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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.

Pasternak A, Kierzek E, Pasternak K, Turner DH, Kierzek R - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-714 Poznan, Noskowskiego 12/14, Poland.

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.

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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.
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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.

Mentions: The derivative of LNA-2,6-diaminopurine was synthesized with an approach similar to that described for synthesis of natural LNA nucleosides (15,28,29) (Figure 1). The derivative of pentafuranose (1) (33) was condensed with trimethylsilylated 2,6-diaminopurine in 1,2-dichloroethane in the presence of trimethylsilyl trifluoromethanesulfonate as catalyst (34). Treatment of derivative (2) with lithium hydroxide resulted in the 5′-O-methanesulfonyl derivative (3), which was converted with lithium benzoate into the 5′-O-benzoyl derivative (4). The application of lithium benzoate instead of sodium benzoate very significantly improved solubility of the benzoate salt in N,N-dimethylformamide. Treatment of 5′-O-benzoyl derivative (4) with aqueous ammonia resulted in formation of (5). Removal of the 3′-O-benzyl with ammonium formate in the presence of Pd/C (35) resulted in formation of LNA-2,6-diaminopurine riboside (6). Derivative (6) was treated with acetyl chloride to produce (7), which was converted into LNA-N2,N6-diacetyl-2,6-diaminopurine riboside (8), using classical Khorana's procedure (36), and later into the 5′-O-dimethoxytrityl derivative (9). The overall yield of synthesis up to this step was 18%. In reaction of LNA-5′-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside (9) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite was converted into LNA-5′-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside-3′-O-phosphoramidite (10) in 93% yield. It was possible to use acetyl instead of isobutyryl to protect the 2,6-amino groups of LNA-2,6-diaminopurine riboside because LNA-5′-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside-3′-O-phosphoramidite (10) is soluble in acetonitrile. This is in contrast to 5′-O-dimethoxytrityl-2′-O-methyl-N2,N6-diacetyl-2,6-diaminopurine riboside-3′-O-phosphoramidite. The details concerning chemical synthesis of derivatives (2–10) are described in Supplementary Data.Figure 1.


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.

Pasternak A, Kierzek E, Pasternak K, Turner DH, Kierzek R - Nucleic Acids Res. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919511&req=5

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.
Mentions: The derivative of LNA-2,6-diaminopurine was synthesized with an approach similar to that described for synthesis of natural LNA nucleosides (15,28,29) (Figure 1). The derivative of pentafuranose (1) (33) was condensed with trimethylsilylated 2,6-diaminopurine in 1,2-dichloroethane in the presence of trimethylsilyl trifluoromethanesulfonate as catalyst (34). Treatment of derivative (2) with lithium hydroxide resulted in the 5′-O-methanesulfonyl derivative (3), which was converted with lithium benzoate into the 5′-O-benzoyl derivative (4). The application of lithium benzoate instead of sodium benzoate very significantly improved solubility of the benzoate salt in N,N-dimethylformamide. Treatment of 5′-O-benzoyl derivative (4) with aqueous ammonia resulted in formation of (5). Removal of the 3′-O-benzyl with ammonium formate in the presence of Pd/C (35) resulted in formation of LNA-2,6-diaminopurine riboside (6). Derivative (6) was treated with acetyl chloride to produce (7), which was converted into LNA-N2,N6-diacetyl-2,6-diaminopurine riboside (8), using classical Khorana's procedure (36), and later into the 5′-O-dimethoxytrityl derivative (9). The overall yield of synthesis up to this step was 18%. In reaction of LNA-5′-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside (9) with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite was converted into LNA-5′-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside-3′-O-phosphoramidite (10) in 93% yield. It was possible to use acetyl instead of isobutyryl to protect the 2,6-amino groups of LNA-2,6-diaminopurine riboside because LNA-5′-O-dimethoxytrityl-N2,N6-diacetyl-2,6-diaminopurine riboside-3′-O-phosphoramidite (10) is soluble in acetonitrile. This is in contrast to 5′-O-dimethoxytrityl-2′-O-methyl-N2,N6-diacetyl-2,6-diaminopurine riboside-3′-O-phosphoramidite. The details concerning chemical synthesis of derivatives (2–10) are described in Supplementary Data.Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioorganic Chemistry, Polish Academy of Sciences, 60-714 Poznan, Noskowskiego 12/14, Poland.

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.

Show MeSH
Related in: MedlinePlus