A versatile Solid Phase Method For the Synthesis of Masked 3′-Thiol Group Containing Oligonucleotides

Kumar, A

doi:10.1080/07328319308016203

Cited by 5 publications

(9 citation statements)

References 25 publications

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“…Synthesis of the Aleuritic Acid CPG. In recent years, the chemical literature has reported numerous methods for synthesizing different phosphoramidite reagents used to introduce single or multiple functional groups at the 3′ or 5′ terminus of a synthesized DNA oligonucleotide (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). In contrast, far fewer methods are reported for utilizing a specifically modified solid support to synthesize 3′-modified oligonucleotides (13,(71)(72)(73)(74)(75)(76)(77)(78)(79)(80)(81)(82), most likely due to the synthetic inconveniences associated with the preparation of such reagents.…”

Section: Resultsmentioning

confidence: 99%

“…The discovery of the phosphoramidite method, which enables automated synthesis of natural and modified DNA molecules (1)(2)(3)(4), has stimulated the development of numerous reagents and methods to introduce a specific modification or functional group at a selected position within a synthesized oligonucleotide (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Some phosphoramidite labeling reagents are commercially available.…”

Section: Introductionmentioning

confidence: 99%

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New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes

Skrzypczynski

Wayland

2003

Bioconjugate Chem.

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An efficient and versatile preparative approach is described, allowing for the preparation of DNA probes modified with an aldehyde group at the 3'- or 5'-end. The developed synthetic strategy allows for the preparation of a new family of phosphoramidites and solid supports compatible with the automated synthesis of modified oligonucleotide probes. These new reagents were prepared from intermediates 3 and 3a, obtained from the commercially available aleuritic acid 1. It was demonstrated that the new phosphoramidite reagents also could be used as new types of cleavable linkers. A new and efficient method for the production of 5' aldehyde-labeled DNA probes was developed.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes

Skrzypczynski

Wayland

2003

Bioconjugate Chem.

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“…Functionalizing the 3′-terminus of synthetic ODNs by this technique requires the preparation of four different solid supports, each individually derivatized with a disulfide-linked nucleotide (i.e., deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine). A simpler method of functionalizing synthetic ODNs involves the derivatization of CPG with mercaptohexyl derivatives reported by Kumar et al (24,25). Another method of synthesizing ODN-peptide hybrids whereby the peptide was ligated to 3′-end of ODNs was by the templatedirected two-step reaction involving site-specific ligation of amine-terminated ODN to ODN-peptide linked by thioester linkage (27).…”

Section: Discussionmentioning

confidence: 99%

“…The DNTP-activated support is then reacted with O-DMT-6-mercaptohexanol, giving rise to 3′-thiolated ODNs that are subsequently used to synthesize DNA. The reactivity of the 3′-thiol-derivatized ODN was demonstrated by its efficient conjugation to pyrenyl maleimide (24,25). The method of native chemical ligation by chemoselective coupling of two protected peptides has been described (26).…”

Section: Introductionmentioning

confidence: 99%

Use of N^α-Fmoc-cysteine(S-thiobutyl) Derivatized Oligodeoxynucleotides for the Preparation of Oligodeoxynucleotide−Peptide Hybrid Molecules

1998

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The chemical modification of antisense oligodeoxynucleotides (ODNs) by conjugating synthetic peptides of known membranotropic activities to the 3' and/or 5' terminus of ODNs may serve two functions that are important for increasing their bioavailability by protecting the ODNs from exonuclease digestion and facilitated delivery into cells. We have previously reported the preparation of ODN-peptide conjugates by the total synthesis approach. However, by such technology the preparation of ODN-peptide conjugates in amounts sufficient for in vitro functional analysis is at present limited to the syntheses of peptides containing residues without acidolytic deprotection. Requisite to the alternative method of site-specific conjugation, the segment coupling approach is the derivatization of an ODN with a nucleophilic moiety. In this paper, we describe a novel method of functionalizing synthetic ODNs by incorporating S-thiobutyl-protected Nalpha-Fmoc-cysteine to aminopropyl-functionalized CPG by standard Nalpha-Fmoc SPPS methodology. The derivatized solid support can be used to synthesize an ODN of any sequence by the phosphoramidite chemistry, and the removal of the S-thiobutyl side chain function can be conveniently affected by the standard amminolytic deprotection of ODNs containing 1 M DTT. The purified cysteine-derivatized ODN was shown to react specifically and efficiently with two types of synthetic peptides corresponding to regions within the glycoprotein (gp) of HIV that have been shown to have membranotropic activities: a 18 residue maleimide-derivatized Tat peptide of the transactivator (tat) of HIV and a 22 residue peptide corresponding to the carboxyl terminus of gp41(Ca-gp41).

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“…The use of various modified phosphoramidites as masked synthons for preparing derivatized oligonucleotides at both terminal and internal sites has been reviewed (Beaucage and Iyer, 1993;Goodchild, 1990). These masked synthons allow incorporation of protected functional groups such as amines (Cruickshank and Stockwell, 1988;Levina et al, 1993;Telser et al, 1989), carboxylic acids (Wang and Bergstrom, 1993), thiols (Bradley and Hanna, 1992;Goodwin and Glick, 1993;Kuijpers and van Boeckel, 1993;Kumar, 1993b), and thiocarbonyls (Coleman and Kesicki, 1994;Waters and Connolly, 1992;Xu et al, 1995). Once incorporated, the analogues are deprotected and modified postsyntheti-cally.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Characterization of a New 5-Thiol-Protected Deoxyuridine Phosphoramidite for Site-Specific Modification of DNA

Meyer

Hanna

1996

Bioconjugate Chem.

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A new nucleotide analogue was developed for site-specific incorporation of a reactive thiol group into DNA. This creates a unique site for the post-synthetic modification of that nucleotide with a variety of molecular tags, such as photo-cross-linkers and fluorescent or spin-label moieties. 5'-O-(4,4'-Dimethoxytrityl)-5-[S-(2,4-dinitrophenyl)thio]-2'-deoxyuridin e 3'-O-(2-cyanoethyl N,N'-diisopropylphosphoramidite) was synthesized and incorporated at internal positions in several oligonucleotides using automated DNA synthesis and standard phosphoramidite chemistry. The coupling yield of the analogue was comparable to the coupling yield for a standard phosphoramidite, and no significant differences were observed in the overall yields of the dinitrophenyl-labeled oligonucleotides compared to the corresponding unmodified oligonucleotides. Characterization of the dinitrophenyl-modified oligonucleotides included enzymatic degradation, HPLC chromatography, and gel electrophoresis. Deprotection of the mercaptan group with beta-mercaptoethanol yielded an oligonucleotide containing 5-mercaptodeoxyuridine which was then selectively modified, without purification, by reaction with 5-(iodoacetamido)fluorescein. Incorporation of the dinitrophenyl-modified oligonucleotide into double-stranded DNA was achieved using the polymerase chain reaction. CHaracterization of the dinitrophenyl-labeled product by immunodetection with anti-dinitrophenyl antibodies confirmed the stability of the protecting group to the thermocycling and thus established the use of this thiol-protected mercaptodeoxyuridine phosphoramidite for preparation of site-specifically modified DNA.

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A versatile Solid Phase Method For the Synthesis of Masked 3′-Thiol Group Containing Oligonucleotides

Cited by 5 publications

References 25 publications

New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes

New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes

Use of N^α-Fmoc-cysteine(S-thiobutyl) Derivatized Oligodeoxynucleotides for the Preparation of Oligodeoxynucleotide−Peptide Hybrid Molecules

Synthesis and Characterization of a New 5-Thiol-Protected Deoxyuridine Phosphoramidite for Site-Specific Modification of DNA

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A versatile Solid Phase Method For the Synthesis of Masked 3′-Thiol Group Containing Oligonucleotides

Cited by 5 publications

References 25 publications

New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes

New Reagents for the Introduction of Reactive Functional Groups into Chemically Synthesized DNA Probes

Use of Nα-Fmoc-cysteine(S-thiobutyl) Derivatized Oligodeoxynucleotides for the Preparation of Oligodeoxynucleotide−Peptide Hybrid Molecules

Synthesis and Characterization of a New 5-Thiol-Protected Deoxyuridine Phosphoramidite for Site-Specific Modification of DNA

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Use of N^α-Fmoc-cysteine(S-thiobutyl) Derivatized Oligodeoxynucleotides for the Preparation of Oligodeoxynucleotide−Peptide Hybrid Molecules