Additions of Thiols to 7‑Vinyl-7-deazaadenine Nucleosides and Nucleotides. Synthesis of Hydrophobic Derivatives of 2′-Deoxyadenosine, dATP and DNA

Additions of alkyl- or arylthiols to 7-vinyl-7-deaza-2′-deoxyadenosine gave a series of 7-[2-(alkyl- or arylsulfanyl)­ethyl]-7-deaza-2′-deoxyadenosines in 45–85% yields. The nucleosides were converted to 5′-<i>O</i>-mono-(<b>dA</b>^<b>SR</b><b>MP</b>) or triphosphates (<b>dA</b>^<b>SR</b><b>TP</b>) by phosphorylation. The modified triphosphates were also prepared by thiol addition to 7-vinyl-7-deaza-dATP. The triphosphates <b>dA</b>^<b>SR</b><b>TP</b> were good substrates for DNA polymerases useful in the enzymatic synthesis of base-modified oligonucleotides (ONs) or DNA containing flexibly linked hydrophobic substituents in the major groove. Primer extension was used for the synthesis of ONs with one or several modifications, PCR was used for the synthesis of heavily modified DNA, whereas terminal deoxynucleotidyl transferase was used for a single-nucleotide labeling of the 3′-end

Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR

Enzymatic synthesis of short (10–22 nt) base-modified oligonucleotides (ONs) was developed by nicking enzyme amplification reaction (NEAR) using Vent­(exo-) polymerase, Nt.BstNBI nicking endonuclease, and a modified deoxyribonucleoside triphosphate (dNTP) derivative. The scope and limitations of the methodology in terms of different nucleobases, length, sequences, and modifications has been thoroughly studied. The methodology including isolation of the modified ONs was scaled up to nanomolar amounts and the modified ONs were successfully used as primers in primer extension and PCR. Two simple and efficient methods for fluorescent labeling of the PCR products were developed, based either on direct fluorescent labeling of primers or on NEAR synthesis of ethynylated primers, PCR, and final click labeling with fluorescent azides

Flexible Alkyne-Linked Thymidine Phosphoramidites and Triphosphates for Chemical or Polymerase Synthesis and Fast Postsynthetic DNA Functionalization through Copper-Catalyzed Alkyne–Azide 1,3-Dipolar Cycloaddition

Two alternative flexible alkyne-linked thymine nucleosides (propargyl-diethylene glycol- or undecyn-linked 5-hydroxymethyluracil derivatives), as well as their phosphoramidites and triphosphates, were designed and synthesized. The nucleoside 3′-<i>O</i>-phosphoramidites were successfully incorporated into oligonucleotides on a solid support, whereas the nucleoside triphosphates served as good substrates for polymerase synthesis of modified DNA, which underwent fast and efficient copper-catalyzed alkyne–azide 1,3-dipolar cycloaddition (CuAAC) reactions

Bodipy-Labeled Nucleoside Triphosphates for Polymerase Synthesis of Fluorescent DNA

New fluorescent nucleosides and nucleoside triphosphate (dNTPs) analogs bearing the F-Bodipy fluorophore linked through a short, flexible nonconjugate tether were synthesized. The Bodipy-labeled dNTPs were substrates for several DNA polymerases which incorporated them into DNA in primer extension, nicking enzyme amplification reaction, and polymerase chain reaction. The fluorescence of F-Bodipy is not quenched upon incorporation in DNA and can be detected both in solutions and on gels

Nucleotides Bearing Red Viscosity-Sensitive Dimethoxy-Bodipy Fluorophore for Enzymatic Incorporation and DNA Labeling

Nucleosides and 2′-deoxyribonucleoside triphosphates (dNTPs) bearing 3,3′-dimethoxy-2,2′-diphenyl-6-(4-hydroxyphenyl)-bodipy fluorophore attached through a propargyl or propargyl-triethylene glycol linker to position 5 of 2′-deoxycytidine were designed and synthesized. They exerted bright red fluorescence and good sensitivity to viscosity changing their lifetime from 1.6 to 4.5 ns. The modifed dNTPs were substrates for DNA polymerases and were used in enzymatic synthesis of labeled DNA through primer extension. The modified DNA probes served as viscosity sensors responding to protein binding by changes of lifetime. The nucleotide with longer linker (dCpegMOBTP) was transported to live cells and incorporated into the genomic DNA, which can be useful for staining of DNA and imaging of DNA synthesis

C–H Imidation of 7‑Deazapurines

We developed and presented here a ferrocene-catalyzed C–H imidation of 7-deazapurines (pyrrolo­[2,3-<i>d</i>]­pyrimidines) with <i>N</i>-imidyl peroxyesters. The reactions occur regioselectively at position 8 in 7-deazapurines, leading to a series of 8-succinimido-, phtalimido-, or naphthalimido-7-deazapurine derivatives. Attempted hydrazinolysis of resulting 8-imidyl-7-deazapurines led to corresponding 8-amino-7-deazapurine, which was very unstable and quickly decomposed

C–H Imidation of 7‑Deazapurines

We developed and presented here a ferrocene-catalyzed C–H imidation of 7-deazapurines (pyrrolo­[2,3-<i>d</i>]­pyrimidines) with <i>N</i>-imidyl peroxyesters. The reactions occur regioselectively at position 8 in 7-deazapurines, leading to a series of 8-succinimido-, phtalimido-, or naphthalimido-7-deazapurine derivatives. Attempted hydrazinolysis of resulting 8-imidyl-7-deazapurines led to corresponding 8-amino-7-deazapurine, which was very unstable and quickly decomposed

C–H Phosphonation of Pyrrolopyrimidines: Synthesis of Substituted 7- and 9‑Deazapurine-8-phosphonate Derivatives

The Mn­(OAc)₃-promoted C–H phosphonation of 7-deazapurines (pyrrolo­[2,3-<i>d</i>]­pyrimidines) and 9-deazapurines (pyrrolo­[3,2-<i>d</i>]­pyrimidines) with diethylphosphite was developed. The reactions occur regioselectively at position 8 both in 7 and 9-deazapurines, leading to new deazapurine-8-phosphonate derivatives, which can be further modified and transformed to 6-(het)­aryl-deazapurine derivatives or deprotected to free phosphonic acids

Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins

Nucleotides, 2′-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7-deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53)

5‑Substituted Pyrimidine and 7‑Substituted 7‑Deazapurine dNTPs as Substrates for DNA Polymerases in Competitive Primer Extension in the Presence of Natural dNTPs

A complete series of 5-substituted uracil or cytosine, as well as 7-substituted 7-deazaadenine and 7-deazaguanine 2′-deoxyribonucleoside triphosphates (dNTPs) bearing substituents of increasing bulkiness (H, Me, vinyl, ethynyl, and phenyl) were systematically studied in competitive primer extension in the presence of their natural counterparts (nonmodified dNTPs), and their kinetic data were determined. The results show that modified dNTPs bearing π-electron-containing substituents (vinyl, ethynyl, Ph) are typically excellent substrates for DNA polymerases comparable to or better than natural dNTPs. The kinetic studies revealed that these modified dNTPs have higher affinity to the active site of the enzyme–primer–template complex, and the calculations (semiempirical quantum mechanical scoring function) suggest that it is due to the cation−π interaction of the modified dNTP with Arg629 in the active site of Bst DNA polymerase