Methyl 6-azido-6-de­oxy-α-d-galactoside

The structure of the title compound, C7H13N3O5, was solved using data from a multiple fragment crystal. The galactoside ring adopts a 4 C 1 chair conformation. In the crystal, the molecules are linked by strong O—H⋯O hydrogen bonds, which build linkages around the screw axis of the cell in a similar way to the iodo analogue. These C-5 and C-6 packing motifs expand to R 2 2(10), C 2 2(7) and C 2 2 2(8) motifs, as found in closely related compounds

Methyl 6-de­oxy-6-iodo-α-d-galactoside

In the crystal of the title compound, C7H13IO5, the molecules are linked by O—H⋯O hydrogen bonds, which build linkages around one screw axis of the cell. These C(5) and C(6) packing motifs expand to R 2 2(10) and C2 2(11) motifs and are similar to those found for closely related compounds. The galactoside ring has a 1 C 4 chair conformation

Development of virus-like particles with inbuilt immunostimulatory properties as vaccine candidates

The development of virus-like particle (VLP) based vaccines for human papillomavirus, hepatitis B and hepatitis E viruses represented a breakthrough in vaccine development. However, for dengue and COVID-19, technical complications, such as an incomplete understanding of the requirements for protective immunity, but also limitations in processes to manufacture VLP vaccines for enveloped viruses to large scale, have hampered VLP vaccine development. Selecting the right adjuvant is also an important consideration to ensure that a VLP vaccine induces protective antibody and T cell responses. For diseases like COVID-19 and dengue fever caused by RNA viruses that exist as families of viral variants with the potential to escape vaccine-induced immunity, the development of more efficacious vaccines is also necessary. Here, we describe the development and characterisation of novel VLP vaccine candidates using SARS-CoV-2 and dengue virus (DENV), containing the major viral structural proteins, as protypes for a novel approach to produce VLP vaccines. The VLPs were characterised by Western immunoblot, enzyme immunoassay, electron and atomic force microscopy, and in vitro and in vivo immunogenicity studies. Microscopy techniques showed proteins self-assemble to form VLPs authentic to native viruses. The inclusion of the glycolipid adjuvant, α-galactosylceramide (α-GalCer) in the vaccine formulation led to high levels of natural killer T (NKT) cell stimulation in vitro, and strong antibody and memory CD8+ T cell responses in vivo, demonstrated with SARS-CoV-2, hepatitis C virus (HCV) and DEN VLPs. This study shows our unique vaccine formulation presents a promising, and much needed, new vaccine platform in the fight against infections caused by enveloped RNA viruses

7-Benzyloxymethyl-9-bromo-6-chloro-9-deazapurine

The title compound, C14H11BrClN3O, crystallizes with two independent molecules in the asymmetric unit. In the crystal, the molecules are linked by C—N...Br halogen bonds, as well as weak methylene C—H...π, phenyl C—H...π, C—H...Br and phenyl C—H...O(ether) interactions

Luciferase-Based Assay for Adenosine: Application to <i>S</i>-Adenosyl-l-homocysteine Hydrolase

<i>S</i>-Adenosyl-l-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of <i>S</i>-adenosyl-l-homocysteine (SAH) to adenosine (ADO) and l-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to <i>S</i>-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (<i>K</i>_m, <i>k</i>_cat) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10^–7 unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions

Active Site and Remote Contributions to Catalysis in Methylthioadenosine Nucleosidases

5′-Methylthioadenosine/<i>S-</i>adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5′-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from <i>Vibrio cholerae</i> (<i>Vc</i>MTAN) and <i>Escherichia coli</i> (<i>Ec</i>MTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in <i>Vc</i>MTAN and by enthalpy in <i>Ec</i>MTAN. Catalytic sites of <i>Vc</i>MTAN and <i>Ec</i>MTAN in contact with reactants differ by two residues; Ala113 and Val153 in <i>Vc</i>MTAN are Pro113 and Ile152, respectively, in <i>Ec</i>MTAN. We mutated the <i>Vc</i>MTAN catalytic site residues to match those of <i>Ec</i>MTAN in anticipation of altering its properties toward <i>Ec</i>MTAN. Inhibition of <i>Vc</i>MTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in <i>Vc</i>MTAN. The transition-state analogue affinity and thermodynamics in mutant <i>Vc</i>MTAN became even more unlike those of <i>Ec</i>MTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in <i>Vc</i>MTAN are unable to recapitulate the properties of <i>Ec</i>MTAN. X-ray crystal structures of <i>Ec</i>MTAN, <i>Vc</i>MTAN, and a multiple-site mutant of <i>Vc</i>MTAN most closely resembling <i>Ec</i>MTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences