A chemical synthesis of LNA-2,6-diaminopurine riboside, and the influence of 2'-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

Abstract

Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids. Derivatives of 2,6-diaminopurine riboside (D) are one type of modified nucleotide. The presence of an additional amino group at position 2 relative to adenine results in formation of a third hydrogen bond when interacting with uridine. New method for chemical synthesis of protected 3'-O-phosphoramidite of LNA-2,6-diaminopurine riboside is described. The derivatives of 2'-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides were used to prepare complete 2'-O-methyl RNA and LNA-2'-O-methyl RNA chimeric oligonucleotides to pair with RNA oligonucleotides. Thermodynamic stabilities of these duplexes demonstrated that replacement of a single internal 2'-O-methyladenosine with 2'-O-methyl-2,6-diaminopurine riboside (D(M)) or LNA-2,6-diaminopurine riboside (D(L)) increases the thermodynamic stability (DeltaDeltaG degrees 37) on average by 0.9 and 2.3 kcal/mol, respectively. Moreover, the results fit a nearest neighbor model for predicting duplex stability at 37 degrees C. D-A and D-G but not D-C mismatches formed by D(M) or D(L) generally destabilize 2'-O-methyl RNA/RNA and LNA-2'-O-methyl RNA/RNA duplexes relative to the same type of mismatches formed by 2'-O-methyladenosine and LNA-adenosine, respectively. The enhanced thermodynamic stability of fully complementary duplexes and decreased thermodynamic stability of some mismatched duplexes are useful for many RNA studies, including those involving microarrays.

Figure 1.

Synthesis of LNA-2,6-diaminopurine phosphoramidite. Reagents and conditions: (i) 2,6-diaminopurine, HMDS, TMSOTf, dichloroethane; (ii) LiOH·H₂O, THF, H₂O; (iii) BzOLi, DMF; (iv) conc. NH₄OH, Py; (v) Pd/C, HCOONH₄, MeOH; (vi) AcCl, Py; (vii) KOH, Py, H₂O, EtOH; (viii) DMTrCl, Py; (ix) 4,5-DCI, NC(CH₂)₂OP[N(iPr)₂]₂, CH₃CN.