breast, and lung cancer mouse models upon a single bolus intravenous injection. Furthermore, SH1 can be used to detect small cancerous tissues smaller than 2 mm in diameter in orthotopic lung cancer models. Thus, SH1 could be a promising cancer-targeting agent and have a bright future for intraoperative optical imaging and image-guided cancer surgery.

Aptasensors versus immunosensors-Which will prevail?

Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element.

Antigen epitope-TLR7/8a conjugate as self-assembled carrier-free nanovaccine for personalized immunotherapy

Epitope-based vaccine is a promising personalized cancer immunotherapy; however, a simple and effective approach for its bulk manufacturing is challenging. Current vaccination strategies complicate the process by introducing unnecessary components such as additional delivery carriers, and assembly units. Herein, a type of toll-like receptor 7/8 agonist-epitope conjugate (termed as TLR7/8a-epitope) has been developed as a self-assembled and carrier-free nano vaccine platform, which effectively introduces the antigen and adjuvant with maximum precision, resulting in significantly enhanced dendritic cells (DCs) activation through the MyD88-dependent TLR signaling pathway. TLR7/8a-epitope nanovaccine can prolong the local retention and increase drainage efficiency into the lymph node, eliciting a significantly higher level of CD8 T-cell immunity than those of conventional vaccine formulations. The immunization with TLR7/8a-epitope nanovaccine in mice can not only resist the invasion of B16 cancer cells, but also produce significant therapeutic effects against established B16 melanoma tumors. Therefore, the TLR7/8a-epitope nanovaccine, developed by the direct chemical conjugation of antigen peptide with immunoadjuvant, has great advantages of clear and leanest compositions, controllable and definite preparation process, and remarkable therapeutic effects, representing a new appraoch for personalized cancer immunotherapy. Statement of significanceHerein, a kind of toll-like receptor 7/8 agonist-epitope conjugate was developed and spontaneously self-assemble into nanostructure in aqueous solution without the use of any additional constituents, which can be termed as unique carrier-free nanovaccine platform, providing effectually the leanest vaccine components with maximally and precisely loading of antigen and adjuvant.Significantly, the nanovaccine augmented the immunogenicity of antigenic peptide by increasing DCs activation through MyD88-mediated TLR signaling pathways and promoting T-cell priming. Moreover, nanovaccines could prolong the local retention and further increase the efficiency of drainage into dLNs, which was contributing to efficient initiation of epitope-specific memory and effector T-cell immune responses, leading to effective prophylactic and therapeutic antitumor effects.

Functionalization of Magnetic Beads with Biotinylated Nanobodies for MALDI-TOF/MS-Based Quantitation of Small Analytes.

Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound.

Template-directed conjugation of heterogeneous oligonucleotides to a homobifunctional molecule for programmable supramolecular assembly.

Nanoscience aspires to mimic nature's control over functional molecular assemblies. Here we present a templating technique for the efficient attachment of two different oligonucleotides to a homobifunctional molecule, enabling its controlled and programmable placement within a DNA nanostructure. We demonstrate its application to a range of organic molecules with different conjugation chemistries and water solubilities. We show that the two oligonucleotide adapters can be used to integrate a bifunctional cyanine dye into a self-assembled three-dimensional DNA origami nanostructure, giving control of both position and orientation. We also demonstrate the use of both adapters to exert dynamic control over the environment of the target molecule by means of a series of strand-displacement reactions.

Adding a diazo-transfer reagent to culture to generate secondary metabolite probes for click chemistry.

Converting discrete microbial metabolites into chemical probes for chemical biology and medicinal chemistry studies is typically preceded by lengthy purification and chemical derivatization processes. Standard practice involves purifying the target microbial metabolite from culture, followed by derivatization and/or conjugation chemistry to convert the pure metabolite into a tagged species. This multistep approach can pose difficulties in generating useful yields of chemical probes, particularly in the case of low-abundant metabolites, as common in metabolomes. This chapter describes a methodological approach to simplify the steps towards generating chemical probes from complex mixtures, that combines: (a) tailored purification processes; (b) compound identification using state-of-the-art tandem mass spectrometry and data-dependent fragmentation; and (c) in situ bioorthogonal bioconjugation chemistries. The combination of these methods, as illustrated by the conversion of a set of amine-bearing metabolites to the cognate azide analogs suitable for biotinylation through azide-alkyne cycloaddition, describes a powerful approach to access new chemical probes of low-abundant metabolites that might otherwise be inaccessible using traditional methods.

Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen.

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.

Engineering bacterial surface interactions using DNA as a programmable material.

The diverse surface interactions and functions of a bacterium play an important role in cell signaling, host infection, and colony formation. To understand and synthetically control the biological functions of individual cells as well as the whole community, there is growing attention on the development of chemical and biological tools that can integrate artificial functional motifs onto the bacterial surface to replace the native interactions, enabling a variety of applications in biosynthesis, environmental protection, and human health. Among all these functional motifs, DNA emerges as a powerful tool that can precisely control bacterial interactions at the bio-interface due to its programmability and biorecognition properties. Compared with conventional chemical and genetic approaches, the sequence-specific Watson-Crick interaction enables almost unlimited programmability in DNA nanostructures, realizing one base-pair spatial control and bio-responsive properties. This highlight aims to provide an overview on this emerging research topic of DNA-engineered bacterial interactions from the aspect of synthetic chemists. We start with the introduction of native bacterial surface ligands and established synthetic approaches to install artificial ligands, including direct modification, metabolic engineering, and genetic engineering. A brief overview of DNA nanotechnology, reported DNA-bacteria conjugation chemistries, and several examples of DNA-engineered bacteria are included in this highlight. The future perspectives and challenges in this field are also discussed, including the development of dynamic bacterial surface chemistry, assembly of programmable multicellular community, and realization of bacteria-based theranostic agents and synthetic microbiota as long-term goals.

A promising discovery of an anti-aging chemical conjugate derived from lipoic acid and sesamol established in Drosophila melanogaster

Phytonutrients, lipoic acid and sesamol, were chemically combined to yield the medically important lipoic acid–sesamol conjugate (LSC). The results from these investigations offer a perspective on the utilization of LSC as anti-aging agents.

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins.

The bioconjugation of proteins—that is, the creation of a covalent link between a protein and any other molecule—has been studied for decades, partly because of the numerous applications of protein conjugates, but also due to the technical challenge it represents. Indeed, proteins possess inner physico-chemical properties—they are sensitive and polynucleophilic macromolecules—that make them complex substrates in conjugation reactions. This complexity arises from the mild conditions imposed by their sensitivity but also from selectivity issues, viz the precise control of the conjugation site on the protein. After decades of research, strategies and reagents have been developed to address two aspects of this selectivity: chemoselectivity—harnessing the reacting chemical functionality—and site-selectivity—controlling the reacting amino acid residue—most notably thanks to the participation of synthetic chemistry in this effort. This review offers an overview of these chemical bioconjugation strategies, insisting on those employing native proteins as substrates, and shows that the field is active and exciting, especially for synthetic chemists seeking new challenges.

Conjugate of ibrutinib with a TLR7 agonist suppresses melanoma progression and enhances antitumor immunity.

The use of large molecules for immunotherapy has led to exciting developments in cancer treatment, such as the development of PD-1/PD-L1 antibodies. However, small molecule targeted therapies still lack effective immune-functional classes. Ideal anticancer drugs should simultaneously generate immune memory when killing cancer cells to prevent tumor relapse and metastasis. To this end, we carried out a rationally designed strategy to develop novel classes of small molecule compounds with bifunctional targeting and immunostimulatory abilities by conjugating targeting compounds with TLR7 agonists, generating immune-targeting conjugates (ImmunTacs). GY161, as a representative ImmunTac, was synthesized via chemical conjugation of ibrutinib with a TLR7 agonist. In vitro, GY161 stimulated the production of cytokines by mouse spleen lymphocytes, promoted the maturation of dendritic cells (DCs), and inhibited the growth and induced the apoptosis of B16 melanoma cells by regulating the c-Met/β-catenin pathway. In vivo, GY161 enhanced the frequency of CD8+ T cells in spleens and tumors, suppressed the growth of B16 melanoma cell-derived tumors and prolonged the survival time of mice. In summary, GY161 could prevent melanoma progression through direct tumor killing and by triggering specific immunity. These results strongly suggest that ImmunTacs are a reliable and promising strategy for developing small molecule immunogenic anticancer drugs.

Alpha-Fetoprotein Binds Toxins and Can Be Used to Treat Cancer

The inefficiency of the immune system to kill cancer cells leads to the disease. Like in pregnancy, oncofetal proteins counteract the immune attack. Alpha-fetoprotein is an immunosuppressive protein generated by the embryo and in insignificant amounts by adults. It delivers nutrients to embryo cells and to the monocytes that suppress both innate and adaptive immunity during pregnancy and cancer. The small subpopulation of suppressor monocytes and cancer cells absorb the alpha-fetoprotein-nutrient complex through the specific receptor that is mostly absent in normal adult cells. It comes out that suppressor monocytes are the main targets in cancer prophylactic or treatment, not cancer cells. By delivering toxins instead of nutrients alpha-fetoprotein kills suppressor monocytes canceling immune suppression, as well as killing cancer cells directly. It is the perfect synergy of the most powerful cancer immunotherapy with targeted chemotherapy. Alpha-fetoprotein chemical conjugates, as well as the complexes of this oncofetal protein with binding toxins, have shown promising results in cancer treatments. Oral porcine alpha-fetoprotein complexes with toxins can prevent and/or treat cancer, although their immunotherapy mechanism of action is undiscovered.

Potential roles of hyaluronic acid in in vivo CAR T cell reprogramming for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has recently shown unprecedented clinical efficacy for cancer treatment, particularly of hematological malignancies. However, the complex manufacturing processes that involve ex vivo genetic modification of autologous T cells limits its therapeutic application. CAR T cells generated in vivo provide a valid alternative immunotherapy, "off-the-shelf", for cancer treatment. This approach requires carriers for the delivery of CAR-encoding constructs, which are plasmid DNA or messenger RNA, to T cells for CAR expression to help eradicate the tumor. As such, there are a growing number of studies reporting gene delivery systems for in vivo CAR T cell therapy based on viral vectors and polymeric nanoparticles. Hyaluronic acid (HA) is a natural biopolymer that can serve for gene delivery, because of its inherent properties of cell recognition and internalization, as well as its biodegradability, biocompatibility, and presence of functional groups for the chemical conjugation of targeting ligands. In this review, the potential of HA in the delivery of CAR constructs is discussed on the basis of previous experience of HA-based nanoparticles for gene therapy. Furthermore, current studies on CAR carriers for in vivo-generated CAR T cells are included, giving an idea of a rational design of HA-based systems for the more efficient delivery of CAR to circulating T cells.

Self-Reporting Polysaccharide Polymersome for Doxorubicin and Cisplatin Delivery to Live Cancer Cells.

We report self-reporting fluorescent polysaccharide polymersome nanoassemblies for enzyme-responsive intracellular delivery of two clinical anticancer drugs doxorubicin (DOX) and cisplatin to study the real-time drug-releasing aspects by fluorescent resonance energy transfer (FRET) bioimaging in live cancer cells. Fluorescent polymersomes were tailor-made by tagging an aggregation-induced emission (AIE) optical chromophore, tetraphenylethylene (TPE), and a plant-based vesicular directing hydrophobic unit through enzyme-biodegradable aliphatic ester chemical linkages in the polysaccharide dextran. The blue-luminescent polymersome self-assembled in water and exhibited excellent encapsulation capability for the red-luminescent anticancer drug DOX. FRET between the AIE polymersome host and DOX guest molecules resulted in a completely turn-off probe. At the intracellular level, the lysosomal enzymatic disassembly of the polymersome restored the dual fluorescent signals from DOX and TPE at the nucleus and the lysosomes, respectively. Live-cell confocal microscopy coupled with selective photoexcitation was employed to study the real-time polymersome disassembly by monitoring the turn-on fluorescent signals in human breast cancer cell lines. Alternatively, carboxylic acid-functionalized AIE polymersomes were also tailor-made for cisplatin stitching to directly monitor Pt drug delivery. The polymersome nanoassemblies exhibited excellent structural tolerance for the chemical conjugation of the Pt drugs, and the fluorescence signals were unaltered. An in vitro drug release study confirmed that the cisplatin-stitched fluorescent polymersomes were very stable under physiological conditions and underwent lysosomal enzymatic degradation to inhibit the cancer cell growth. A lysosomal colocalization experiment using confocal microscopy substantiates the enzyme-responsive degradation of these polymersomes to release both the encapsulated and conjugated drugs at the intracellular level. The present design provides a unique opportunity to deliver more than one anticancer drug from a single polymersome platform in cancer research.

GMMA as a ‘plug and play’ technology to tackle infectious disease to improve global health: context and perspectives for the future

ABSTRACT Introduction Generalized-Modules-for-Membrane-Antigens (GMMA) is a technology platform developed to design outer membrane vesicle (OMV)-based vaccines. GMMA are basically OMVs derived from a bacterial strain specifically engineered to obtain a fit-for-purpose and affordable vaccine by potentiating, or deleting, expression of specific genes. OMVs can be used as a carrier for antigens by inducing their expression on them, with the aim to improve antigen immunogenicity and design multivalent combination vaccines. Areas Covered We expanded this finding to show that the chemical conjugation of different proteic and/or polysaccharidic antigens, to GMMA, is a methodology complementary to the genetic manipulation to obtain highly effective combination vaccines. Here we discuss our findings with a specific focus on the impact that GMMA technology can have on global health, as this technology platform is particularly suited to support the development of affordable vaccines for low-income countries. Expert Opinion We believe that it is critical to elucidate the mode of action of GMMA immunogenicity and have provided a summarized description of the immunological questions to be addressed in the near future. The improved knowledge of GMMA might lead to designing more effective and safer GMMA-based vaccines to tackle the most serious vaccine-preventable diseases.

Side-by-Side Comparison of Five Chelators for 89Zr-Labeling of Biomolecules: Investigation of Chemical/Radiochemical Properties and Complex Stability.

Simple SummaryThe positron emitter 89Zr4+ is an important radionuclide for the preparation of radiolabeled antibodies, being applied in highly specific and sensitive positron emission tomography (PET) imaging of malignancies. The introduction of 89Zr4+ into biomolecules is performed using chelating agents, wrapping up the radiometal and preventing its release from the antibody by forming so-called complexes. Desferrioxamine B (DFO) is the clinical gold standard chelator for the preparation of 89Zr antibodies despite its known inability to stably encapsulate the radiometal, resulting in 89Zr release and associated challenges such as decreased image quality and radiation dose to healthy tissues. Therefore, several research groups have been working to develop new chelating agents able to stably encapsulate the 89Zr4+ ion. However, there are no data available directly comparing the stability of the formed 89Zr complexes of the most promising chelating agents developed so far. Here, we report on the comparison of five different chelators with high potential for stable complexation of 89Zr and determined two of them—DFO* and 3,4,3-(LI-1,2-HOPO)—to be highly interesting for the preparation of 89Zr-based radiolabeled agents and routine clinical application.In this work, five different chelating agents, namely DFO, CTH-36, DFO*, 3,4,3-(LI-1,2-HOPO) and DOTA-GA, were compared with regard to the relative kinetic inertness of their corresponding 89Zr complexes to evaluate their potential for in vivo application and stable 89Zr complexation. The chelators were identically functionalized with tetrazines, enabling a fully comparable, efficient, chemoselective and biorthogonal conjugation chemistry for the modification of any complementarily derivatized biomolecules of interest. A small model peptide of clinical relevance (TCO-c(RGDfK)) was derivatized via iEDDA click reaction with the developed chelating agents (TCO = trans-cyclooctene and iEDDA = inverse electron demand Diels-Alder). The bioconjugates were labeled with 89Zr4+, and their radiochemical properties (labeling conditions and efficiency), logD(7.4), as well as the relative kinetic inertness of the formed complexes, were compared. Furthermore, density functional theory (DFT) calculations were conducted to identify potential influences of chelator modification on complex formation and geometry. The results of the DFT studies showed—apart from the DOTA-GA derivative—no significant influence of chelator backbone functionalization or the conjugation of the chelator tetrazines by iEDDA. All tetrazines could be efficiently introduced into c(RGDfK), demonstrating the high suitability of the agents for efficient and chemoselective bioconjugation. The DFO-, CTH-36- and DFO*-modified c(RGDfK) peptides showed a high radiolabeling efficiency under mild reaction conditions and complete 89Zr incorporation within 1 h, yielding the 89Zr-labeled analogs as homogenous products. In contrast, 3,4,3-(LI-1,2-HOPO)-c(RGDfK) required considerably prolonged reaction times of 5 h for complete radiometal incorporation and yielded several different 89Zr-labeled species. The labeling of the DOTA-GA-modified peptide was not successful at all. Compared to [89Zr]Zr-DFO-, [89Zr]Zr-CTH-36- and [89Zr]Zr-DFO*-c(RGDfK), the corresponding [89Zr]Zr-3,4,3-(LI-1,2-HOPO) peptide showed a strongly increased lipophilicity. Finally, the relative stability of the 89Zr complexes against the EDTA challenge was investigated. The [89Zr]Zr-DFO complex showed—as expected—a low kinetic inertness. Unexpectedly, also, the [89Zr]Zr-CTH-36 complex demonstrated a high susceptibility against the challenge, limiting the usefulness of CTH-36 for stable 89Zr complexation. Only the [89Zr]Zr-DFO* and the [89Zr]Zr-3,4,3-(LI-1,2-HOPO) complexes demonstrated a high inertness, qualifying them for further comparative in vivo investigation to determine the most appropriate alternative to DFO for clinical application.

In Situ Visualizing Nascent RNA by Exploring DNA-Templated Oxidative Amination of 4-Thiouridine.

Tracking and mapping the nascent RNA molecules in cells is essential for deciphering embryonic development and neuronal differentiation. Here, we utilized 4-thiouridine (s4U) as a metabolic tag to label nascent RNA and developed a fluorescence imaging method based on the DNA-templated oxidative amination (DTOA) reaction of s4U. The DTOA reaction occurred between amine-modified DNA and s4U-containing RNA with high sequence specificity and chemical selectivity. Target nascent mRNAs in HeLa cells, including those encoding green fluorescent proteins (GFPs) and endogenous BAG-1, were thus lit up selectively by DTOA-based fluorescence in situ hybridization (DTOA FISH). We believe the DTOA conjugation chemistry shown in this study could be generally applied to investigate the spatial distribution of nascent transcription dynamics in cellular processes.