The p-nitrophenylethyl (NPE) group

Himmelsbach, Frank; Schulz, B.; Trichtinger, Thomas; Charubala, Ramamurthy; Pfleiderer, Wolfgang

doi:10.1016/0040-4020(84)85103-0

Cited by 221 publications

(123 citation statements)

References 60 publications

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“…RNA Gel Blot Analysis with Oligonucleotides Containing 5-Fluorodeoxyuridine Residues. The calculated td (dissociation temperature of hybrids in solution) for probe 1 to pyruvate carboxylase mRNA was 57.80C (17). DNA melting data yielded a tm of 60TC for probe 1 and 52TC for probe 2.…”

Section: Resultsmentioning

confidence: 99%

“…Because the unprotected 0-4 group of the uracil ring is reactive toward nucleophilic displacement resulting in chain branching during synthesis, we used the p-nitrophenylethyl group to protect the 0-4 atom of 5-fluorodeoxyuridine (16,17 Step 1. 5-Dimethoxytrityl-5-fluorodeoxyuridine: 5-Fluorodeoxyuridine (42 mmol, 9.74 g) was suspended in 100 ml of dry pyridine in a 200-ml round-bottom flask attached to a drying tube.…”

Section: Methodsmentioning

confidence: 99%

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5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.

Habener

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic complementary oligonucleotides are useful hybridization probes for the detection of mRNAs and genes encoding proteins for which only a partial amino acid sequence is known. Usually this involves the synthesis of mixtures of oligonucleotides complementary to all possible bases in degenerate positions of codons. As an alternative we have prepared and characterized a series of unique oligonucleotides containing a pyrimidine analog, 5-fluorodeoxyuridine (F). Thermodynamic parameters and the melting temperatures of hybrid duplexes containing A-F and G-F base pairs showed that they are considerably more stable than duplexes containing APT and GOT base pairs. The stability of a duplex decreased linearly with the number ofmismatches introduced at positions at least a codon apart. A 5-fluorodeoxyuridine-substituted oligonucleotide cDNA detects rat liver pyruvate carboxylase mRNA on a RNA gel blot with a dissociation temperature only 10'C below the measured melting temperature in solution. We suggest that the complexity of oligonudeotide cDNAs used for screening gene libraries can be reduced by the design of single hybridization probes containing the substituted bases-5-fluorodeoxyuridine to pair with adenosine or guanosine, guanosmne to pair with cytidine or thymidine, and deoxyinosine to pair with adenosine or cytidine at positions of codon degeneracy-and still retain near-maximum stability of hybrid duplexes.Recombinant DNA technology allows the isolation of genes encoding specific proteins. When the partial amino acid sequence of the protein is known, an approach to isolation of the genes is the synthesis of mixtures of 8-64 oligonucleotides of 14-17 bases that are complementary in sequence to all possible codons predicted for the corresponding amino acid sequence (1-3)J A difficulty in these syntheses, however, is that coupling efficiencies of the nucleotides at each step are not uniform resulting in unequal representations of the oligonucleotides in the mixture; certain of the sequences may be practically absent (4). In general the successful detection of the correct sequence has been possible only when it is present in the screened library at a relatively high abundance (>0.1-1% of sequences). A complex mixture of oligonucleotide probes may lead to spurious hybridization to incorrect sequences, particularly in attempts to identify a correct single-copy sequence in mammalian gene libraries with a complexity approaching 109 base pair.To circumvent these problems, base analogs have been designed that can potentially base pair with any of the four natural bases (adenosine, thymidine, guanosine, and cytidine) in DNA. For example, deoxyinosine (5), 2-amino-2'-deoxyadenosine (4), and phenyl derivatives (5) have been used to prepare unique probes at positions of base degeneracy, but base pairing is significantly less stable than the normal Watson-Crick base pairing (6). Similarly, deoxyguanosine has been used in positions opposite to cytidine or thymidine ambiguities because the GOT wobble base pair is be...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

5-Fluorodeoxyuridine as an alternative to the synthesis of mixed hybridization probes for the detection of specific gene sequences.

Habener

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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“…Figure 12). [54][55][56] The oligonucleotides are assembled on o-nitrophenylcarbonate derivatized resin (9) and then two-step deprotection procedure is carried out. The cyanoethyl groups of the phosphates are first selectively removed by a mixture of 40% triethylamine in pyridine and then the resins are subjected to a mixture of 0.5 M DBU in pyridine, which cleaves the p-nitrophenylethyl protections and concomitantly releases the target oligonucleotides.…”

Section: Protecting Groups Removable With β-Eliminationmentioning

confidence: 99%

“…Demethylation of the phosphotriesters is accomplished with a treatment of disodium-2-carbamoyl-2-cyanoethylene-1,1,-dithiolate trihydrate (dccdt) (57), the cyanoethyl carbamates are removed by a mixture of triethylamine in DMF (1:1, v/v, 16 h at 55°C) (58) and then the fully deprotected oligonucleotide is released by photolysis. It may be notable that the unsubstituted cyanoethyloxycarbonyl group may be used for the guanine base (54), but the more base labile α,α-dimethylated derivative is required for adenine (53) and cytosine (52). …”

Section: Protecting Groups Removable With β-Eliminationmentioning

confidence: 99%

Solid-phase synthesis of base-sensitive oligonucleotides

Virta¹

2008

Arkivoc

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Oligonucleotides bearing N-acylated and O-alkylated nucleobases, biodegradable phosphate protections, cap-structures, internucleotidic H-phosphonates, methylphosphates, Omethylphosphorothioates and phosphotriesters are sensitive to nucleophiles, nucleopeptides are prone to β-elimination and some of the dihydropyrimidines undergo retro condensation. None of them withstand the standard ammonolytic deprotection, but an alternative synthetic procedure should be adopted. In this review, orthogonal protecting group schemes and the N-unprotected methods for the solid-phase synthesis of these important oligonucleotides are presented.

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“…Thus, 2'-deoxy-N 2 -[2-(4-nitrophenyl)ethoxycarbonyl]guanosine (5) was synthesized by the transient protection method [14] using trimethylsilyl chloride for intermediary blocking of the sugar OH groups, followed by acylation with 2-(4-nitrophenyl)ethyl carbonochloridate ( 2-(4-nitrophenyl)ethyl chloroformate) (cf. the corresponding 3 and 4) [12]. The preparation of 2'-deoxy-O 6 -[2-(4-nitrophenyl)ethyl]-N 2 -[2-(4-nitrophenyl)ethoxycarbonyl]guanosine (7) could be achieved in a one-pot reaction starting from 3', 5'-di-O-acetyl-2'-deoxyguanosine [15] which was first subject to a Mitsunobu reaction [16] leading, under O -butyl derivative 8, 10, 12, and 14, respectively, of which 10 and 12 were isolated as intermediates before hydrolysis to 11 and 13, whereas 8 and 14 were converted without isolation into 2'-deoxy-N 2 -isobutyryl-O 6 -methylguanosine (9) and the corresponding O 6 -butyl derivative 15.…”

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confidence: 99%