The mechanism of the chemical synthesis of oligonucleotides and its synthetic consequences

Abstract

The data obtained mainly by pulsed NMR spectroscopy on phosphorus nuclei on the mechanism of the internucleotide phosphodiester (PDE) group formation are summarised. With arylsulphonyl chloride as condensing reagent monomeric nucleotide derivative B (nucleoside metaphosphate or its pyridinium adduct) is the highly reactive intermediate. In the presence of PDE groups in nucleoside or nucleotide component the significantly less reactive derivatives with trisubstituted pyrophosphoryl residues are formed both with arylsulphonyl chloride and dicyclohexylcarbodiimide (DCC). The reactive B form of nucleotide component may be obtained using greater excess of arylsulphonyl chloride with simultaneous convertion of PDE groups to tetrasubstituted pyrophosphates amenable to side reactions. The convertion of PDE groups to easily hydrolysable dicyclohexylurea derivatives by reaction with DCC is proposed to reversible blocking of PDE groups of nucleoside component. The B type derivatives of mononucleotides or oligonucleotides with blocked PDE groups seems to be the best nucleotide components.

Rapid synthesis of oligodeoxyribonucleotides IV. Improved solid phase synthesis of oligodeoxy-ribonucleotides through phosphotriester intermediates

“…10 ml of solvent was used per wash per 0.35 g resin. Resin was swollen in IIMF and treated with (1) 5 x t-amyl alcohol, 2 min, (2) 5 x acetic acid, 2 min, (3) 2 x 1.5 M hydrogen chloride in acetic acid, 5 + 25 min, (4) 5 x acetic acid, 2 min, (5) 10 x F, 2 min, (6) 3 x 10% diisopropylethylamine/ ITF, 2 min, (7) 5 x IMF, 2 min, (8) 1 x 2 equivalents of the symmetrical anhydride of the succinate derivative (ir 1822 cm 1, C=0 stretch; formed from 4 equivalents of the succinate by the same procedure as for Boc gly), 120 min, (9) 5 x DMF, 2 min. A sample of resin was washed with 5 x dichloromethane, 2 min, and then 5 x diethylether, 2 min, and dried in vacuo.…”

“…An orange colour is immediately liberated into solution on addition of the acid.3 For oligonucleotides containing adenine steps 3 and 4 are repeated (usually once) until no further orange colour is seen. A sample of dried resin can also be assayed for trityl content 4. The nucleoside or oligonucleotide 3'-phosphate (5 equivs) is dried by coevaporation three times with anhydrous pyridine and in a dry box triisopropylbenzene sulphonyltetrazole (8.75-10 equivs) added.…”

Affinity sorbents containing nucleic acids and their fragments

“…The substituted O-phosphorylisoureas 11 can also be transformed into side products, e.g., be hydrolysed with the liberation of the original phosphate group and urea or undergo consecutive conversions leading to pyrophosphates, polyphosphates and O-phosphorylisourea derivatives. 75 The latter, in turn, produce non-reactive N-pyrophosphorylurea derivatives 13 75,76 or N-phosphorylurea derivatives 14. 66, 77 ± 79 In the activation of oligonucleotides by carbodiimides resulting in compounds 11 ± 14, modification of internucleotide phosphate groups 76,79 and heterocyclic bases 69,80 is observed.…”

“…75 The latter, in turn, produce non-reactive N-pyrophosphorylurea derivatives 13 75,76 or N-phosphorylurea derivatives 14. 66, 77 ± 79 In the activation of oligonucleotides by carbodiimides resulting in compounds 11 ± 14, modification of internucleotide phosphate groups 76,79 and heterocyclic bases 69,80 is observed. In the case of oligodeoxyribonucleotides, the reaction with the internucleotide phosphate groups results in the urea derivatives 15, 76 whereas the reaction with oligoribonucleotides leads to the isomerisation and cleavage of the internucleotide bonds.…”