Synthetic Studies toward 2,3-di-N-Acyl-2,4,6-Trideoxy-L-Altropyranoses as Synthetic Precursors to Pseudaminic Acid

With the growing therapeutic inefficiency of traditional antibiotics by rapidly spreading antimicrobial resistance (AMR) through different mechanisms, and a significant slow-down in the development of novel antimicrobials, especially in the pharmaceutical industry during recent years, it is of utmost importance to maintain research to address this global challenge. The chemical synthesis of carbohydrate antigens that are unique to pathogenic bacteria can benefit the search for antibacterial therapeutics with the development of prophylactic vaccines such as polysaccharide conjugates. Bacterial nonulosonic acids (NonAs) that include pseudaminic (Pse) and legionaminic (Leg) acids are found in important structural components that contribute to certain pathogens’ virulence, like Pseudomonas aeruginosa and Campylobacter jejuni: they have been recently shown to be good candidates for use as antigen epitopes in vaccination, and their biosynthetic precursors can also be used towards the development of other types of antibacterial therapeutics. The research presented here begins with preliminary investigations into a synthesis from L-arabinose that has the potential to produce 5 different NonA structures with only a few appropriate variations in the scheme. The synthesis of two C5-(R)/(S) hexose diastereomers was achieved with different selectivity, and those can further undergo an inversion and installation of nitrogen functionalities on C-2 and C-4, before the final three-carbon extension with a phosphoenolpyruvate (PEP) equivalent to produce the target NonA. The work showed promise, justifying future development. Next, a short, mild and scalable synthetic scheme towards 2,4-di-acetamido-2,4,6-trideoxy-L-altrose (Alt-diNAc), the biosynthetic precursor to Pse, is presented: the desired product was obtained from commercially available L-fucose in 10 steps and 23% overall yield, making it the most efficient synthesis published to-date. A further optimized shorter version of synthesis is described as well through regioselective sulfonyl activation to form a key epoxide intermediate, ultimately giving Alt-diNAc in 7 steps and 27% overall yield. Based on these achievements, a new and elegant methodology for the differentiable functionalization of the N2/N4 amide groups of Alt-diNAc was developed, which relies on a regiospecific O→N migration of acyl groups during a Staudinger reduction of the O-acylated di-azido precursor. The new methodology was proved to have broad scope and provides unprecedented versatility to introduce different N-acyl functionalities to the N5 and N7 positions of Pse. Finally, preliminary work towards a potentially stereoselective three-carbon extension of hexose precursors to NonAs is described, with the synthesis of a phenol-based cleavable linker containing an α-methyl ketone that can potentially undergo aldol addition intramolecularly, and then ruthenium-catalyzed oxidation to produce the required carboxylic functionalities for Pse. A successful selective coupling of this linker to one of the two amido groups on the L-altro-configuration precursor was then achieved, paving the way to investigate the diastereoselectivity of intramolecular aldol additions with this strategy in the future. Several possible variations to the linker functional groups and length can easily be incorporated in this synthetic plan , and provide an exciting prospect for future developments

COVID-19 Unmasked Global Collaboration Protocol: longitudinal cohort study examining mental health of young children and caregivers during the pandemic

Background: Early empirical data shows that school-aged children, adolescents and adults are experiencing elevated levels of anxiety and depression during the COVID-19 pandemic. Currently, there is very little research on mental health outcomes for young children. Objectives: To describe the formation of a global collaboration entitled, ‘COVID-19 Unmasked’. The collaborating researchers aim to (1) describe and compare the COVID-19 related experiences within and across countries; (2) examine mental health outcomes for young children (1 to 5 years) and caregivers over a 12-month period during the COVID-19 pandemic; (3) explore the trajectories/time course of psychological outcomes of the children and parents over this period and (4) identify the risk and protective factors for different mental health trajectories. Data will be combined from all participating countries into one large open access cross-cultural dataset to facilitate further international collaborations and joint publications. Methods: COVID-19 Unmasked is an online prospective longitudinal cohort study. An international steering committee was formed with the aim of starting a global collaboration. Currently, partnerships have been formed with 9 countries (Australia, Cyprus, Greece, the Netherlands, Poland, Spain, Turkey, the UK, and the United States of America). Research partners have started to start data collection with caregivers of young children aged 1–5 years old at baseline, 3-months, 6-months, and 12-months. Caregivers are invited to complete an online survey about COVID-19 related exposure and experiences, child’s wellbeing, their own mental health, and parenting. Data analysis: Primary study outcomes will be child mental health as assessed by scales from the Patient-Reported Outcomes Measurement Information System–Early Childhood (PROMIS-EC) and caregiver mental health as assessed by the Depression Anxiety Stress Scale (DASS-21). The trajectories/time course of mental health difficulties and the impact of risk and protective factors will be analysed using hierarchical linear models, accounting for nested effects (e.g. country) and repeated measures

Regiospecific O → N Acyl Migration as a Methodology to Access l‑Altropyranosides with an <i>N</i>2,<i>N</i>4‑Differentiation

Pseudaminic acid and its biosynthetic altropyranoside precursors are bacterial components currently being investigated toward novel antibacterial strategies. One structural feature associated with these naturally occurring flagellar carbohydrates is the different N-acylation patterns on the two amido functionalities, posing a synthetic challenge. A new one-pot methodology is reported and a scope of diverse N2/N4-differentiated analogs are presented via a Staudinger reduction-mediated regiospecific O3 → N4 acyl migration, followed by an autonomous N2-acylation

Specificity of recognition of mRNA 5′ cap by human nuclear cap-binding complex

The heterodimeric nuclear cap-binding complex (CBC) binds to the mono-methylated 5′ cap of eukaryotic RNA polymerase II transcripts such as mRNA and U snRNA. The binding is important for nuclear maturation of mRNAs and possibly in the first round of translation and nonsense-mediated decay. It is also essential for nuclear export of U snRNAs in metazoans. We report characterization by fluorescence spectroscopy of the recognition of 5′ capped RNA by human CBC. The association constants (K(as)) for 17 mono- and dinucleotide cap analogs as well as for the oligomer m(7)GpppA(m2′) pU(m2′)pA(m2′) cover the range from 1.8 × 10(6) M(−1) to 2.3 × 10(8) M(−1). Higher affinity for CBC is observed for the dinucleotide compared with mononucleotide analogs, especially for those containing a purine nucleoside next to m(7)G. The mRNA tetramer associates with CBC as tightly as the dinucleotide analogs. Replacement of Tyr138 by alanine in the CBP20 subunit of CBC reduces the cap affinity except for the mononucleotide analogs, consistent with the crystallographic observation of the second base stacking on this residue. Our spectroscopic studies showed that contrary to the other known cap-binding proteins, the first two nucleotides of a capped-RNA are indispensable for its specific recognition by CBC. Differences in the cap binding of CBC compared with the eukaryotic translation initiation factor 4E (eIF4E) are analyzed and discussed regarding replacement of CBC by eIF4E

Novel “anti-reverse” cap analogs with superior translational properties

Synthetic analogs of the 5′-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m(7)Gp(3)G. Unfortunately, because of the presence of a 3′-OH on both the m(7)Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two “anti-reverse” cap analogs (ARCAs), m(7)3′dGp(3)G and m(2)(7,3′-)(O)Gp(3)G, that cannot be incorporated in the reverse orientation because of modifications at the C3′ position of m(7)Guo. In the present study, we have synthesized seven new cap analogs modified in the C2′ and C3′ positions of m(7)Guo and in the number of phosphate residues, m(2)(7,2′-)(O)Gp(3)G, m(7)2′dGp(3)G, m(7)2′dGp(4)G, m(2)(7,2′-)(O)Gp(4)G, m(2)(7,3′-)(O)Gp(4)G, m(7)Gp(5)G, and m(2)(7,3′-)(O)Gp(5)G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2′, like those at C3′, prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs