Double sugar and phosphate backbone-constrained nucleotides: synthesis, structure, stability, and their incorporation into oligodeoxynucleotides.
Two diastereomerically pure carba-LNA dioxaphosphorinane nucleotides [(S(p))- or (R(p))-D(2)-CNA], simultaneously conformationally locked at the sugar and the phosphate backbone, have been designed and synthesized. Structural studies by NMR as well as by ab initio calculations showed that in (S(p))- and (R(p))-D(2)-CNA the following occur: (i) the sugar is locked in extreme North-type conformation with P = 11 degrees and Phi(m) = 54 degrees ; (ii) the six-membered 1,3,2-dioxaphosphorinane ring adopts a half-chair conformation; (iii) the fixed phosphate backbone delta, epsilon, and zeta torsions were found to be delta [gauch(+)], epsilon (cis), zeta [anticlinal(+)] for (S(p))-D(2)-CNA, and delta [gauche(+)], epsilon (cis), zeta [anticlinal(-)] for (R(p))-D(2)-CNA. It has been found that F(-) ion can catalyze the isomerization of pure (S(p))-D(2)-CNA or (R(p))-D(2)-CNA to give an equilibrium mixture (K = 1.94). It turned out that at equilibrium concentration the (S(p))-D(2)-CNA isomer is preferred over the (R(p))-D(2)-CNA isomer by 0.39 kcal/mol. The chemical reactivity of the six-membered dioxaphosphorinane ring in D(2)-CNA was found to be dependent on the internucleotidic phosphate stereochemistry. Thus, both (S(p))- and (R(p))-D(2)-CNA dimers (17a and 17b) were very labile toward nucleophile attack in concentrated aqueous ammonia [t(1/2) = 12 and 6 min, respectively] to give carba-LNA-6',5'-phosphodiester (21) approximately 70-90%, carba-LNA-3',5'-phosphodiester (22) approximately 10%, and carba-LNA-6',3'-phosphodiester (23) <10%. In contrast, the (S(p))-D(2)-CNA was about 2 times more stable than (R(p))-D(2)-CNA under hydrazine hydrate/pyridine/AcOH (pH = 5.6) [t(1/2) = 178 and 99 h, respectively], which was exploited in the deprotection of pure (S(p))-D(2)-CNA-incorporated antisense oligodeoxynucleotides (AON). Thus, after removal of the solid supports from the (S(p))-D(2)-CNA-modified AONs by BDU/MeCN, they were treated with hydrazine hydrate in pyridine/AcOH to give pure AONs in 35-40% yield, which was unequivocally characterized by MALDI-TOF to show that they have an intact six-membered dioxaphosphorinane ring. The effect of pure (S(p))-D(2)-CNA modification in the AONs was estimated by complexing to the complementary RNA and DNA strands by the thermal denaturation studies. This showed that this cyclic phosphotriester modification destabilizes the AON/DNA and AON/RNA duplex by about -6 to -9 degrees C/modification. Treatment of (S(p))-D(2)-CNA-modified AON with concentrated aqueous ammonia gave carba-LNA-6',5'-phosphodiester modified AON ( approximately 80%) plus a small amount of carba-LNA-3',5'-phosphodiester-modified AON ( approximately 20%). It is noteworthy that Carba-LNA-3',5'-phosphodiester modification stabilized the AON/RNA duplex by +4 degrees C/modification (J. Org. Chem. 2009, 74, 118), whereas carba-LNA-6', 5'-phosphodiester modification destabilizes both AON/RNA and AON/DNA significantly (by -10 to -19 degrees C/modification), which, as shown in our comparative CD studies, that the cyclic phosphotriester modified AONs as well as carba-LNA-6',5'-phosphodiester modified AONs are much more weakly stacked than carba-LNA-3',5'-phosphodiester-modified AONs.
Zhou
Chuanzheng
C
Department of Bioorganic Chemistry, Box 581, ICM, Biomedical Center, Uppsala University, SE-751 23 Uppsala, Sweden.
Plashkevych
Oleksandr
O
Chattopadhyaya
Jyoti
J
D001483:Base Sequence; D002241:Carbohydrates / Q000737:chemistry*; D002384:Catalysis; D002942:Circular Dichroism; D003851:Deoxyribonucleases / Q000378:metabolism; D019281:Dimerization; D005459:Fluorides / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics; D010710:Phosphates / Q000737:chemistry*; D011789:Quantum Theory; D012313:RNA / Q000737:chemistry / Q000235:genetics; D013237:Stereoisomerism; D044366:Transition Temperature
D002241:Carbohydrates; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D010710:Phosphates; C477371:locked nucleic acid; D012313:RNA; D003851:Deoxyribonucleases; D005459:Fluorides