Search

Studying the complex and intricate retinoids metabolic pathways by chemical biology approaches requires design and synthesis of biologically functional molecular probes. Only few of such molecular retinoid probes could be found in literature, most of them bearing a molecular structure quite different from natural retinoids. To provide close-to-native retinoid probes, we have developed a versatile late-stage method for the insertion of azide function at the C4 position of several retinoids. This one-step process opens straightforward access to different retinoid and carotenoid probes from commercially available precursors. We have further demonstrated that the different molecular probes retain ability of the original compound to activate genes' transcription, despite azide insertion, highlighting biological activities that were further validated in zebrafish in vivo model. The present work paves the way to future studies on vitamin A's metabolism., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H 2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications., (© 2024 Wiley-VCH GmbH.)

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs. In this study, we report the design, synthesis, and evaluation of novel analytical tools suitable to monitor the activity of GPL biosynthetic enzymes in vitro. Our synthetic targets, namely, azide-modified PA, azide-modified DAG, and azide-modified CDP-DAG, were successfully obtained from solketal as their common starting material. Moreover, using CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT), an enzyme that catalyzed the final reaction step in synthesizing phosphatidylinositol, we demonstrated that azide-modified CDP-DAG worked as a substrate for CDIPT., (© 2023 Wiley-VCH GmbH.)

Nonheme iron enzymes are versatile biocatalysts for a broad range of unique and powerful transformations, such as hydroxylation, chlorination, and epimerization as well as cyclization/ring-opening of organic molecules. Beyond their native biological functions, these enzymes are robust for engineering due to their structural diversity and high evolvability. Based on enzyme promiscuity and directed evolution as well as inspired by synthetic organic chemistry, nonheme iron enzymes can be repurposed to catalyze reactions previously only accessible with synthetic catalysts. To this end, our group has engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for new-to-nature radical transformations. In particular, we have demonstrated that a nonheme iron enzyme, (4-hydroxyphenyl)pyruvate dioxygenase from streptomyces avermitilis (SavHppD), can be repurposed to enable abiological radical-relay process to access C(sp 3 )-H azidation products. This represents the first known instance of enzymatic radical relay azidation reactions. In this chapter, we describe the detailed experimental protocol to convert promiscuous nonheme iron enzymes into efficient and selective biocatalyst for radical relay azidation reactions. One round of directed evolution is described in detail, which includes the generation and handling of site-saturation mutagenesis, protein expression and whole-cell reactions screening in a 96-well plate. These protocol details might be useful to engineer various nonheme iron enzymes for other applications., (Copyright © 2024. Published by Elsevier Inc.)

Bromodomain-PHD finger protein 1 (BRPF1) belongs to the BRPF family of bromodomain-containing proteins. Bromodomains are exclusive reader modules that recognize and bind acetylated histones and non-histone transcription factors to regulate gene expression. The biological functions of acetylated histone recognition by BRPF1 bromodomain are well characterized; however, the function of BRPF1 regulation via non-histone acetylation is still unexplored. Therefore, identifying the non-histone interactome of BRPF1 is pivotal in deciphering its role in diverse cellular processes, including its misregulation in diseases like cancer. Herein, we identified the non-histone interacting partners of BRPF1 utilizing a protein engineering-based approach. We site-specifically introduced the unnatural photo-cross-linkable amino acid 4-azido-L-phenylalanine into the bromodomain of BRPF1 without altering its ability to recognize acetylated histone proteins. Upon photoirradiation, the engineered BRPF1 generates a reactive nitrene species, cross-linking interacting partners with spatio-temporal precision. We demonstrated the robust cross-linking efficiency of the engineered variant with reported histone ligands of BRPF1 and further used the variant reader to cross-link its interactome. We also characterized novel interacting partners by proteomics, suggesting roles for BRPF1 in diverse cellular processes. BRPF1 interaction with interleukin enhancer-binding factor 3, one of these novel interacting partners, was further validated by isothermal titration calorimetry and co-IP. Lastly, we used publicly available ChIP-seq and RNA-seq datasets to understand the colocalization of BRPF1 and interleukin enhancer-binding factor 3 in regulating gene expression in the context of hepatocellular carcinoma. Together, these results will be crucial for full understanding of the roles of BRPF1 in transcriptional regulation and in the design of small-molecule inhibitors for cancer treatment., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Methemoglobin (metHb), the oxidized form of hemoglobin, lacks the ability of reversible oxygen binding; however, it has a high binding affinity to toxic substances such as cyanide, hydrosulfide, and azide. This innate property of metHb offers the clinical option to treat patients poisoned with these toxins, by oxidizing the endogenous hemoglobin in the red blood cells (RBCs). The binding properties of naked metHb (isolated from RBC) with these toxins has been studied; however, the binding behaviors of metHb under the intracellular conditions of RBC are unclear because of the difficulty in detecting metHb status changes in RBC. This study aimed to elucidate the binding properties of metHb in RBC under physiological and poisoned conditions using artificial RBC, which was hemoglobin encapsulated in a liposome. The mimic-circumstances of metHb in RBC (metHb-V) was prepared by oxidizing the hemoglobin in artificial RBC. Spectroscopic analysis indicated that the metHb in metHb-V exhibited a binding behavior different from that of naked metHb, depending on the toxic substance: When the pH decreased, (i) the cyanide binding affinity of metHb-V remained unchanged, but that of naked metHb decreased (ii) the hydrosulfide binding affinity was increased in metHb-V but was decreased in naked metHb. (iii) Azide binding was increased in metHb-V, which was similar to that in naked metHb, irrespective of the pH change. Thus, the binding behavior of intracellular metHb in the RBC with cyanide, hydrosulfide, and azide under physiological and pathological conditions were partly elucidated using the oxidized artificial RBC., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuto Suzuki reports financial support were provided by JST SPRING and Keio University Doctorate Student Grant-in-Aid Program from the Ushioda Memorial Fund. Kazuaki Taguchi reports relationships with Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, and Takahashi Industrial and Economic Research Foundation that includes: funding grants. Yuto Suzuki, Kazuaki Taguchi, Hiromi Sakai, and Kazuaki Matsumoto has patent #PCT/JP2021/ 31,458., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Glycan recognition by glycan-binding proteins is central to the biology of all living organisms. The efficient capture and characterization of relatively weak non-covalent interactions remains an important challenge in various fields of research. Photoaffinity labeling strategies can create covalent bonds between interacting partners, and photoactive scaffolds such as benzophenone, diazirines and aryl azides have proved widely useful. Since their first introduction, relatively few improvements have been advanced and products of photoaffinity labeling remain difficult to detect. We report a fluorinated azido-coumarin scaffold which enables photolabeling under fast and mild activation, and which can leave a fluorescent tag on crosslinked species. Coupling this scaffold to an α-fucoside, we demonstrate fluorogenic photolabeling of glycan-protein interactions over a wide range of affinities. We expect this strategy to be broadly applicable to other chromophores and we envision that such "fluoro-crosslinkers" could become important tools for the traceable capture of non-covalent binding events., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The human gut microbiome modulates physiological functions and pathologies; however, a mechanistic understanding of microbe-host and microbe-microbe interactions remains elusive owing to a lack of suitable approaches to monitor obligate anaerobic bacterial populations. Common genetically encoded fluorescent protein reporters, derived from the green fluorescent protein, require an oxidation step for fluorescent light emission and therefore are not suitable for use in anaerobic microbes residing in the intestine. Fluorescence in situ hybridization is a useful alternative to visualize bacterial communities in their natural niche; however, it requires tissue fixation. We therefore developed an approach for the real-time detection and monitoring of live communities of anaerobic gut commensals in their natural environment. We leverage the bacterial cells' reliance on sugars for macromolecule synthesis in combinatorial click chemistry labeling, where the addition of azide-modified sugars to the culturing media enables the fluorescence labeling of newly synthesized molecules via the addition of combinations of exogenous fluorophores conjugated to cyclooctynes. This process is suitable for labeling communities of live anaerobic gut bacteria with combinations of fluorophores that do not require oxygen to mature and fluoresce, and that can be detected over time in their natural environments. The labeling procedure requires 4-9 d, depending on the varying growth rates of different bacterial strains, and an additional 1-2 d for the detection and monitoring steps. The protocol can be completed by users with basic expertise in bacterial culturing., (© 2023. Springer Nature Limited.)

This work features the use of amber suppression-mediated unnatural amino acid (UAA) incorporation into proteins for various imaging purposes. The site-specific incorporation of the UAA, p-azido-L-phenylalanine (pAzF), provides an azide handle that can be used to complete the strain promoted azide-alkyne click cycloaddition (SPAAC) reaction to introduce an imaging modality such as a fluorophore or a positron emission tomography (PET) tracer on the protein of interest (POI). Such methodology can be pursued directly in mammalian cell lines or on proteins expressed in vitro, thereby conferring a homogeneous pool of protein conjugates. A general procedure for UAA incorporation to use with a site-specific protein labeling method is provided allowing for in vitro and in vivo imaging applications based on the representative proteins PTEN and PD-L1. This approach would help elucidate the cellular or in vivo biological activities of the POI., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Primary hemostasis (platelet plug formation) and secondary hemostasis (fibrin clot formation) are intertwined processes that occur upon vascular injury. Researchers have sought to target wounds by leveraging cues specific to these processes, such as using peptides that bind activated platelets or fibrin. While these materials have shown success in various injury models, they are commonly designed for the purpose of treating solely primary or secondary hemostasis. In this work, a two-component system consisting of a targeting component (azide/GRGDS PEG-PLGA nanoparticles) and a crosslinking component (multifunctional DBCO) is developed to treat internal bleeding. The system leverages increased injury accumulation to achieve crosslinking above a critical concentration, addressing both primary and secondary hemostasis by amplifying platelet recruitment and mitigating plasminolysis for greater clot stability. Nanoparticle aggregation is measured to validate concentration-dependent crosslinking, while a 1:3 azide/GRGDS ratio is found to increase platelet recruitment, decrease clot degradation in hemodiluted environments, and decrease complement activation. Finally, this approach significantly increases survival relative to the particle-only control in a liver resection model. In light of prior successes with the particle-only system, these results emphasize the potential of this technology in aiding hemostasis and the importance of a holistic approach in engineering new treatments for hemorrhage., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.)

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hydrazoic acid (HN 3 ) and its deprotonated form azide ion (N 3 - ) (AHA) are toxic because they inhibit the cytochrome c oxidase complex IV (CoX IV) embedded in the inner mitochondrial membrane that forms part of the enzyme complexes involved in cellular respiration. Critical to its toxicity is the inhibition of CoX IV in the central nervous system and cardiovascular system. Hydrazoic acid is an ionizable species and its affinity for membranes, and the associated permeabilities, depend on the pH values of aqueous media on both sides of the membranes. In this article, we address the permeability of AHA through the biological membrane. In order to understand the affinity of the membrane for the neutral and ionized form of azide, we measured the octanol/water partition coefficients at pH values of 2.0 and 8.0, which are 2.01 and 0.00034, respectively. Using a Parallel Artificial Membrane Permeability Assay (PAMPA) experiment, we measured the effective permeability through the membrane, which is logP e  - 4.97 and - 5.26 for pH values of 7.4 and pH 8.0, respectively. Experimental permeability was used to validate theoretical permeability, which was estimated by numerically solving a Smoluchowski equation for AHA diffusion through the membrane. We demonstrated that the rate of permeation through the cell membrane of 8.46·10 4  s -1 is much higher than the rate of the chemical step of CoX IV inhibition by azide of 200 s -1 . The results of this study show that transport through the membrane does not represent the rate-limiting step and therefore does not control the rate of CoX IV inhibition in the mitochondria. However, the observed dynamics of azide poisoning is controlled by circulatory transport that takes place on a time scale of minutes., (© 2023. The Author(s).)

Context: The azide anion (N 3 -) is highly toxic. It exists most commonly as sodium azide, which is used widely and is readily available, raising the potential for occupational incidents and use as a weapon of mass destruction. Azide-poisoned patients present with vomiting, seizures, hypotension, metabolic acidosis, and coma; death can occur. No specific azide antidote exists, with treatment being solely supportive. Azide inhibits mitochondrial cytochrome c oxidase and is likely oxidized to nitric oxide in vivo . Cytochrome c oxidase inhibition depletes intracellular adenosine triphosphate and increases oxidative stress, while increased nitric oxide causes hypotension and exacerbates oxidative damage. Here, we tested whether the cobalamin (vitamin B 12 ) analog cobinamide, a strong and versatile antioxidant that also neutralizes nitric oxide, can reverse azide toxicity in mammalian cells, Drosophila melanogaster , and mice., Results: We found cobinamide bound azide with a moderate affinity (K a 2.87 × 10 5 M -1 ). Yet, cobinamide improved growth, increased intracellular adenosine triphosphate, and reduced apoptosis and malondialdehyde, a marker of oxidative stress, in azide-exposed cells. Cobinamide rescued Drosophila melanogaster and mice from lethal exposure to azide and was more effective than hydroxocobalamin. Azide likely generated nitric oxide in the mice, as evidenced by increased serum nitrite and nitrate, and reduced blood pressure and peripheral body temperature in the animals; the reduced temperature was likely due to reflex vasoconstriction in response to the hypotension. Cobinamide improved recovery of both blood pressure and body temperature., Conclusion: We conclude cobinamide likely acted by neutralizing both oxidative stress and nitric oxide, and that it should be given further consideration as an azide antidote.

An activity-based labeling (ABL) approach was investigated for the phenol-oxidizing bacterium, Pseudomonas sp. CF600. Phenol-grown cells were exposed to several different terminal diynes, and following cell breakage, extracts of these cells were added to copper-catalyzed alkyne/azide cycloaddition reactions containing Alexa Fluor 647 azide. Analysis of total cell proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and near-infrared scanning demonstrated covalent fluorescent labeling of a 58- and a 34-kDa polypeptide in all diyne-treated cell types. Further studies using 1,4-diethynylbenzene (DEB) demonstrated that these labeled polypeptides were consistently detected in cells grown on substrates that exhibited phenol-dependent O2 uptake activity but not observed when cells were grown on substrates such as dextrose or catechol that did not support this activity. Fluorescent labeling of the two polypeptides in DEB-treated, phenol-grown cells was time dependent and was inhibited by several known substrates for phenol hydroxylase. These results suggest that diverse diynes act as mechanism-based inactivators of phenol hydroxylase in Pseudomonas sp. CF600 and that this effect can be exploited by ABL approaches to selectively label the major 58- and 34-kDa subunits of the hydroxylase component of this complex enzyme., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Azide, the most used photo-crosslinking group, facilitates the analysis of protein structure and function. This group is particularly useful when photochemically label antibodies and examine protein-protein interactions. The use of the expanded genetic code technique allows the special labeling of the functional azide group in proteins by adding the unnatural amino acid (UAA), p-azido-l-phenylalanine (AzF), in response to the amber codon during translation. However, a low UAA uptake rate due to mass transfer resistance in the cell membrane may lead to the early termination of the full-length protein. This study reports a general method for the efficient in vivo incorporation of AzF into the target protein by improving cell permeability using organic solvents. As expected, the yield of the full-length protein was significantly increased, which indicated that the AzF uptake was greatly improved due to the addition of organic solvents. Our method can serve as a good reference for improving the genetic incorporation of other kinds of UAAs into proteins., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hydroxyhydrocarbon (Hhc) moieties in the backbone of peptidic natural products can exert a substantial influence on the bioactivities of the products, making Hhc units an attractive class of building blocks for de novo peptides. However, despite advances in in vitro genetic code reprogramming, the ribosomal incorporation of Hhc units remains challenging. Here we report a method for in vitro ribosomal synthesis of natural-product-like peptides bearing Hhc units. A series of azide/hydroxy acids were designed as chemical precursors of Hhc units and incorporated into the nascent peptide chain by means of genetic code reprogramming. Post-translational reduction of the azide induced an O-to-N acyl shift to rearrange the peptide backbone. This method allows for one-pot ribosomal synthesis of designer macrocycles bearing various β-, γ- and δ-type Hhc units. We also report the synthesis of a statine-containing peptidomimetic inhibitor of β-secretase 1 as a showcase example., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O 2 ). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC 50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

Sodium azide is a strong mutagen that has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolically converted to the proximate mutagen azidoalanine, which requires further bioactivation to a putative ultimate mutagen that remains elusive. The nature of the DNA modifications induced by azides leading to mutations is also unknown. Other mutagenic organic azido compounds seem to share the same bioactivation pathway to the ultimate mutagenic species as they induce point mutations dependent on the same DNA repair pathways. We investigated mutations induced by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) in the human TK6 cell line. Until now, azides have been considered to be non-mutagens and non-carcinogens in mammals, including humans, as judged only by the conventional clastogenicity chromosomal aberration types of bioassays. Here, we show the potent mutagenicity of AZG in cultured human cells, comparable to alkylating agents such as methyl methanesulfonate at concentrations with similar lethality. The potent ability of an organic azide to induce base substitutions in a mammalian system raises an alert with respect to human exposure to organic and inorganic azido compounds., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems., Competing Interests: Declaration of interests S.K. is an inventor on patents related to mTAG labeling and TOP-seq mapping., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The disaccharide trehalose is essential for viability of Mycobacterium tuberculosis, which synthesizes trehalose de novo but can also utilize exogenous trehalose. The mycobacterial cell wall encompasses two permeability barriers, the cytoplasmic membrane and the outer mycolic acid-containing mycomembrane. The ABC transporter LpqY-SugA-SugB-SugC has previously been demonstrated to mediate the specific uptake of trehalose across the cytoplasmic membrane. However, it is still unclear how the transport of trehalose molecules across the mycomembrane is mediated. In this study, we harnessed the antimycobacterial activity of the analogue 6-azido trehalose to select for spontaneous resistant M. tuberculosis mutants in a merodiploid strain harbouring two LpqY-SugA-SugB-SugC copies. Mutations mediating resistance to 6-azido trehalose mapped to the proline-proline-glutamate (PPE) family member PPE51 (Rv3136), which has recently been shown to be an integral mycomembrane protein involved in uptake of low-molecular weight compounds. A site-specific ppe51 gene deletion mutant of M. tuberculosis was unable to grow on trehalose as the sole carbon source. Furthermore, bioorthogonal labelling of the M. tuberculosis Δppe51 mutant incubated with 6-azido trehalose corroborated the impaired internalization. Taken together, the results indicate that the transport of trehalose and trehalose analogues across the mycomembrane of M. tuberculosis is exclusively mediated by PPE51., (© 2022. The Author(s).)

Bacteria in human milk contribute to the establishment of the infant gut microbiome. As such, numerous studies have characterized the human milk microbiome using DNA sequencing technologies, particularly 16S rRNA gene sequencing. However, such methods are not able to differentiate between DNA from viable and non-viable bacteria. The extent to which bacterial DNA detected in human milk represents living, biologically active cells is therefore unclear. Here, we characterized both the viable bacterial content and the total bacterial DNA content (derived from viable and non-viable cells) of fresh human milk ( n = 10). In order to differentiate the living from the dead, a combination of propidium monoazide (PMA) and full-length 16S rRNA gene sequencing was used. Our results demonstrate that the majority of OTUs recovered from fresh human milk samples (67.3%) reflected DNA from non-viable organisms. PMA-treated samples differed significantly in their bacterial composition compared to untreated samples (PERMANOVA p < 0.0001). Additionally, an OTU mapping to Cutibacterium acnes had a significantly higher relative abundance in PMA-treated (viable) samples. These results demonstrate that the total bacterial DNA content of human milk is not representative of the viable human milk microbiome. Our findings raise questions about the validity of conclusions drawn from previous studies in which viability testing was not used, and have broad implications for the design of future work in this field.

To quantify viable probiotic Lacticaseibacillus paracasei (L. paracasei) in fermented milk accurately and quickly, propidium monoazide combined with quantitative loop-mediated isothermal amplification (PMA-qLAMP) was applied. The optimal PMA treatment conditions for treating a L. paracasei suspension were determined using an orthogonal test to eliminate the DNA amplification of 108 CFU/mL of dead L. paracasei. Primers were designed based on the species-specific gyrB gene of L. paracasei. A phylogenetic tree based on the gyrB gene showed that L. paracasei clustered on the same branch with 91% support. Compared with the 16 strains commonly found in fermented milk, three strains of L. paracasei showed positive PMA-qLAMP results, and the melting temperature was approximately 82.4°C. There was a linear relationship (R2 = 0.9983) between the Ct values and the logarithm of the concentration of viable bacteria. The PMA-qLAMP detection limit for the L. paracasei artificially added to fermented milk was 7.3 × 102 CFU/mL. There was no significant difference between the logarithm values of the concentration of viable L. paracasei of 50 fermented milk samples within shelf life using the PMA-qLAMP and plate count methods (P > 0.01). PMA-qLAMP is specific and accurate for obtaining reliable results faster than when using plate counts., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The mammalian sirtuins are a group of posttranslational modification enzymes that remove acyl modifications from lysine residues in an NAD + -dependent manner. Although initially proposed as histone deacetylases (HDACs), they are now known to target other cellular enzymes and proteins as well. Sirtuin-catalyzed simple amide hydrolysis has profound biological consequences including suppression of gene expression, promotion of DNA damage repair, and regulation of glucose and lipid metabolism. Human sirtuins have been intensively pursued by both academia and industry as potential therapeutic targets for the treatment of diseases such as cancer and neurodegeneration. To gain a better understanding of their roles in various cellular events, innovative chemical probes are highly sought after. This current study focuses on the development of activity-based chemical probes (ABPs) for the profiling of sirtuin activity in biological samples. Cyclooctyne-containing and azido-containing probes were synthesized to enable the subsequent copper-free "click" conjugation to either a fluorophore or biotin. The two groups of structurally related ABPs demonstrated different labeling efficiency and selectivity: the cyclooctyne-containing probes failed to label recombinant sirtuins to any appreciable level, while the azido-containing ABPs showed good isoform selectivity. The azido-containing ABPs were further analyzed for their ability to label an individual sirtuin isoform in protein mixtures and cell lysates. These biocompatible ABPs allow the study of dynamic cellular protein activity change to become possible., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A facile and convergent procedure for the synthesis of azobenzene-based probe was reported, which could selectively release interested proteins conducted with sodium dithionite. Besides, the cleavage efficiency is closely associated with the structural features, in which an ortho-hydroxyl substituent is necessary for reactivity. In addition, the azobenzene tag applied in the Ac 4 GlcNAz-labled proteins demonstrated high efficiency and selectivity in comparison with Biotin-PEG 4 -Alkyne, which provides a useful platform for enrichment of any desired bioorthogonal proteomics., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We present a chemical approach to profile fatty acid uptake in single cells. We use azide-modified analogues to probe the fatty acid influx and surface-immobilized dendrimers with dibenzocyclooctyne (DBCO) groups for detection. A competition between the fatty acid probes and BHQ2-azide quencher molecules generates fluorescence signals in a concentration-dependent manner. By integrating this method onto a microfluidics-based multiplex protein analysis platform, we resolved the relationships between fatty acid influx, oncogenic signaling activities, and cell proliferation in single glioblastoma cells. We found that p70S6K and 4EBP1 differentially correlated with fatty acid uptake. We validated that cotargeting p70S6K and fatty acid metabolism synergistically inhibited cell proliferation. Our work provided the first example of studying fatty acid metabolism in the context of protein signaling at single-cell resolution and generated new insights into cancer biology.

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3'-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand-protein contacts and its signaling complexes.

While two-dimensional infrared (2D-IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active-site dynamics is limited due to the lack of site-specific 2D-IR probes. We demonstrate the genetic incorporation of a novel 2D-IR probe, m-azido-L-tyrosine (N3Y) in the active-site of DddK, an iron-dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active-site iron to Fe III , and the addition of denaturation reagents, result in significant decrease in enzyme activity and active-site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D-IR probe N3Y should be broadly applicable to investigate how the enzyme active-site motions at the fs-ps time scale direct reaction pathways to facilitating specific chemical reactions., (© 2021 Wiley-VCH GmbH.)

Detection of azide-tagged biomolecules ( e.g. , azido sugars) inside living cells using "click" chemistry has been revolutionary to the field of chemical biology. However, we currently still lack suitable synthetic biology tools to autonomously and rapidly detect azide ions. Here, we have developed an engineered synthetic promoter system called cyn regulon, and complementary Escherichia coli engineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter genes. The engineered cyn azide operon allowed highly tunable reporter green fluorescent protein (GFP) expression over three orders of inducer azide ion concentrations (0.01-5 mM) and rapidly induced GFP expression by over 600-fold compared to the uninduced control. Next, we showcase the superior performance of this engineered cyn -operon over the classical lac -operon for recombinant protein production. Finally, we highlight how this synthetic biology toolkit can enable glycoengineering-based applications by facilitating in vivo activity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor substrates.

Here were report the combination of biocompatible click chemistry of ω-azidosphinganine with fluorescence microscopy and mass spectrometry as a powerful tool to elaborate the sphingolipid metabolism. The azide probe was efficiently synthesized over 13 steps starting from l-serine in an overall yield of 20% and was used for live-cell fluorescence imaging of the endoplasmic reticulum in living cells by bioorthogonal click reaction with a DBCO-labeled fluorophore revealing that the incorporated analogue is mainly localized in the endoplasmic membrane like the endogenous species. A LC-MS(/MS)-based microsomal in vitro assay confirmed that ω-azidosphinganine mimics the natural species enabling the identification and analysis of metabolic breakdown products of sphinganine as a key starting intermediate in the complex sphingolipid biosynthetic pathways. Furthermore, the sphinganine-fluorophore conjugate after click reaction was enzymatically tolerated to form its dihydroceramide and ceramide metabolites. Thus, ω-azidosphinganine represents a useful biofunctional tool for metabolic investigations both by in vivo fluorescence imaging of the sphingolipid subcellular localization in the ER and by in vitro high-resolution mass spectrometry analysis. This should reveal novel insights of the molecular mechanisms sphingolipids and their processing enzymes have e.g. in infection.

Conjugation of nanoparticles (NP) to the surface of living cells is of interest in the context of exploiting the tissue homing properties of ex vivo engineered T cells for tumor-targeted delivery of drugs loaded into NP. Cell surface conjugation requires either a covalent or non-covalent reaction. Non-covalent conjugation with ligand-decorated NP (LNP) is challenging and involves a dynamic equilibrium between the bound and unbound state. Covalent NP conjugation results in a permanently bound state of NP, but the current routes for cell surface conjugation face slow reaction kinetics and random conjugation to proteins in the glycocalyx. To address the unmet need for alternative bioorthogonal strategies that allow for efficient covalent cell surface conjugation, we developed a 2-step click conjugation sequence in which cells are first metabolically labeled with azides followed by reaction with sulfo-6-methyl-tetrazine-dibenzyl cyclooctyne (Tz-DBCO) by SPAAC, and subsequent IEDDA with trans-cyclooctene (TCO) functionalized NP. In contrast to using only metabolic azide labeling and subsequent conjugation of DBCO-NP, our 2-step method yields a highly specific cell surface conjugation of LNP, with very low non-specific background binding., (© 2020 Wiley-VCH GmbH.)

Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD + ) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD + enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD + provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.

Halohydrin dehalogenases (HHDHs) are valuable biocatalysts for the synthesis of enantiopure benzyl glycidyl ether (BGE) and its derivatives, which are important synthetic intermediates for anti-cancer and anti-obesity drugs. However, all the reported HHDHs exhibit low enantioselectivity. In this study, we screened site-saturation mutagenesis libraries of AbHHDH at positions R89, A136, V137, P178, N179, F180, I181, Y186 and F187 for mutants with enhanced enantioselectivity toward BGE. The four improved variant R89V, R89Y, R89K and V137I were identified, and the double mutant R89Y/V137I showed 2.9-fold higher enantioselectivity than the wild type. The regions of HHDH containing the identified mutations were analyzed by homology modeling to explain the changes of enantioselectivity. Kinetic resolution of 20 to 100 mM BGE using whole cells of Escherichia coli expressing the mutant R89Y/V137I resulted in (R)-BGE yields of 42 to 32.5%, with ee >99%. This study improves our understanding of the enantioselectivity of HHDHs and contributes improved biocatalysts for the kinetic resolution of BGE., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest related to this work., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We disclose our studies on a copper-mediated reaction of alkynes with trimethylsilyl azide to afford nitriles, and proposed a reaction mechanism, which involves an iodoalkyne and an iodotriazole as intermediates.

The virus SARS CoV-2, which causes the respiratory infection COVID-19, continues its spread across the world and to date has caused more than a million deaths. Although COVID-19 vaccine development appears to be progressing rapidly, scientists continue the search for different therapeutic options to treat this new illness. In this work, we synthesized five new 1-aryl-5-(3-azidopropyl)indol-4-ones and showed them to be potential inhibitors of the SARS CoV-2 main protease (3CLpro). The compounds were obtained in good overall yields and molecular docking indicated favorable binding with 3CLpro. In silico ADME/Tox profile of the new compounds were calculated using the SwissADME and pkCSM-pharmacokinetics web tools, and indicated adequate values of absorption, distribution and excretion, features related to bioavailability. Moreover, low values of toxicity were indicated for these compounds. And drug-likeness levels of the compounds were also predicted according to the Lipinski and Veber rules., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Metabolic glycoengineering with unnatural sugars provides a powerful tool to label cell membranes with chemical tags for subsequent targeted conjugation of molecular cargos via efficient chemistries. This technology has been widely explored for cancer labelling and targeting. However, as this metabolic labelling process can occur in both cancerous and normal cells, cancer-selective labelling needs to be achieved to develop cancer-targeted therapies. Unnatural sugars can be either rationally designed to enable preferential labelling of cancer cells, or specifically delivered to cancerous tissues. In this Review Article, we will discuss the progress to date in design and delivery of unnatural sugars for metabolic labelling of tumour cells and subsequent development of tumour-targeted therapy. Metabolic cell labelling for cancer immunotherapy will also be discussed. Finally, we will provide a perspective on future directions of metabolic labelling of cancer and immune cells for the development of potent, clinically translatable cancer therapies.

RNA-RNA interactions are essential for biology, but they can be difficult to study due to their transient nature. While crosslinking strategies can in principle be used to trap such interactions, virtually all existing strategies for crosslinking are poorly reversible, chemically modifying the RNA and hindering molecular analysis. We describe a soluble crosslinker design (BINARI) that reacts with RNA through acylation. We show that it efficiently crosslinks noncovalent RNA complexes with mimimal sequence bias and establish that the crosslink can be reversed by phosphine reduction of azide trigger groups, thereby liberating the individual RNA components for further analysis. The utility of the new approach is demonstrated by reversible protection against nuclease degradation and trapping transient RNA complexes of E. coli DsrA-rpoS derived bulge-loop interactions, which underlines the potential of BINARI crosslinkers to probe RNA regulatory networks., (© 2020 Wiley-VCH GmbH.)

Postmortem studies suggest that schizophrenia is associated with abnormal expression of specific GABA A receptor (GABA A R) α subunits, including α5GABA A R. Positron emission tomography (PET) measures of GABA A R availability in schizophrenia, however, have not revealed consistent alterations in vivo. Animal studies using the GABA A R agonist [ 3 H]-muscimol provide evidence that antipsychotic drugs influence GABA A R availability, in a region-specific manner, suggesting a potential confounding effect of these drugs. No such data, however, are available for more recently developed subunit-selective GABA A R radioligands. To address this, we combined a rat model of clinically relevant antipsychotic drug exposure with quantitative receptor autoradiography. Haloperidol (0.5 and 2 mg/kg/day) or drug vehicle were administered continuously to adult male Sprague-Dawley rats via osmotic mini-pumps for 28 days. Quantitative receptor autoradiography was then performed postmortem using the GABA A R subunit-selective radioligand [ 3 H]-Ro15-4513 and the non-subunit selective radioligand [ 3 H]-flumazenil. Chronic haloperidol exposure increased [ 3 H]-Ro15-4513 binding in the CA1 sub-field of the rat dorsal hippocampus (p<0.01; q<0.01; d=+1.3), which was not dose-dependent. [ 3 H]-flumazenil binding also increased in most rat brain regions (p<0.05; main effect of treatment), irrespective of the haloperidol dose. These data confirm previous findings that chronic haloperidol exposure influences the specific binding of non-subtype selective GABA A R radioligands and is the first to demonstrate a potential effect of haloperidol on the binding of a α1/5GABA A R-selective radioligand. Although caution should be exerted when extrapolating results from animals to patients, our data support a view that exposure to antipsychotics may be a confounding factor in PET studies of GABA A R in the context of schizophrenia., Competing Interests: Conflict of interest ACV discloses receipt of research funding from F. Hoffman La Roche Ltd and UBC Biopharma SPRL as part of a research program on early life immune activation. The views expressed are those of the author and not necessarily those of F. Hoffman La Roche or UCB Biopharma SPRL. All other authors declare that they have no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; thus, TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer. However, mechanistic insights into TrKA signaling are lacking due to lack of site-dependent phosphorylation control. Here we engineer two light-sensitive tyrosine analogues, namely p-azido-L-phenylalanine (AzF) and the caged-tyrosine (ONB), through amber codon suppression to optically manipulate the phosphorylation state of individual intracellular tyrosines in TrkA. We identify TrkA-AzF and ONB mutants, which can activate the ERK pathway in the absence of NGF ligand binding through light control. Our results not only reveal how TrkA site-dependent phosphorylation controls the defined signaling process, but also extend the genetic code expansion technology to enable regulation of receptor-type kinase activation by optical control at the precision of a single phosphorylation site. It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening in a site-directed phosphorylation pathway for targeted therapy.

Targeted immunomodulation of dendritic cells (DCs) in vivo will enable manipulation of T-cell priming and amplification of anticancer immune responses, but a general strategy has been lacking. Here we show that DCs concentrated by a biomaterial can be metabolically labelled with azido groups in situ, which allows for their subsequent tracking and targeted modulation over time. Azido-labelled DCs were detected in lymph nodes for weeks, and could covalently capture dibenzocyclooctyne (DBCO)-bearing antigens and adjuvants via efficient Click chemistry for improved antigen-specific CD8 + T-cell responses and antitumour efficacy. We also show that azido labelling of DCs allowed for in vitro and in vivo conjugation of DBCO-modified cytokines, including DBCO-IL-15/IL-15Rα, to improve priming of antigen-specific CD8 + T cells. This DC labelling and targeted modulation technology provides an unprecedented strategy for manipulating DCs and regulating DC-T-cell interactions in vivo.

Methyllysine sites in proteins are recognized by an array of reader domains that mediate protein-protein interactions for controlling cellular processes. Herein, we engineer a chromodomain, an essential methyllysine reader, to carry 4-azido-l-phenylalanine (AzF) via amber suppressor mutagenesis and demonstrate its potential to bind and crosslink methylated proteins in human cells. We further develop a first-of-its kind chromodomain variant bearing two AzF units with enhanced crosslinking potential suitable for profiling the transient methyllysine interactome.

RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling. This technique, termed P hotoaffinity E v a luation of R NA L igation- Seq uencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enable the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.

Herein we describe a method for inducing cancer cell death, which relies on the use of a H2O2-responsive glycan metabolic precursor in conjunction with antibody-dependent cellular cytotoxicity (ADCC) or photodynamic therapy (PDT).

Azospirillum brasilense is a plant growth promoting bacteria used as an inoculant in diverse crops. Accurate analytical methods are required to enumerate viable cells in inoculant formulations or in planta. We developed a quantitative polymerase chain reaction (qPCR) assay associated to propidium monoazide (PMA) to evaluate the cell viability of A. brasilense in inoculant and in maize roots. A. brasilense was grown in culture medium and was exposed to 50 ℃. Maize roots were grown in vitro and harvested 7 days after inoculation. Quantification was performed by qPCR, PMA-qPCR, and plate counting. Standard curves efficiency values ranged from 85 to 99%. The limit of detection was 10 4  CFU per gram of fresh root. Enumeration obtained in maize roots by qPCR where higher than enumeration by PMA-qPCR and by plate counting. PMA-qPCR assay was efficient in quantifying inoculant viable cells and provides reliable results in a quickly and accurately way compared to culture-dependent methods.

Understanding how a cell membrane protein functions on living cells remains a challenge for cell biology. Specific placement of functional molecules on specific proteins in their native environment would allow comprehensive study of proteins' dynamic functions. Existing methods cannot facilely achieve multiple modifications on specific membrane proteins. In this report, we describe an aptamer-induced, protein-specific bio-orthogonal modification technology for precise nongenetic immobilization of multiple small functional molecules on target membrane glycoproteins by combining metabolic technology and aptamer targeting. In brief, DNA probes were designed by modifying aptamers, which bind to target proteins on the surfaces of living cells pretreated with N -azidoacetylmannosamine-tetraacylated (Ac 4 ManNAz). The cyclooctynes tagged of DNA probes will approach the azide groups to trigger the bio-orthogonal reactions. After UV irradiation and hybridization with cDNA (complementary DNA), the aptamers can be removed, and the process can be repeated to achieve multiple modifications for multicolor imaging and cell surface nanoengineering on specific proteins.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an in vivo neuronal tracing set-up. Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin . functionalized with an azide-containing Cy5 dye ( N 3 -Cy5-WGA ). A Cy7 quencher dye ( Cy7-DBCO ) was subsequently used to yield Cy7-Cy5-WGA , a compound wherein the Cy5 emission is quenched by Förster resonance energy transfer to Cy7. The photophysical properties of N 3 -Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture) , ex vivo (muscle tissue from mice; used for dose optimization), and in vivo ( nervus ischiadicus in THY-1 YFP mice) . Results: In situ , conjugation of Cy7-DBCO to N 3 -Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N 3 -Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N 3 -Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo, Cy7-DBCO effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site. Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Endo/lysosomal escape and the subsequent controllable/precise release of drugs and genes are key challenges for efficient synergistic cancer therapy. Herein, we report a photoactivated polyprodrug nanoparticle system (PPNPsiRNA) centered on effective light-controlled codelivery of Pt(iv) prodrug and siRNA for synergistic cancer therapy. Under green-light irradiation, PPNPsiRNA can sustainedly generate oxygen-independent azidyl radicals to facilitate endo/lysosomal escape through the photochemical internalization (PCI) mechanism. Besides, concurrent Pt(ii) release and siRNA unpacking could occur in a controllable manner after the decomposition of Pt(iv), main chain shattering of photoactivated polyprodrug and the PPNPsiRNA disassociation. Based on these innovative features, excellent synergistic therapeutic efficacy of chemo- and RNAi therapies of PPNPsiBcl-2 could be achieved on ovarian cancer cells under light irradiation. The facile synthesized and prepared photoactivatable polyprodrug nanoparticle system provides a new strategy for effective gene/drug codelivery, where controllable endo/lysosomal escape and the subsequent drug/gene release/unpacking play vital roles, which could be adopted as a versatile codelivery nanoplatform for the treatment of various cancers.

Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose ( 5-AR ), as well as the synthesis of an inactive control derivative, 1-azidoribose ( 1-AR ), and their application toward understanding protein ribose-glycation in vitro and in cellulo . With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe's utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.

Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients' cells to develop their own cure. One key advancement to develop so-called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne- and/or azide-modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)