Efficient Access to Deuterated and Tritiated Nucleobase Pharmaceuticals and Oligonucleotides using Hydrogen‐Isotope Exchange

A general approach for the efficient hydrogen‐isotope exchange of nucleobase derivatives is described. Catalyzed by ruthenium nanoparticles, using mild reaction conditions, and involving either D₂ or T₂ as isotopic sources, this reaction possesses a wide substrate scope and a high solvent tolerability. This novel method facilitates the access to essential diagnostic tools in drug discovery and development: tritiated pharmaceuticals with high specific activities and deuterated oligonucleotides suitable for use as internal standards during LC‐MS quantification.     

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP,NAD+, NADH,NADP+, and NADPH

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3‐d]pyrimidine analogue of adenosine ( ^tz A ), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and ^tz ATP to yield N^tzAD⁺ , which can be enzymatically phosphorylated by NAD⁺ kinase and ATP or ^tz ATP to the corresponding N^tzADP⁺ . The latter can be engaged in NADP‐specific coupled enzymatic transformations involving conversion to N^tzADPH by glucose‐6‐phosphate dehydrogenase and reoxidation to N^tzADP⁺ by glutathione reductase. The N^tzADP⁺ / N^tzADPH cycle can be monitored in real time by fluorescence spectroscopy.     

N2‐Substituted 2′‐Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ

N²‐Alkyl‐2′‐deoxyguanosine triphosphate (N²‐alkyl‐dGTP) derivatives with methyl, butyl, benzyl, or 4‐ethynylbenzyl substituents were prepared and tested as substrates for human DNA polymerases. N²‐Benzyl‐dGTP was equal to dGTP as a substrate for DNA polymerase κ (pol κ), but was a poor substrate for pols β, δ, η, ι, or ν. In vivo reactivity was evaluated through incubation of N²‐4‐ethynylbenzyl‐dG with wild‐type and pol κ deficient mouse embryonic fibroblasts. CuAAC reaction with 5(6)‐FAM‐azide demonstrated that only cells containing pol κ were able to incorporate N²‐4‐ethynylbenzyl‐dG into the nucleus. This is the first instance of a Y‐family‐polymerase‐specific dNTP, and this method could be used to probe the activity of pol κ in vivo.     

Ribonucleotides and RNA Promote Peptide Chain Growth

All known forms of life use RNA‐mediated polypeptide synthesis to produce the proteins encoded in their genes. Because the principal parts of the translational machinery consist of RNA, it is likely that peptide synthesis was achieved early in the prebiotic evolution of an RNA‐dominated molecular world. How RNA attracted amino acids and then induced peptide formation in the absence of enzymes has been unclear. Herein, we show that covalent capture of an amino acid as a phosphoramidate favors peptide formation. Peptide coupling is a robust process that occurs with different condensation agents. Kinetics show that covalent capture can accelerate chain growth over oligomerization of the free amino acid by at least one order of magnitude, so that there is no need for enzymatic catalysis for peptide synthesis to begin. Peptide chain growth was also observed on phosphate‐terminated RNA strands. Peptide coupling promoted by ribonucleotides or ribonucleotide residues may have been an important transitional form of peptide synthesis that set in when amino acids were first captured by RNA.