Nonionic nucleic acid analogs. Synthesis and characterization of dideoxyribonucleoside methylphosphonates

Abstract

A series of dideoxyribonucleoside methylphosphonate analogues, dNpN and dNpNp, which contain a nonionic 3'--5' methylphosphonyl internucleoside linkage were prepared. The two diastereoisomers, designated isomers 1 and 2, of each dimer differ in configuration of the methylphosphonate group and were separated by column chromatography. The diastereoisomers of each dimer have different conformations in solution as shown by ultraviolet hypochromicity data and their circular dichroism spectra. For example, dApA isomer 1 is more highly stacked than isomer 2, although both isomers are less stacked than the dinucleoside monophosphate, dApA. The circular dichroism spectrum of isomer 1 is very similar to that of dApA, while the CD spectrum of isomer 2 shows a loss of molecular ellipticity, [theta], at 270 nm and a greatly diminished [theta] at 250 nm. These results suggest that the stacked bases of dApA isomer 1 tend to orient in an oblique manner, while those in isomer 2 tend to orient in a parallel manner. This interpretation is verified by the 1H NMR study of these dimers (L. S. Kan, D. M. Cheng, P. S. Miller, J. Yano, and P. O. P. Ts'o, unpublished experiments). Both diastereoisomers of dAaA form 2U:1A and 2T:1A complexes with poly(U) and poly(dT), respectively. The higher Tm (Tm of poly(U)--isomer 1, 15.4 degrees C; Tm of poly(U)--isomer 2, 19.8 degrees C; Tm of poly(dT)--isomer 1, 18.7 degrees C; Tm of poly(dT)--isomer 2, 18.4 degrees C) values of these complexes vs. those of the corresponding dApA--polynucleotide complexes (Tm of poly(U)--dApA, 7.0 degrees C; Tm of poly(dT)--DApA, 9.2 degrees C) result from decreased charge repulsion between the nonionic dimer backbone and the negatively charged polymer backbone. The difference in conformations between dApA isomer 1 and dApA isomer 2 is reflected in the Tm of the isomer 1-poly(U) complex which is 4.4 degrees C lower than that of the isomer 2-poly(U) complex. Since these nonionic oligonucleotide analogues are taken up by cells in culture, they show promise as molecular probes for the function and structure of nucleic acids inside living cells.

Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure

“…Preparation of oligonucleotides Oligonucleotides (1)(2)(3)(4)(5) and the dT3N2T3 (N = dA, dC, dG or T) model sequences described in the text were prepared on a 15 ,umole scale using a Biosearch 8750 DNA synthesizer as described elsewhere. 19 (5 mg), followed by 1 ml of EDA.…”

“…NH40H for two hours followed by EDA (hereafter referred to as the two step method), yielding clean material with no polar modifications (data not shown). The Deprotection of oligonucleotides using the improved procedure A series of methylphosphonate oligonucleotides (1-5) were synthesized on a 15 Amole scale (Table 1). An aliquot of each (1 zmole) was subjected to either the two step method, or the new one-pot procedure described above.…”

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

“…Methylphosphonate (MP) oligonucleotides act RNase Hindependent and have been investigated intensively, since they show reduced nonspecific mRNA interactions and offer high stability in cellular environment [13]. The 2′-position of the sugar moiety can also serve as a modification site.…”

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2′-O-methylribonucleosides

“…In addition to the expected nucleosides, a peak whose mobility corresponded to one of the diastereoisomers of the diimer, d-ApT, was also observed. The dimer obtained from the first peak corresponded to authentic Rp-d-ApT, whereas the dimer obtained from the second peak corresponded to authentic Sp-d-ApT (25)(26)(27). The retention times of these two diastereomers differed by 0.22 min on the C-18 column using a linear gradient of 2-3 % acetonitrile for the first 12 min of elution followed by a linear gradient of 3-30% acetonitrile for the next 8 min.…”