Synthesis of deoxynucleoside methylphosphonates via a phosphonamidite approach

Jäger, Alfred; Engels, Joachim W.

doi:10.1016/s0040-4039(01)80180-5

Cited by 71 publications

(38 citation statements)

References 8 publications

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“…Synthesis and Characterization of the Analogue Operator Sequences. A single racemic methylphosphonate linkage can be introduced into a DNA sequence (Botfield & Weiss, 1994) using a solid-phase-based protocol (Miller et al, 1991) and the methylphosphonamidite nucleoside building block (Dorman et al, 1984;Jager & Engels, 1984;Agrawal & Goodchild, 1987). However, the resulting diastereomeric mixture can only be resolved in the case of relatively short sequences.…”

Section: Resultsmentioning

confidence: 99%

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Smith

McLaughlin

1997

Biochemistry

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Fourteen analogue DNA sequences containing the trp operator sequence and a single diastereomeric methylphosphonate linkage are each prepared from the stereochemically pure nucleoside methylphosphonate dimer building block, prepared as a phosphoramidite. The analogue sequences are shown to be single diastereomers on the basis of HPLC analysis of the digestion mixture; in each case, only a single diastereomeric dimer is present. These analogue sequences can be used effectively to probe for interactions to either of the prochiral phosphate oxygens as illustrated by their use to identify critical interactions in the trp repressor-operator complex. In a number of cases, the pairs of diastereomeric analogue sequences exhibit variable binding affinities that can be used to identify one of the prochiral phosphate oxygens as a critical site for complex-stabilizing interactions. Upon the basis of dissociation constants, apparent incremental binding energies are assigned to specific interactions. In all but one example, these identified sites for interactions to the phosphate backbone can be correlated with contacts implicated by the crystal structure analysis of the trp repressor-operator complex.

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Section: Resultsmentioning

confidence: 99%

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Smith

McLaughlin

1997

Biochemistry

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“…Synthesis of Oligodeoxyribonucleoside Methylphosphonates. The methylphosphonate oligomers were synthesized on a 1% cross-linked polystyrene support using 5'-(dimethoxytrityl)nucleoside 3'-(methylphosphonic imidazolide) intermediates (Miller et al, 1986) or on a controlled-pore glass support using 5/-(dimethoxytrityl)nucleoside 3,-[[(iV,7V-diisopropylamino)methyl]phosphonamidite] intermediates (Dorman et al, 1984;Jager & Engels, 1984;Marcus-Sekura et al, 1987) which were purchased from American Bionetics Inc. To suppress formation of 4-(2-aminoethyl)cytosine during the deblocking procedure (Miller et al, 1986), the support was treated overnight with a solution containing 1.2 mL of pyridine, 0.3 mL of glacial acetic acid, and 0.05 mL of 85% hydrazine hydrate to remove benzoyl groups from cytosine and adenine residues of the protected oligomer (Letsinger et al, 1968). The partially deprotected oligomer was then further deprotected and cleaved from its support by sequential treatment with 50% ethylenediamine in ethanol and with 80% acetic acid.…”

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confidence: 99%

Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

Lee

Murakami

Blake³

et al. 1988

Biochemistry

118

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Oligodeoxyribonucleoside methylphosphonates derivatized at the 5' end with 4'-(amino-alkyl)-4,5',8-trimethylpsoralen were prepared. The interaction of these psoralen-derivatized methylphosphonate oligomers with synthetic single-stranded DNAs 35 nucleotides in length was studied. Irradiation of a solution containing the 35-mer and its complementary methylphosphonate oligomer at 365 nm gave a cross-linked duplex produced by cycloaddition between the psoralen pyrone ring of the derivatized methylphosphonate oligomer and a thymine base of the DNA. Photoadduct formation could be reversed by irradiation at 254 nm. The rate and extent of cross-linking were dependent upon the length of the aminoalkyl linker between the trimethylpsoralen group and the 5' end of the methylphosphonate oligomer. Methylphosphonate oligomers derivatized with 4'-[[N-(2-aminoethyl)amino]methyl]- 4,5',8-trimethylpsoralen gave between 70% and 85% cross-linked product when irradiated for 20 min at 4 degrees C. Further irradiation did not increase cross-linking, and preirradiation of the psoralen-derivatized methylphosphonate oligomer at 365 nm reduced or prevented cross-linking. These results suggest that the methylphosphonate oligomers undergo both cross-linking and deactivation reactions when irradiated at 365 nm. The extent of cross-linking increased up to 10 microM oligomer concentration and dramatically decreased at temperatures above the estimated Tm of the methylphosphonate oligomer-DNA duplex. The cross-linking reaction was dependent upon the fidelity of base-pairing interactions between the methylphosphonate oligomers and the single-stranded DNA. Noncomplementary oligomers did not cross-link, and the extent of cross-linking of oligomers containing varying numbers of noncomplementary bases was greatly diminished or eliminated.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…with the 5'-hydroxyl group of an oligonucleotide followed by oxidation (the last method is suitable for a solid phase synthesis) result in a mixture of P-diastereoisomers [37][38][39][40]. Although the latter monomers can be separated into P-diastereomers, they are known to undergo a rapid epimerisation in the presence of tetrazole [41].…”

Section: Methylphosphonatesmentioning

confidence: 99%

P-Chiral Oligonucleotides in Biological Recognition Processes

Guga

2007

CTMC

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Internucleotide phosphodiester linkages in non-modified oligonucleotides are quickly degraded by nucleolytic enzymes present in the cells and this feature practically eliminates natural DNA and RNA molecules from medical applications and from many structural and mechanistic studies. P-chiral oligonucleotide analogs, in which one of the non-bridging phosphate oxygen atoms is substituted with another heteroatom (e.g. S, Se) or a chemical group (e.g. CH3, BH3(-)), have significantly greater nuclease resistance and also offer important possibilities for detailed studies of interactions with other biomolecules at the molecular level. Notably, these substitutions do not disrupt hydrogen bonding between nucleobases and affect the overall geometry of the oligomers to only low or moderate extent, although important changes of hydration patterns and changes of interactions with metal ions are observed. Such the probes, including isotopomeric species labeled with a heavy oxygen isotope, possessing phosphorus atoms of selected absolute configurations, have been used for elucidation of the mode of action of many enzymes (nucleases, transferases, kinases), ribozymes and DNA-zymes, as well as for investigations on thermodynamic stability of nucleic acids complexes (duplexes, triplexes, i-motif) and for studies on a mechanism of conformational changes of B-Z type. They are also useful tools for analysis of interactions of the phosphoryl oxygen atoms in natural precursors with functional groups of proteins. The synthetic routes to stereodefined forms of selected types of P-chiral oligonucleotides are presented, as well as recently developed methods for their configurational analysis at micromolar concentration. Selected examples of application of diastereomerically pure P-chiral oligonucleotides for structural, biochemical and biological experiments are discussed.

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Synthesis of deoxynucleoside methylphosphonates via a phosphonamidite approach

Cited by 71 publications

References 8 publications

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Probing Contacts to the DNA Backbone in the trp Repressor−Operator Sequence-Specific Protein−Nucleic Acid Complex Using Diastereomeric Methylphosphonate Analogues

Interaction of psoralen-derivatized oligodeoxyribonucleoside methylphosphonates with single-stranded DNA

P-Chiral Oligonucleotides in Biological Recognition Processes

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