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Mononuclear to Polynuclear U IV Structural Units: Effects of Reaction Conditions on U‐Furoate Phase Formation
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Abstract This article contains detailed synthetic protocols for preparation of 5‐cyanomethyluridine (cnm5U) and 5‐cyanouridine (cn5U) phosphoramidites. The synthesis of the cnm5U phosphoramidite building block starts with commercially available 5‐methyluridine (m5C), followed by bromination of the 5‐methyl group to install the cyano moiety using TMSCN/TBAF. The cn5U phosphoramidite is obtained by regular Vorbrüggen glycosylation of the protected ribofuranose with silylated 5‐cyanouracil. These two modified phosphoramidites are suitable for synthesis of RNA oligonucleotides on solid phase using conventional amidite chemistry. Our protocol provides access to two novel building blocks for constructing RNA‐based therapeutics. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cnm5U and cn5U phosphoramidites Basic Protocol 2: Synthesis, purification, and characterization of cnm5U‐ and cn5U‐modified RNA oligonucleotidesmore » « less
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Henkin, Tina M (Ed.)The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Aspcontains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negativeEscherichia coliK12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence ofmnmCorthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in bothBacillus subtilisandStreptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U inB. subtilis. Analysis of tRNA modifications of bothS. mutansandStreptococcus pneumoniaeshows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.more » « less
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Abstract Improvements in analytical procedures in parallel with intercalibration of40Ar/39Ar and U–Pb methods and laboratories, spurred since 2003 by the EarthTime geochronology community initiative, have led to ±2σuncertainties of the order of 50–100 ka, or better, for Cretaceous ash beds. Assembled here are 5740Ar/39Ar ages and 17238U–206Pb ages of volcanic ash beds in strata of the Western Interior Basin of North America determined during the last 15 years since these improvements have been made. These age determinations span from 108 Ma in the middle Albian to 66 Ma in the latest Maastrichtian. Five of the40Ar/39Ar ages from Campanian and Maastrichtian strata are newly reported here, whereas the remainder are from the literature. Building on the pioneering work of John Obradovich and Bill Cobban, where possible these age determinations are tied to ammonite and inoceramid biostratigraphy. For most ash beds, the temporal uncertainties, unlike earlier timescales for the Western Interior Basin, are much shorter than the duration of fossil biozones. Proposed ages for stage boundaries based on this review of the radioisotopic ages include: Maastrichtian–Danian, 66.02 ± 0.08 Ma; Campanian–Maastrichtian, 72.20 ± 0.20 Ma; Santonian–Campanian, 84.19 ± 0.38 Ma; Coniacian–Santonian, 86.49 ± 0.44 Ma; Turonian–Coniacian, 89.75 ± 0.38 Ma; Cenomanian–Turonian, 93.95 ± 0.05 Ma; Albian–Cenomanian, 100.00 ± 0.40 Ma. Six bentonites that occur within theVascoceras diartianum, Neocardiocerus juddi, Prionocylus macombi, Scaphites preventricosus, Scaphites depressusandDesmoscaphites bassleriammonite zones, dated using both40Ar/39Ar and U–Pb methods, yield ages in agreement to within 150 ka and form the backbone of the Western Interior Basin timescale. In parallel, improvements in the taxonomy of ammonites, inoceramids and foraminifera, and recent field work, are better establishing the biostratigraphic framework for these age determinations. Each of these efforts contributes to the progressive refinement of the chronostratigraphic framework of the Western Interior Basin, and enhances its utility for global correlation.more » « less
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- https://doi.org/10.1002/chem.201905759
