A mitochondria-targeted fluorescent probe based on biocompatible RBH-U for the enhanced response of Fe3+ in living cells and quenching of Cu2+ in vitro.

A rhodamine hydrazide conjugating uridine moiety (RBH-U) is firstly synthesized by screening different synthetic routes, and then developed as a fluorescence probe for selective detection of Fe3+ ions in an aqueous solution, accompanied by visual color change with naked eyes. Upon the addition of Fe3+ in a 1:1 stoichiometry, a 9-fold enhancement in the fluorescence intensity of the RBH-U was observed with an emission wavelength of 580 nm. In the presence of other metal ions, the "turn-on" fluorescent probe with pH-independent (value 5.0 to 8.0) is remarkably specific for Fe3+ with a detection limit as low as 0.34 µM. Further, the enhanced fluorescence intensity of RBH-U- Fe3+ can be quenched as a switch-off sensor to assist in the recognition of Cu2+ ions. Additionally, the colocalization assay demonstrated that RBH-U containing uridine residue can be used as a novel mitochondria-targeted fluorescent probe with rapid reaction time. Cytotoxicity and cell imaging of RBH-U probe in live NIH-3T3 cells suggest that it can be a potential candidate for clinical diagnosis and Fe3+ tracking toll for the biological system due to its biocompatibility and nontoxicity in NIH-3T3 cells even up to 100 µM.

Cytidine Alleviates Dyslipidemia and Modulates the Gut Microbiota Composition in ob/ob Mice.

Cytidine and uridine are endogenous metabolites in the pyrimidine metabolism pathway, and cytidine is a substrate that can be metabolized into uridine via cytidine deaminase. Uridine has been widely reported to be effective in regulating lipid metabolism. However, whether cytidine could ameliorate lipid metabolism disorder has not yet been investigated. In this research, ob/ob mice were used, and the effect of cytidine (0.4 mg/mL in drinking water for five weeks) on lipid metabolism disorder was evaluated in terms of an oral glucose tolerance test, serum lipid levels, liver histopathological analysis and gut microbiome analysis. Uridine was used as a positive control. Our findings reveal that cytidine could alleviate certain aspects of dyslipidemia and improve hepatic steatosis via modulating the gut microbiota composition in ob/ob mice, especially increasing the abundance of short-chain fatty acids-producing microbiota. These results suggest that cytidine supplementation could be a potential therapeutic approach for dyslipidemia.

Cytidine deaminases catalyze the conversion of N(S,O)4-substituted pyrimidine nucleosides.

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2'-deoxycytidine to uridine and 2'-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N4-acyl-cytidines, N4-alkyl-cytidines, and N4-alkyloxycarbonyl-cytidines, and S4-alkylthio-uridines and O4-alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible ß3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.

Identification of a novel 5-aminomethyl-2-thiouridine methyltransferase in tRNA modification.

The uridine at the 34th position of tRNA, which is able to base pair with the 3'-end codon on mRNA, is usually modified to influence many aspects of decoding properties during translation. Derivatives of 5-methyluridine (xm5U), which include methylaminomethyl (mnm-) or carboxymethylaminomethyl (cmnm-) groups at C5 of uracil base, are widely conserved at the 34th position of many prokaryotic tRNAs. In Gram-negative bacteria such as Escherichia coli, a bifunctional MnmC is involved in the last two reactions of the biosynthesis of mnm5(s2)U, in which the enzyme first converts cmnm5(s2)U to 5-aminomethyl-(2-thio)uridine (nm5(s2)U) and subsequently installs the methyl group to complete the formation of mnm5(s2)U. Although mnm5s2U has been identified in tRNAs of Gram-positive bacteria and plants as well, their genomes do not contain an mnmC ortholog and the gene(s) responsible for this modification is unknown. We discovered that MnmM, previously known as YtqB, is the methyltransferase that converts nm5s2U to mnm5s2U in Bacillus subtilis through comparative genomics, gene complementation experiments, and in vitro assays. Furthermore, we determined X-ray crystal structures of MnmM complexed with anticodon stem loop of tRNAGln. The structures provide the molecular basis underlying the importance of U33-nm5s2U34-U35 as the key determinant for the specificity of MnmM.

Assessment of the role of nucleoside transporters, P-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in the placental transport of entecavir using in vitro, ex vivo, and in situ methods.

The nucleoside analog entecavir (ETV) is a first-line pharmacotherapy for chronic hepatitis B in adult and pediatric patients. However, due to insufficient data on placental transfer and its effects on pregnancy, ETV administration is not recommended for women after conception. To expand knowledge of safety, we focused on evaluating the contribution of nucleoside transporters (NBMPR sensitive ENTs and Na+ dependent CNTs) and efflux transporters, P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), and multidrug resistance-associated transporter 2 (ABCC2), to the placental kinetics of ETV. We observed that NBMPR and nucleosides (adenosine and/or uridine) inhibited [3H]ETV uptake into BeWo cells, microvillous membrane vesicles, and fresh villous fragments prepared from the human term placenta, while Na+ depletion had no effect. Using a dual perfusion study in an open-circuit setup, we showed that maternal-to-fetal and fetal-to-maternal clearances of [3H]ETV in the rat term placenta were decreased by NBMPR and uridine. Net efflux ratios calculated for bidirectional transport studies performed in MDCKII cells expressing human ABCB1, ABCG2, or ABCC2 were close to the value of one. Consistently, no significant decrease in fetal perfusate was observed in the closed-circuit setup of dual perfusion studies, suggesting that active efflux does not significantly reduce maternal-to-fetal transport. In conclusion, ENTs (most likely ENT1), but not CNTs, ABCB1, ABCG2, and ABCC2, contribute significantly to the placental kinetics of ETV. Future studies should investigate the placental/fetal toxicity of ETV, the impact of drug-drug interactions on ENT1, and interindividual variability in ENT1 expression on the placental uptake and fetal exposure to ETV.

Synthesis of oligonucleotides containing 5'-homo-4'-selenouridine derivative and its increased resistance against nuclease.

As technologies using RNA or DNA have been developed, various chemical modifications of nucleosides have been attempted to increase the stability of oligonucleotides. Since it is known that 2'-OMe-modification greatly contributes to increasing the stability of oligonucleotides, we added 2'-OMe to our previously developed 4'-selenonucleoside and 5'-homo-4'-selenonucleoside as the modified monomers for oligonucleotide: 2'-methoxy-4'-selenouridine (2'-OMe-4'-Se-U) and 5'-homo-2'-methoxy-4'-selenouridine (5'-homo-2'-OMe-4'-Se-U). We synthesized oligonucleotides containing the chemically modified 4'-selenouridine and evaluated their thermal stability and nuclease resistance. In conclusion, the nuclease stability of the oligonucleotide containing 5'-homo-2'-OMe-4'-selenouridine increased while its thermal stability decreased.

5-Ethynyluridine: A Bio-orthogonal Uridine Variant for mRNA-Based Therapies and Vaccines.

The identification of pseudo- and N1 -methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues has propelled the development of mRNA-based vaccines and therapeutics. Here, we have characterised another uridine analogue, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it does not induce interleukin-6 secretion or expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression, as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU-modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity to the other uridine analogues. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent of RNA folding. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics.

Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome.

The ribosome serves as the universally conserved translator of the genetic code into proteins and supports life across diverse temperatures ranging from below freezing to above 120°C. Ribosomes are capable of functioning across this wide range of temperatures even though the catalytic site for peptide bond formation, the peptidyl transferase center, is nearly universally conserved. Here we find that Thermoproteota, a phylum of thermophilic Archaea, substitute cytidine for uridine at large subunit rRNA positions 2554 and 2555 (Escherichia coli numbering) in the A loop, immediately adjacent to the binding site for the 3'-end of A-site tRNA. We show by cryo-EM that E. coli ribosomes with uridine to cytidine mutations at these positions retain the proper fold and post-transcriptional modification of the A loop. Additionally, these mutations do not affect cellular growth, protect the large ribosomal subunit from thermal denaturation, and increase the mutational robustness of nucleotides in the peptidyl transferase center. This work identifies sequence variation across archaeal ribosomes in the peptidyl transferase center that likely confers stabilization of the ribosome at high temperatures and develops a stable mutant bacterial ribosome that can act as a scaffold for future ribosome engineering efforts.

Nucleoside-Based Cross-Linkers for Hydrogels with Tunable Properties.

Herein, we report bioderived cross-linkers to create biopolymer-based hydrogels with tunable properties. Nucleosides (inosine and uridine) and ribose (pentose sugar lucking the nitrogenous base) were partially oxidized to yield inosine dialdehyde (IdA), uridine dialdehyde (UdA), and ribose dialdehyde (RdA). The dialdehydes were further used as cross-linkers with polysaccharide chitosan to form hydrogels. Depending on the cross-linker type and concentration, the hydrogels showed tunable rheological, mechanical, and liquid holding properties allowing the preparation of injectable, soft, and moldable hydrogels. Computational modeling and molecular dynamics simulations shed light on hydrogel formation and revealed that, in addition to covalent bonding, noncovalent interactions (π-π stacking, cation-π, and H-bonding) also significantly contributed to the cross-linking process. To demonstrate various application possibilities, the prepared hydrogels were used as a growth platform for plant cells, as injectable inks for layer-by-layer 3D printing applications, and as moldable hydrogels for soft lithography to replicate the microstructure of the plant. These findings suggest that the obtained tunable biocompatible hydrogels have the potential to be good candidates for various biotechnological applications.

Circulating uridine dynamically and adaptively regulates food intake in humans.

Feeding behavior must be continuously adjusted to match energy needs. Recent discoveries in murine models identified uridine as a regulator of energy balance. Here, we explore its contribution to the complex control of food intake in humans by administering a single dose of uridine monophosphate (UMP; 0.5 or 1 g) to healthy participants in two placebo-controlled studies designed to assess food behavior (registration: DRKS00014874). We establish that endogenous circulating uridine correlates with hunger and ensuing food consumption. It also dynamically decreases upon caloric ingestion, prompting its potential role in a negative feedback loop regulating energy intake. We further demonstrate that oral UMP administration temporarily increases circulating uridine and-when within the physiological range-enhances hunger and caloric intake proportionally to participants' basal energy needs. Overall, uridine appears as a potential target to tackle dysfunctions of feeding behavior in humans.

The regulatory effects of specific polyphenols on Akkermansia are dependent on uridine.

We examined the microbial regulatory capacity of four polyphenols with different structure in healthy mice and explore the mechanism according to exogenous metabolites and microbial metabolites. Oral administration of four polyphenols, including caffeic acid (CA), procyanidin (PA), puerarin (Pue), and resveratrol (Res), did not lead to metabolic disorder in healthy mice. Gut microbiota analysis revealed that CA, PA, and Pue administration significantly enhanced the abundance of Akkermansia and Ruminococcaceae UCG-014 while Res supplement mainly promoted the growth of Lactobacillus and Bacteroides. Furthermore, correlation analysis and exogenous metabolite prediction revealed that the effects of polyphenols, including CA, PA, and Pue, on Akkermansia have strong relationship with uridine while the regulation of Res on microbiota might be dependent on the decrease on petroselinic acid. These investigations considerably suggest the importance of exploration of exogenous metabolites and reveal the similarity of effects of polyphenols on microbiota and metabolites.

Evolutionary Diversity of Dus2 Enzymes Reveals Novel Structural and Functional Features among Members of the RNA Dihydrouridine Synthases Family.

Dihydrouridine (D) is an abundant modified base found in the tRNAs of most living organisms and was recently detected in eukaryotic mRNAs. This base confers significant conformational plasticity to RNA molecules. The dihydrouridine biosynthetic reaction is catalyzed by a large family of flavoenzymes, the dihydrouridine synthases (Dus). So far, only bacterial Dus enzymes and their complexes with tRNAs have been structurally characterized. Understanding the structure-function relationships of eukaryotic Dus proteins has been hampered by the paucity of structural data. Here, we combined extensive phylogenetic analysis with high-precision 3D molecular modeling of more than 30 Dus2 enzymes selected along the tree of life to determine the evolutionary molecular basis of D biosynthesis by these enzymes. Dus2 is the eukaryotic enzyme responsible for the synthesis of D20 in tRNAs and is involved in some human cancers and in the detoxification of ß-amyloid peptides in Alzheimer's disease. In addition to the domains forming the canonical structure of all Dus, i.e., the catalytic TIM-barrel domain and the helical domain, both participating in RNA recognition in the bacterial Dus, a majority of Dus2 proteins harbor extensions at both ends. While these are mainly unstructured extensions on the N-terminal side, the C-terminal side extensions can adopt well-defined structures such as helices and beta-sheets or even form additional domains such as zinc finger domains. 3D models of Dus2/tRNA complexes were also generated. This study suggests that eukaryotic Dus2 proteins may have an advantage in tRNA recognition over their bacterial counterparts due to their modularity.

Aspergillus fumigatus Elongator complex subunit 3 affects hyphal growth, adhesion and virulence through wobble uridine tRNA modification.

The eukaryotic multisubunit Elongator complex has been shown to perform multiple functions in transcriptional elongation, histone acetylation and tRNA modification. However, the Elongator complex plays different roles in different organisms, and the underlying mechanisms remain unexplored. Moreover, the biological functions of the Elongator complex in human fungal pathogens remain unknown. In this study, we verified that the Elongator complex of the opportunistic fungal pathogen Aspergillus fumigatus consists of six subunits (Elp1-6), and the loss of any subunit results in similarly defective colony phenotypes with impaired hyphal growth and reduced conidiation. The catalytic subunit-Elp3 of the Elongator complex includes a S-adenosyl methionine binding (rSAM) domain and a lysine acetyltransferase (KAT) domain, and it plays key roles in the hyphal growth, biofilm-associated exopolysaccharide galactosaminogalactan (GAG) production, adhesion and virulence of A. fumigatus; however, Elp3 does not affect H3K14 acetylation levels in vivo. LC-MS/MS chromatograms revealed that loss of Elp3 abolished the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification of tRNA wobble uridine (U34), and the overexpression of tRNAGlnUUG and tRNAGluUUC, which normally harbor mcm5s2U modifications, mainly rescues the defects of the Δelp3 mutant, suggesting that tRNA modification rather than lysine acetyltransferase is responsible for the primary function of Elp3 in A. fumigatus. Strikingly, global proteomic comparison analyses showed significantly upregulated expression of genes related to amino acid metabolism in the Δelp3 mutant strain compared to the wild-type strain. Western blotting showed that deletion of elp3 resulted in overexpression of the amino acid starvation-responsive transcription factor CpcA, and deletion of CpcA markedly reversed the defective phenotypes of the Δelp3 mutant, including attenuated virulence. Therefore, the findings of this study demonstrate that A. fumigatus Elp3 functions as a tRNA-modifying enzyme in the regulation of growth, GAG production, adhesion and virulence by maintaining intracellular amino acid homeostasis. More broadly, our study highlights the importance of U34 tRNA modification in regulating cellular metabolic states and virulence traits of fungal pathogens.

RNADSN: Transfer-Learning 5-Methyluridine (m5U) Modification on mRNAs from Common Features of tRNA.

One of the most abundant non-canonical bases widely occurring on various RNA molecules is 5-methyluridine (m5U). Recent studies have revealed its influences on the development of breast cancer, systemic lupus erythematosus, and the regulation of stress responses. The accurate identification of m5U sites is crucial for understanding their biological functions. We propose RNADSN, the first transfer learning deep neural network that learns common features between tRNA m5U and mRNA m5U to enhance the prediction of mRNA m5U. Without seeing the experimentally detected mRNA m5U sites, RNADSN has already outperformed the state-of-the-art method, m5UPred. Using mRNA m5U classification as an additional layer of supervision, our model achieved another distinct improvement and presented an average area under the receiver operating characteristic curve (AUC) of 0.9422 and an average precision (AP) of 0.7855. The robust performance of RNADSN was also verified by cross-technical and cross-cellular validation. The interpretation of RNADSN also revealed the sequence motif of common features. Therefore, RNADSN should be a useful tool for studying m5U modification.

A Simple High-Throughput Method for the Analysis of Vicine and Convicine in Faba Bean.

The faba bean is one of the earliest domesticated crops, with both economic and environmental benefits. Like most legumes, faba beans are high in protein, and can be used to contribute to a balanced diet, or as a meat substitute. However, they also produce the anti-nutritional compounds, vicine and convicine (v-c), that when enzymatically degraded into reactive aglycones can potentially lead to hemolytic anemia or favism. Current methods of analysis use LC-UV, but are only suitable at high concentrations, and thus lack the selectivity and sensitivity to accurately quantitate the low-v-c genotypes currently being developed. We have developed and fully validated a rapid high-throughput LC-MS method for the analysis of v-c in faba beans by optimizing the extraction protocol and assessing the method of linearity, limit of detection, limit of quantitation, accuracy, precision and matrix effects. This method uses 10-times less starting material; removes the use of buffers, acids and organic chemicals; and improves precision and accuracy when compared to current methods.

Amauroderma rugosum Extract Suppresses Inflammatory Responses in Tumor Necrosis Factor Alpha/Interferon Gamma-Induced HaCaT Keratinocytes.

Keratinocytes form the physical barrier of the skin and play an important role in the inflammatory process. Amauroderma rugosum is an edible mushroom; however, its pharmacological properties have seldom been studied. Although the anti-inflammatory effect of the organic solvent extract of Amauroderma rugosum has been previously reported, it is not known whether the aqueous extract has a similar effect. In addition, the effect of Amauorderma rugosum extract on skin has never been explored. Therefore, the objectives of the present study were to evaluate the anti-inflammatory effects of the aqueous extract of Amauroderma rugosum on HaCaT keratinocytes, to explore its mechanisms of action, and to study the possible active ingredients involved. The results showed that the aqueous extract of Amauroderm rugosum at a concentration of 1.5 mg/mL was non-toxic to HaCaT cells and inhibited the release of cytokine interleukin-1ß, and chemokines interleukin-8 and monocyte chemoattractant protein-1 in tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-stimulated HaCaT cells. Amauroderma rugosum extract reduced the intracellular levels of reactive oxygen species. In addition, Amauroderma rugosum extract reduced the total protein expression of nuclear factor-kappa B (NF-κB) and B-cells inhibitor alpha in HaCaT keratinocytes and inhibited the phosphorylation of mitogen-activated protein kinase kinase (MEK) 1/2, extracellular signal-regulated kinase (ERK) 1/2, protein kinase B (Akt), and mammalian target of rapamycin (mTOR) in TNF-α- and INF-γ-stimulated HaCaT keratinocytes. Chemical analysis revealed that the aqueous extract of Amauroderma rugosum contains polysaccharides, triterpenes, and phenolic compounds. Anti-inflammatory compounds, such as gallic acid, guanosine, and uridine, were also present. The anti-inflammatory effect of Amauroderma rugosum could be mimicked by a combination of gallic acid, guanosine, and uridine. In conclusion, our study suggests that the aqueous extract of Amauroderma rugosum exerts anti-inflammatory effects on keratinocytes through its antioxidant and inhibitory effects on MEK/ERK-, Akt/mTOR-, and NF-κB-dependent signaling pathways.

Elucidation of the substrate of tRNA-modifying enzymes MnmEG leads to in vitro reconstitution of an evolutionarily conserved uridine hypermodification.

The evolutionarily conserved bacterial proteins MnmE and MnmG collectively install a carboxymethylaminomethyl (cmnm) group at the fifth position of wobble uridines of several tRNA species. While the reaction catalyzed by MnmEG is one of the central steps in the biosynthesis of the methylaminomethyl (mnm) posttranscriptional tRNA modification, details of the reaction remain elusive. Glycine is known to be the source of the carboxy methylamino moiety of cmnm, and a tetrahydrofolate (THF) analog is thought to supply the one carbon that is appended to the fifth position of U. However, the nature of the folate analog remains unknown. This article reports the in vitro biochemical reconstitution of the MnmEG reaction. Using isotopically labeled methyl and methylene THF analogs, we demonstrate that methylene THF is the true substrate. We also show that reduced FAD is required for the reaction and that DTT can replace the NADH in its role as a reductant. We discuss the implications of these methylene-THF and reductant requirements on the mechanism of this key tRNA modification catalyzed by MnmEG.

The Novel hDHODH Inhibitor MEDS433 Prevents Influenza Virus Replication by Blocking Pyrimidine Biosynthesis.

The pharmacological management of influenza virus (IV) infections still poses a series of challenges due to the limited anti-IV drug arsenal. Therefore, the development of new anti-influenza agents effective against antigenically different IVs is therefore an urgent priority. To meet this need, host-targeting antivirals (HTAs) can be evaluated as an alternative or complementary approach to current direct-acting agents (DAAs) for the therapy of IV infections. As a contribution to this antiviral strategy, in this study, we characterized the anti-IV activity of MEDS433, a novel small molecule inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 exhibited a potent antiviral activity against IAV and IBV replication, which was reversed by the addition of exogenous uridine and cytidine or the hDHODH product orotate, thus indicating that MEDS433 targets notably hDHODH activity in IV-infected cells. When MEDS433 was used in combination either with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, or with an anti-IV DAA, such as N4-hydroxycytidine (NHC), synergistic anti-IV activities were observed. As a whole, these results indicate MEDS433 as a potential HTA candidate to develop novel anti-IV intervention approaches, either as a single agent or in combination regimens with DAAs.

5-Halogenation of Uridine Suppresses Protonation-Induced Tautomerization and Enhances Glycosidic Bond Stability of Protonated Uridine: Investigations via IRMPD Action Spectroscopy, ER-CID Experiments, and Theoretical Calculations.

Uridine (Urd), a canonical nucleoside of RNA, is the most commonly modified nucleoside among those that occur naturally. Uridine has also been an important target for the development of modified nucleoside analogues for pharmaceutical applications. In this work, the effects of 5-halogenation of uracil on the structures and glycosidic bond stabilities of protonated uridine nucleoside analogues are examined using tandem mass spectrometry and computational methods. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and theoretical calculations are performed to probe the structural influences of these modifications. Energy-resolved collision-induced dissociation experiments along with survival yield analyses are performed to probe glycosidic bond stability. The measured IRMPD spectra are compared to linear IR spectra predicted for the stable low-energy conformations of these species computed at the B3LYP/6-311+G(d,p) level of theory to determine the conformations experimentally populated. Spectral signatures in the IR fingerprint and hydrogen-stretching regions allow the 2,4-dihydroxy protonated tautomers (T) and O4- and O2-protonated conformers to be readily differentiated. Comparisons between the measured and predicted spectra indicate that parallel to findings for uridine, both T and O4-protonated conformers of the 5-halouridine nucleoside analogues are populated, whereas O2-protonated conformers are not. Variations in yields of the spectral signatures characteristic of the T and O4-protonated conformers indicate that the extent of protonation-induced tautomerization is suppressed as the size of the halogen substituent increases. Trends in the energy-dependence of the survival yield curves find that 5-halogenation strengthens the glycosidic bond and that the enhancement in stability increases with the size of the halogen substituent.