Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.

N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand.

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.

The Bacillus subtilis ywbD gene encodes RlmQ, the 23S rRNA methyltransferase forming m7G2574 in the A-site of the peptidyl transferase center.

Ribosomal RNA contains many posttranscriptionally modified nucleosides, particularly in the functional parts of the ribosome. The distribution of these modifications varies from one organism to another. In Bacillus subtilis, the model organism for Gram-positive bacteria, mass spectrometry experiments revealed the presence of 7-methylguanosine (m7G) at position 2574 of the 23S rRNA, which lies in the A-site of the peptidyl transferase center of the large ribosomal subunit. Testing several m7G methyltransferase candidates allowed us to identify the RlmQ enzyme, encoded by the ywbD open reading frame, as the MTase responsible for this modification. The enzyme methylates free RNA and not ribosomal 50S or 70S particles, suggesting that modification occurs in the early steps of ribosome biogenesis.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Regioselective Deacetylation in Nucleosides and Derivatives.

Nucleoside analogues are a promising class of natural compounds in the pharmaceutical industry, and many antiviral, antibacterial and anticancer drugs have been created through structural modification of nucleosides scaffold. Acyl protecting groups, especially the acetyl group, play an important role in the protection of hydroxy groups in nucleoside synthesis and modification; consequently, numerous methodologies have been put forth for the acetylation of free nucleosides. However, for nucleosides that contain different O- and N-based functionalities, selective deprotection of the acetyl group(s) in nucleosides has been studied little, despite its practical significance in simplifying the preparation of partially or differentially substituted nucleoside intermediates. In this mini-review, recent approaches for regioselective deacetylation in acetylated nucleosides and their analogues are summarized and evaluated. Different regioselectivities (primary ester, secondary ester, full de-O-acetylation, and de-N-acetylation) are summarized and discussed in each section.

The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry.

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Development of a novel HEK293 cell model lacking SLC29A1 to study the pharmacology of endogenous SLC29A2-encoded equilibrative nucleoside transporter subtype 2.

Equilibrative nucleoside transporters (ENT) mediate the transmembrane flux of endogenous nucleosides and nucleoside analogues used clinically. The predominant subtype, ENT1, has been well characterized. However, the other subtype, ENT2, has been less well characterized in its native milieu due to its relatively low expression and the confounding influence of co-expressed ENT1. We created a cell model where ENT1 was removed from HEK293 cells using CRISPR/cas9 (ENT1KO cells); this cell line has ENT2 as the only functional purine transporter. Transporter function was assessed through measurement of [3H]2-chloroadenosine uptake. ENT1 protein was quantified based on the binding of [3H]nitrobenzylthioinosine, and ENT1/ENT2 protein was detected by immunoblotting. Changes in expression of relevant transporters and enzymes involved in purine metabolism were examined by qPCR. Wildtype HEK293 cells and ENT1KO cells had a similar expression of SLC29A2/ENT2 transcript/protein and ENT2-mediated [3H]2-chloroadenosine transport activity (Vmax values of 1.02 {plus minus} 0.06 and 1.50 {plus minus} 0.22 pmol/µl/s, respectively). Of the endogenous nucleosides/nucleobases tested, adenosine had the highest affinity (Ki) for ENT2 (2.6 µM), while hypoxanthine was the only nucleobase with a sub-millimolar affinity (320 µM). A range of nucleoside/nucleobase analogues were also tested for their affinity for ENT2 in this model, with affinities (Ki) ranging from 8.6 µM for ticagrelor to 2,300 µM for 6-mercaptopurine. Our data suggest that the removal of endogenous ENT1 from these cells does not change the expression or function of ENT2. This cell line should prove useful for the analysis of novel drugs acting via ENT2 and to study ENT2 regulation. Significance Statement We have created a cell line whereby endogenous ENT2 can be studied in detail in the absence of the confounding influence of ENT1. Loss of ENT1 has no impact on the expression and function of ENT2. This novel cell line will provide an ideal model for studying drug interactions with ENT2 as well as the cellular regulation of ENT2 expression and function.

Synthesis and Development of Inverse-Type Nucleoside Diarylethene Photoswitches.

Nucleosidic diarylethenes (DAEs) have evolved from an emerging class of photochromes into a well-established option for integrating photochromic functionalities into biological systems. However, a comprehensive understanding of how chemical structure influences their photochromic properties remains essential. While structural features, such as an inverse connection between the aryl residues and the ethene bridge, are well-documented for classical DAEs, their application to nucleosidic DAEs has been underexplored. In this study, we address this gap by developing three distinct types of inverse nucleosidic DAEs - semi-inverse thiophenes, semi-inverse uridines and inverse uridines. We successfully synthesized these compounds and conducted comprehensive analyses of their photostationary states, thermal stability, reversibility, and reaction quantum yields. Additionally, we conducted an in-depth comparison of their photochromic properties with those of their normal-type counterparts. Among the synthesized compounds, seven semi-inverse thiophenes exhibited the most promising characteristics. Notably, these compounds demonstrated excellent fatigue resistance, with up to 96% retention of photochromic activity over 40 switching cycles, surpassing the performance of all comparable nucleosidic DAEs reported to date. These findings hold significant promise for future applications in various fields.

Exogenous Substrates Prevent the Decline in the Cellular ATP Content of Primary Rat Astrocytes During Glucose Deprivation.

Brain astrocytes are well known for their broad metabolic potential. After glucose deprivation, cultured primary astrocytes maintain a high cellular ATP content for many hours by mobilizing endogenous substrates, but within 24 h the specific cellular ATP content was lowered to around 30% of the initial ATP content. This experimental setting was used to test for the potential of various exogenous substrates to prevent a loss in cellular ATP in glucose deprived astrocytes. The presence of various extracellular monocarboxylates, purine nucleosides or fatty acids prevented the loss of ATP from glucose-deprived astrocytes. Of the 20 proteinogenic amino acids, only alanine, aspartate, glutamate, glutamine, lysine or proline maintained high ATP levels in starved astrocytes. Among these amino acids, proline was found to be the most potent one to prevent the ATP loss. The astrocytic consumption of proline as well as the ability of proline to maintain a high cellular ATP content was prevented in a concentration-dependent manner by the proline dehydrogenase inhibitor tetrahydro-2-furoic acid. Analysis of the concentration-dependencies obtained by considering the different carbon content of the applied substrates revealed that fatty acids and proline are more potent than glucose and monocarboxylates as exogenous substrates to prevent ATP depletion in glucose-deprived astrocytes. These data demonstrate that cultured astrocytes can utilise a wide range of extracellular substrates as fuels to support mitochondrial ATP regeneration and identify proline as potent exogenous substrate for the energy metabolism of starved astrocytes.

The impact of purine nucleosides on neuroplasticity in the adult brain.

Neuroplasticity refers to the nervous system's ability to adapt and reorganize its cell structures and neuronal networks in response to internal and external stimuli. In adults, this process involves neurogenesis, synaptogenesis, and synaptic and neurochemical plasticity. Several studies have reported the significant impact of the purinergic system on neuroplasticity modulation. And, there is considerable evidence supporting the role of purine nucleosides, such as adenosine, inosine, and guanosine, in this process. This review presents extensive research on how these nucleosides enhance the neuroplasticity of the adult central nervous system, particularly in response to damage. The mechanisms through which these nucleosides exert their effects involve complex interactions with various receptors and signaling pathways. Adenosine's influence on neurogenesis involves interactions with adenosine receptors, specifically A1R and A2AR. A1R activation appears to inhibit neuronal differentiation and promote astrogliogenesis, while A2AR activation supports neurogenesis, neuritogenesis, and synaptic plasticity. Inosine and guanosine positively impact cell proliferation, neurogenesis, and neuritogenesis. Inosine seems to modulate extracellular adenosine levels, and guanosine might act through interactions between purinergic and glutamatergic systems. Additionally, the review discusses the potential therapeutic implications of purinergic signaling in neurodegenerative and neuropsychiatric diseases, emphasizing the importance of these nucleosides in the neuroplasticity of brain function and recovery.

Urinary phenotyping of SARS-CoV-2 infection connects clinical diagnostics with metabolomics and uncovers impaired NAD+ pathway and SIRT1 activation.

OBJECTIVES: The stratification of individuals suffering from acute and post-acute SARS-CoV-2 infection remains a critical challenge. Notably, biomarkers able to specifically monitor viral progression, providing details about patient clinical status, are still not available. Herein, quantitative metabolomics is progressively recognized as a useful tool to describe the consequences of virus-host interactions considering also clinical metadata. METHODS: The present study characterized the urinary metabolic profile of 243 infected individuals by quantitative nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LC-MS). Results were compared with a historical cohort of noninfected subjects. Moreover, we assessed the concentration of recently identified antiviral nucleosides and their association with other metabolites and clinical data. RESULTS: Urinary metabolomics can stratify patients into classes of disease severity, with a discrimination ability comparable to that of clinical biomarkers. Kynurenines showed the highest fold change in clinically-deteriorated patients and higher-risk subjects. Unique metabolite clusters were also generated based on age, sex, and body mass index (BMI). Changes in the concentration of antiviral nucleosides were associated with either other metabolites or clinical variables. Increased kynurenines and reduced trigonelline excretion indicated a disrupted nicotinamide adenine nucleotide (NAD+) and sirtuin 1 (SIRT1) pathway. CONCLUSIONS: Our results confirm the potential of urinary metabolomics for noninvasive diagnostic/prognostic screening and show that the antiviral nucleosides could represent novel biomarkers linking viral load, immune response, and metabolism. Moreover, we established for the first time a casual link between kynurenine accumulation and deranged NAD+/SIRT1, offering a novel mechanism through which SARS-CoV-2 manipulates host physiology.

Novel drimane-type sesquiterpenoids and nucleosides from the Helicoma septoconstrictum suppress the growth of ovarian cancer cells.

Four new drimane-type sesquiterpenoids and two new nucleoside derivatives (1-6), were isolated from the fungus Helicoma septoconstrictum. Their structures were determined based on the combination of the analysis of their HR-ESI-MS, NMR, ECD calculations data and acid hydrolysis. All the isolated compounds were detected for their bio-activities against MDA-MB-231, A549/DDP, A2780 and HepG2 cell lines. Helicoside C (4) exhibited superior cytotoxicity against the A2780 cell line with IC50 7.5 ± 1.5 µM. The analysis of reactive oxygen species (ROS) revealed that Helicoside C induced an increase in intracellular ROS. Furthermore, the flow cytometry and mitochondrial membrane potential (MMP) analyses unveiled that Helicoside C mediated mitochondrial-dependent apoptosis in A2780 cells. The western blotting test showed that Helicoside C could suppress the STAT3's phosphorylation. These findings offered crucial support for development of H. septoconstrictum and highlighted the potential application of drimane-type sesquiterpenoids in pharmaceuticals.

Developments in the chemistry and biology of 1,2,3-triazolyl-C-nucleosides.

In the last 50 years, nucleoside analogs have been introduced to drug therapy as antivirals for different types of cancer due to their interference in cellular proliferation. Among the first line of nucleoside treatment drugs, ribavirin (RBV) is a synthetic N-nucleoside with a 1,2,4-triazole moiety that acts as a broad-spectrum antiviral. It is on the World Health Organization (WHO) list of essential medicines. However, this important drug therapy causes several side effects due to its nonspecific mechanism of action. There is thus a need for a continuous study of its scaffold. A particular approach consists of connecting  d-ribose to the nitrogen-containing base with a C-C bond. It provides more stability against enzymatic action and a better pharmacologic profile. The coronavirus disease (COVID) pandemic has increased the need for more solutions for the treatment of viral infections. Among these solutions, remdesivir, the first C-nucleoside, has been approved by the Food and Drug Administration (FDA) for clinical use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It drew attention to the study of the C-nucleoside scaffold. Different C-nucleoside patterns have been synthesized over the years. They show many important activities against viruses and cancer cell lines. 1,2,3-Triazolyl-C-nucleoside derivatives are a prolific and efficient subclass of RBV analogs close to the already-known RBV with a C-C bond modification. These compounds are often prepared by alkynylation of the  d-ribose ring followed by azide-alkyne cycloaddition. They are reported to be active against the Crimean-Congo hemorrhagic fever virus and several tumoral cell lines, showing promising biological potential. In this review, we explore such approaches to 1,2,3-triazolyl-C-nucleosides and their evolution over the years.

Discovery of novel benzimidazole acyclic C-nucleoside DNA intercalators halting breast cancer growth.

Breast cancer continues to be the most frequent cancer worldwide. In practice, successful clinical outcomes were achieved via targeting DNA. Along with the advances in introducing new DNA-targeting agents, the "sugar approach" design was employed herein to develop new intercalators bearing pharmacophoric motifs tethered to carbohydrate appendages. Accordingly, new benzimidazole acyclic C-nucleosides were rationally designed, synthesized and assayed via MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay to evaluate their cytotoxicity against MCF-7 and MDA-MB-231 breast cancer cells compared to normal fibroblasts (Wi-38), compared to doxorubicin. (1S,2R,3S,4R)-2-(1,2,3,4,5-Pentahydroxy)pentyl-1H-5,6-dichlorobenzimidazole 7 and (1S,2R,3S,4R)-2-(1,2,3,4,5-pentahydroxy)pentyl-1H-naphthimidazole 13 were the most potent and selective derivatives against MCF-7 (half-maximal inhibitory concentration [IC50 ] = 0.060 and 0.080 µM, selectivity index [SI] = 9.68 and 8.27, respectively) and MDA-MB-231 cells (IC50 = 0.299 and 0.166 µM, SI = 1.94 and 3.98, respectively). Thus, they were identified as the study hits for mechanistic studies. Both derivatives induced DNA damage at 0.24 and 0.29 µM, respectively. The DNA damage kinetics were studied compared to doxorubicin, where they both induced faster damage than doxorubicin. This indicated that 7 and 13 showed a more potent DNA-damaging effect than doxorubicin. Docking simulations within the DNA double strands highlighted the role of both the heterocyclic core and the sugar side chain in exhibiting key H-bond interactions with DNA bases.

Platelet hyperactivity: a comparison of water-soluble, bioactive compound levels in commercial tomato products and water-soluble tomato concentrate, a supplement with an approved EFSA antiplatelet health effect.

The aim of this study was to evaluate and compare the concentration of water-soluble bioactive compounds in tomato products (polyphenols profile, water-soluble vitamins and nucleophilic substances) with the concentration of the same bioactive molecules existing in a water-soluble patented tomato extract, water-soluble tomato extract (WSTC), commercially available as FruitFlow®. This patented tomato extract has been recognised by EFSA (European Food Safety Authority) in a specific Health Claim declaration as having an "Antiplatelet health effect". More than 100 commercial tomato samples, coming from 18 different processing tomato companies worldwide, were analysed and compared with the FruitFlow® supplement. According to the multivariate statistical analyses applied to the data matrix, it is possible to conclude that the commercial tomato products measured (pastes, purees, others) show a significantly higher concentration of water-soluble bioactive molecules (nucleosides/nucleotides and polyphenols) responsible for an anti-platelet aggregation effect than the FruitFlow® dietary supplement.

Structure-Function Insights into the Dual Role in Nucleobase and Nicotinamide Metabolism and a Possible Use in Cancer Gene Therapy of the URH1p Riboside Hydrolase.

The URH1p enzyme from the yeast Saccharomyces cerevisiae has gained significant interest due to its role in nitrogenous base metabolism, particularly involving uracil and nicotinamide salvage. Indeed, URH1p was initially classified as a nucleoside hydrolase (NH) with a pronounced preference for uridine substrate but was later shown to also participate in a Preiss-Handler-dependent pathway for recycling of both endogenous and exogenous nicotinamide riboside (NR) towards NAD+ synthesis. Here, we present the detailed enzymatic and structural characterisation of the yeast URH1p enzyme, a member of the group I NH family of enzymes. We show that the URH1p has similar catalytic efficiencies for hydrolysis of NR and uridine, advocating a dual role of the enzyme in both NAD+ synthesis and nucleobase salvage. We demonstrate that URH1p has a monomeric structure that is unprecedented for members of the NH homology group I, showing that oligomerisation is not strictly required for the N-ribosidic activity in this family of enzymes. The size, thermal stability and activity of URH1p towards the synthetic substrate 5-fluoruridine, a riboside precursor of the antitumoral drug 5-fluorouracil, make the enzyme an attractive tool to be employed in gene-directed enzyme-prodrug activation therapy against solid tumours.

New Biocides Based on N4-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.

Synthesis of Fluorinated Nucleosides/Nucleotides and Their Antiviral Properties.

The FDA has approved several drugs based on the fluorinated nucleoside pharmacophore, and numerous drugs are currently in clinical trials. Fluorine-containing nucleos(t)ides offer significant antiviral and anticancer activity. The insertion of a fluorine atom, either in the base or sugar of nucleos(t)ides, alters its electronic and steric parameters and transforms the lipophilicity, pharmacodynamic, and pharmacokinetic properties of these moieties. The fluorine atom restricts the oxidative metabolism of drugs and provides enzymatic metabolic stability towards the glycosidic bond of the nucleos(t)ide. The incorporation of fluorine also demonstrates additional hydrogen bonding interactions in receptors with enhanced biological profiles. The present article discusses the synthetic methodology and antiviral activities of FDA-approved drugs and ongoing fluoro-containing nucleos(t)ide drug candidates in clinical trials.

Bond Formation at C8 in the Nucleoside and Nucleotide Purine Scaffold: An Informative Selection.

This paper presents methods for the introduction and exchange of substituents in a nucleobase and its nucleosides and nucleotides with emphasis on the C8-position in the purine skeleton. The nucleobase is open for electrophilic and nucleophilic chemistry. The nucleophilic chemistry consists mainly of displacement reactions when the C8-substituent is a good leaving group such as a halogen atom. The heteroatom in amines, sulfides, or oxides is a good nucleophile. Halides are good reaction partners. Metal-promoted cross-coupling reactions are important for carbylations. Direct oxidative metalation reactions using sterically hindered metal amides offer chemo- and regio-selectivity besides functional tolerance and simplicity. The carbon site is highly nucleophilic after metalation and adds electrophiles resulting in chemical bond formation. Conditions for metal-assisted reactions are described for nucleobases and their glycosides.