(3,4-Dihydroxybut-1-ynyl)uracil, -cytosine and -7-deazaadenine 2'-deoxyribonucleoside triphosphates (dNTPs) were prepared by direct aqueous Sonogashira cross-coupling of halogenated dNTPs with dihydroxybut-1-yne and converted to 3,4-dihydroxybutyl dNTPs through catalytic hydrogenation. Sodium periodate oxidative cleavage of dihydroxybutyl-dUTP gave the desired aliphatic aldehyde-linked dUTP, whereas the oxidative cleavage of the corresponding deazaadenine dNTP gave a cyclic aminal. All dihydroxyalkyl or -alkynyl dNTPs and the formylethyl-dUTP were good substrates for DNA polymerases and were used for synthesis of diol- or aldehyde-linked DNA. The aldehyde linked DNA was used for the labelling or bioconjugations through hydrazone formation or reductive aminations.
A new chemoselective synthesis of pyrrolo[2,3-d]pyrimidines (7-deazapurines) bearing two different aryl groups at positions 4 and 5 was developed based on two orthogonal crosscouplings. Starting from 7-benzyl-protected 4-(phenylsulfanyl)-5-iodopyrrolo[2,3-d]pyrimidine, the Pd-catalyzed
Glyoxal-linked 2'-deoxyuridine 5'-O-mono-and triphosphates were synthesized through a CuAAC click reaction of 4-azidophenylglyoxal or a Sonogashira reaction of 4-bromophenylglyoxal with 5-ethynyl-dUMP or -dUTP. The triphosphates were used as substrates for enzymatic synthesis of modified DNA probes with KOD XL DNA polymerase. The glyoxal-linked nucleotides reacted with arginine-containing peptides to form stable imizadolone-linked conjugates. This reactive glyoxal modification in DNA was used for efficient bioconjugations and crosslinking with Arg-containing peptides or proteins (e. g., histones) and was found to be more reactive than previously reported 1,3-diketone-linked DNA probes.
Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiolcontaining molecules as well as with cysteine-or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.
Abstract2‐Formyl‐2′‐deoxyadenosine triphosphate (dCHOATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHOATP was based on the preparation of protected 2‐dihydroxyethyl‐2′‐deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHOATP triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31‐mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single‐nucleotide extension followed by PEX was used for site‐specific incorporation of one aldehyde‐linked adenosine into a longer 49‐mer sequence. The reactive formyl group was used for cross‐linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647‐linked hydroxylamine.
Six‐valent osmium (osmate) complexes with nitrogenous ligands have previously been used for the modification and redox labeling of biomolecules involving vicinal diol moieties (typically, saccharides or RNA). In this work, aliphatic (3,4‐dihydroxybutyl and 3,4‐dihydroxybut‐1‐ynyl) or cyclic (6‐oxo‐6‐(cis‐3,4‐dihydroxypyrrolidin‐1‐yl)hex‐2‐yn‐1‐yl, PDI) vicinal diols are attached to nucleobases to functionalize DNA for subsequent redox labeling with osmium(VI) complexes. The diol‐linked 2′‐deoxyribonucleoside triphosphates were used for the polymerase synthesis of diol‐linked DNA, which, upon treatment with K2OsO3 and bidentate nitrogen ligands, gave the desired Os‐labeled DNA, which were characterized by means of the gel‐shift assay and ESI‐MS. Through ex situ square‐wave voltammetry at a basal plane pyrolytic graphite electrode, the efficiency of modification/labeling of individual diols was evaluated. The results show that the cyclic cis‐diol (PDI) was a better target for osmylation than that of the flexible aliphatic ones (alkyl‐ or alkynyl‐linked). The osmate adduct‐specific voltammetric signal obtained for OsVI‐treated DNA decorated with PDI showed good proportionality to the number of PDI per DNA molecule. The OsVI reagents (unlike OsO4) do not attack nucleobases; thus offering specificity of modification on the introduced glycol targets.
Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiolcontaining molecules as well as with cysteine-or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.
A facile approach has been developed for enzymatic synthesis of DNA modified by reactive aldehyde groups which can be used for post‐synthetic decoration of DNA through bioconjugations with peptides or other molecules. More information can be found in the Communication by M. Hocek, et al. on page 11890.
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