7‐Aryl‐7‐deazaadenine 2′‐Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

A series of 7‐substituted 7‐deazaadenine and 5‐substituted cytosine 2′‐deoxyribonucleoside triphosphates (dNTPs) were tested for their competitive incorporations (in the presence of dATP and dCTP) into DNA by several DNA polymerases by using analysis based on cleavage by restriction endonucleases. 7‐Aryl‐7‐deazaadenine dNTPs were more efficient substrates than dATP because of their higher affinity for the active site of the enzyme, as proved by kinetic measurements and calculations.     

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.     

Polymerase‐Catalysed Incorporation of Glucose Nucleotides into a DNA Duplex

Active but unselective : Nucleoside triphosphates possessing glucose moieties (such as those depicted) instead of the natural furanose rings are recognised by the active sites of polymerases. Polymerases therefore seem to be very unspecific in their recognition patterns.

     

High‐Density DNA Functionalization by a Combination of Cu‐Catalyzed and Cu‐Free Click Chemistry

We report the regioselective Cu‐free click modification of styrene functionalized DNA with nitrile oxides. A series of modified oligodeoxynucleotides (nine base pairs) was prepared with increasing styrene density. 1,3‐Dipolar cycloaddition with nitrile oxides allows the high density functionalization of the styrene modified DNA directly on the DNA solid support and in solution. This click reaction proceeds smoothly even directly in the DNA synthesizer and gives exclusively 3,5‐disubstituted isoxazolines. Additionally, PCR products (300 and 900 base pairs) were synthesized with a styrene triphosphate and KOD XL polymerase. The click reaction on the highly modified PCR fragments allows functionalization of hundreds of styrene units on these large DNA fragments simultaneously. Even sequential Cu‐free and Cu‐catalyzed click reaction of PCR amplicons containing styrene and alkyne carrying nucleobases was achieved. This new approach towards high‐density functionalization of DNA is simple, modular, and efficient.     

Design,Synthesis, and Evaluation of Bis‐Benzothiazole Derivatives as DNA Minor Groove Binding Agents

A series of novel bis‐benzothiazole derivatives with head‐to‐head orientation were designed, synthesized, and evaluated for their anti‐proliferative activities on U937, HL60, and HeLa cells. A significant part of the targets exhibited considerable in vitro anticancer activity, and some of them were more potent than the reference 5‐FU . Molecular modeling, fluorescence, and viscosimetry studies revealed that these compounds could bind into the minor groove of DNA. Amongst them, the most active compound 7 with IC₅₀ in a range of 6.76 to 8.67 μM against the tested three cell lines, which was 1.46–2.47 and 3.48–4.93 times more potent than 5‐FU and Hoechst 33258 , respectively, warrant further investigations.     

Acetophenyl-thienyl-aniline-Linked Nucleotide for Construction of Solvatochromic Fluorescence Light-Up DNA Probes Sensing Protein-DNA Interactions

The synthesis of 2′-deoxycytidine and its 5′-O-triphosphate bearing solvatochromic acetophenyl-thienyl-aniline fluorophore was developed using the Sonogashira cross-coupling reaction as the key step. The triphosphate was used for polymerase synthesis of labelled DNA. The labelled nucleotide or DNA exerted weak red fluorescence when excited at 405 nm, but a significant colour change (to yellow or green) and light-up (up to 20 times) was observed when the DNA probes interacted with proteins or lipids.     

Discrimination Between 8‐Oxo‐2′‐Deoxyguanosine and 2′‐Deoxyguanosine in DNA by the Single Nucleotide Primer Extension Reaction with Adap Triphosphate

The adenosine derivative of 2‐oxo‐1,3‐diazaphenoxazine (Adap) exhibits a superb ability to recognize and form base pairs with 8‐oxo‐2′‐deoxyguanosine (8‐oxo‐dG) in duplex DNA. In this study, the triphosphate of Adap (dAdapTP) was synthesized and tested for single nucleotide incorporation into primer strands using the Klenow Fragment. The efficiency of dAdapTP incorporation into 8‐oxo‐dG‐containing templates was more than 36‐fold higher than with dG‐containing templates, and provides better discrimination than does the incorporation of natural 2′‐deoxyadenosine triphosphate (dATP). The selective incorporation of dAdapTP into 8‐oxo‐dG templates was therefore applied to the detection of 8‐oxo‐dG in human telomeric DNA sequences extracted from H₂O₂‐treated HeLa cells. The enzymatic incorporation of dAdapTP into 8‐oxo‐dG‐containing templates may provide a novel basis for sequencing oxidative DNA damage in the genome.     

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP‐releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with K_m values averaging 13‐fold higher than those of natural dNTPs, and k_cat values within 1.5‐fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.     

Synthesis and Reactivity Comparisons of 1‐Methyl‐3‐Substituted Cyclopropene Mini‐tags for Tetrazine Bioorthogonal Reactions

Substituted cyclopropenes have recently attracted attention as stable “mini‐tags” that are highly reactive dienophiles with the bioorthogonal tetrazine functional group. Despite this interest, the synthesis of stable cyclopropenes is not trivial and their reactivity patterns are poorly understood. Here, the synthesis and comparison of the reactivity of a series of 1‐methyl‐3‐substituted cyclopropenes with different functional handles is described. The rates at which the various substituted cyclopropenes undergo Diels–Alder cycloadditions with 1,2,4,5‐tetrazines were measured. Depending on the substituents, the rates of cycloadditions vary by over two orders of magnitude. The substituents also have a dramatic effect on aqueous stability. An outcome of these studies is the discovery of a novel 3‐amidomethyl substituted methylcyclopropene tag that reacts twice as fast as the fastest previously disclosed 1‐methyl‐3‐substituted cyclopropene while retaining excellent aqueous stability. Furthermore, this new cyclopropene is better suited for bioconjugation applications and this is demonstrated through using DNA templated tetrazine ligations. The effect of tetrazine structure on cyclopropene reaction rate was also studied. Surprisingly, 3‐amidomethyl substituted methylcyclopropene reacts faster than trans‐cyclooctenol with a sterically hindered and extremely stable tert‐butyl substituted tetrazine. Density functional theory calculations and the distortion/interaction analysis of activation energies provide insights into the origins of these reactivity differences and a guide to the development of future tetrazine coupling partners. The newly disclosed cyclopropenes have kinetic and stability advantages compared to previously reported dienophiles and will be highly useful for applications in organic synthesis, bioorthogonal reactions, and materials science.     

The Structure of an Archaeal B‐Family DNA Polymerase in Complex with a Chemically Modified Nucleotide

Archaeal B‐family DNA polymerases (DNA pols) are the driving force of cutting‐edge biotechnological applications like next‐generation sequencing. The acceptance of chemically modified nucleotides by DNA pols is key to these technologies. Until now, no structural data have been available for these DNA pols in complex with modified substrates, which could build the basis for understanding interactions between the enzyme and the chemically modified nucleotide and for the further development of next‐generation nucleotides. For the first time, we crystallized an exonuclease‐deficient variant of the wild‐type B‐family KOD DNA pol with a modified nucleotide in a closed, ternary complex. We also crystalized the A‐family DNA pol KlenTaq with the same nucleotide. The reported structural data reveal how the protein and the DNA modulate two distinct conformations of the appended moiety in the A‐ and B‐family DNA pols and how these influence the processing of the modified nucleotide. Overall, this study provides first insight into the interplay between B‐family DNA pols and relevant modified substrates.     

Synthesis of Organofluoro Compounds Using Methyl Perfluoroalk‐2‐ynoates as Building Blocks

This review provides an overview of several synthetic applications of methyl perfluoroalk‐2‐ynoates, leading to convenient preparation of many perfluoroalkylated compounds. The use of these important substrates in the synthesis of various five‐, six‐, and seven‐membered heterocycles, cyclopentadienes, and biphenyls is described, alongside a discussion of the mechanistic aspects of these reactions.     

meso‐Pyrrole‐Substituted 22‐Oxacorroles: Building Blocks for the Synthesis of BODIPY‐Bridged 22‐Oxacorrole Dyads

Novel boron‐dipyrromethene (BODIPY)‐bridged 22‐oxacorrole dyads, using meso‐pyrrolyl 22‐oxacorrole as a key synthon, have been synthesized. The reactivity of the meso‐pyrrolyl group of 22‐oxacorrole was exploited to synthesize the first examples of BODIPY‐bridged 22‐oxacorrole dyads in ≈40 % yield. The dyads are stable and exhibited interesting spectral and electrochemical properties.     

Differently Glycosidated 2‐Amino‐2‐deoxy‐d‐glucopyranosiduronic Acids as Building Blocks in Peptide Synthesis

Seven differently glycosidated sugar amino acids (SSAs) derived from glucosamine have been prepared. Following standard solution‐phase peptide‐coupling procedures, the glycosidated 2‐amino‐2‐deoxy‐D ‐glucopyranosiduronic acids were condensed with natural amino acids to furnish useful heterodi‐ and ‐trimeric building blocks to be used in peptide synthesis. Combinations of these building blocks yielded hetero‐oligomeric peptides with two sugar amino acid units in different distances to each other. These were prepared to evaluate the influence of glycosidic side chains on the peptide backbone. Conformations of selected examples were examined by means of ROESY spectroscopy in combination with molecular dynamics (MD) simulations and circular‐dichroism (CD) studies.