Relative reactivity of ribosyl 2 '-OH vs. 3 '-OH in concentrated aqueous solutions of phosphoimidazolide activated nucleotides

Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are u seful substrates for the non-enzymatic synthesis of oligonucleotides. In th e presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN(2)'pN and pN(3)'pN) and the pyrophosphate-linked (N-5'ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2'-OH vs. 3'-OH was determined from the ratio of the yields of pN(2)'pN vs. pN(3)'pN. Experime nts were performed at 23 degrees C in the range 7.2 less than or equal to p H less than or equal to 8.4 with substrates that differ in nucleobase (guan osine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4- diMeIm)). Two metal ions (Mg2+ or Mn2+) were employed as catalysts. The con ditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-sta cking interactions. The ratio pN(2)'pN: pN(3)'pN = 2'-5': 3'-5' was found i ndependent of nucleobase and typically varied between 2 to 3 indicating tha t the 2'-OH is about 2 to 3 times more reactive than the 3'-OH. *pN with Im , compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN(2)'pN: pN(3)'pN ratio. Trend s in the data, observed with all three leaving groups, suggest an increase in pN(2)'pN: pN(3)'pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studie s reporting a relative reactivity 2'-5': 3'-5' = 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions tha t disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stac king that enhances selectively the proximity of the 3'-OH of one molecule t o the reactive P-N bond of an other molecule. The implication of these conc lusions for oligomerization/ligation reactions is discussed.