Oligodeoxynucleotides containing 2'-amino-LNA nucleotides as constrained morpholino phosphoramidate and phosphorodiamidate monomers.

TLDR: Thermal denaturation studies showed that the novel 2'-amino-LNA-based morpholino monomers exert a destabilizing effects on duplexes formed with complementary DNA and RNA.

About: This article is published in Bioorganic & Medicinal Chemistry Letters.The article was published on 2017-07-15 and is currently open access. It has received 3 citations till now. The article focuses on the topics: Phosphoramidate & Oligonucleotide.

Figures

Fig. 2. Snapshots from molecular dynamics simulations of a 9-mer duplex [(50-d (CTGAXATGC):50-d(GCATATCAG)] modified at the central position (X) with a) 30-Obenzyl-2-amino-LNA phosphoramidate monomer 6, and b) 20-amino-LNA-T monomer 5. See ESIy Fig. S7 for a larger display including a snapshot of the dinucleotide structure showing more clearly the sugar pucker of the modified monomers 5 and 6.

Fig. 1. A) Chemical structures of phosphorodiamidate morpholino oligonucleotides (PMO, 1) and the morpholino building blocks (2 and 3) used to synthesize PMO in a 60 ? 30 direction. B) Chemical structures of phosphordiester monomers of LNA, 4 and 20-amino LNA, 5. Structure 6 shows the 20-amino-LNA-T phosphoramidate monomer and structure 7 the 20-amino-LNA-T dimethylamino phosphorodiamidate monomer. B = nucleobases and T = thymin-1-yl.

Fig. 3. Synthetic cycle for incorporation of the phosphorodiamidite 9 with the optimal coupling condition of 0.1 M tetrazole for two rounds of 900 s. Route A enables the formation of phosphoramidate monomer 6 using standard detritylation, oxidation with aqueous iodine and cleavage from resin using sat. aq NH3. Route B enables the formation of chimeric PMO-DNA oligomers containing N,N-dimethylamino phosphorodiamidate monomer 7 using the trityl-scavenger mixture TMPB:CF3COOH:CHCl3 10:0.5:89.5 v/v/v, boronation using 0.05 M BH3 in THF and the post-synthetic transformation followed by cleavage from resin using sat. aq NH3. B = Nucleobases and T = Thymin-1-yl monomer.

Table 1 Thermal denaturation studies for synthesized oligonucleotidesa.

Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Oligodeoxynucleotides containing 20-amino-lna nucleotides as constrained morpholino phosphoramidate and phosphorodiamidate monomers" ?

50 direction of a phosphorodiamidite 20-amino-LNA-T nucleotide as the morpholino phosphoramidate and N, N-dimethylamino phosphorodiamidate monomers into six oligonucleotides is reported. This site in oligonucleotides therefore provides a convenient handle when Nacylated and N-alkylated for appending amino acids residues, fluorescence probes, nucleobases and a piperizino group while preserving the LNA-type high-affinity hybridization with complementary DNA and RNA strands. In order to complete this study, a 9-mer duplex consisting of DNA: RNA [ 50-d ( CTGATATGC ) :50-r ( GCAUAUCAG ) ] was downloaded from the protein data bank ( PDB entry pdb 1HG9 ), the RNA strand was converted to DNA and 30-O-benzyl-20-amino-LNA phosphoramidate monomer 6 was inserted in d ( CTGAXATGC ) as monomer X. An AMBER⁄ force field in Macro Model 9. 1 was used to generate representative low energy structures. This modelling study indicated that the 30-O-benzyl-20-aminoLNA-T is locked into a 30-endo ( N-type ) conformation ( Fig. 2a ) in a manner similar to 20-amino-LNA-T monomer 5 inserted into the same duplex ( Fig. 2b ). 

Q2. What was the reaction reaction used for the synthesis of 7?

Synthon 9 was then activated and coupled with the growing oligonucleotide in order to generate the phosphoramidite internucleotide linkage. 

Q3. What was the effect of one incorporation of monomer 6 on the stability of ON2?

When ON2-ON5 were hybridized to DNA, one incorporation of monomer 6 induced a drastic drop in Tm value of 14.5 C for ON2 while no detectable transition above 5 C was observed for ON3 with four incorporations of monomer 6. 

Q4. What is the way to prepare a morpholino monomer?

Using synthon 9 and the standard 20-deoxynucleoside 30-phosphoramidites, oligonucleotides ON2-ON5 and ON7-ON8 were synthesized, characterized by LC-MS, and used to evaluate the effect of the novel constrained morpholino monomers on duplex stability. 

Q5. What is the method for synthesis of a phosphoramidite?

Following oxidation and capping, the cycle can be repeated using synthon 9 or the standard 20-deoxynucleoside 30-phosphoramidites. 

Q6. What was the first step in the synthesis of 7?

This post-synthetic transformation consisted of 1) detritylation, 2) removal of the cyanoethyl substituent with MeCN:NEt3 (1:1; v/v) (thereby also oxidizing P(III) to P(V)), 3) oxidative substitution by reaction with I2 and dimethylamine, and 4) cleavage from the solid support using sat. 

Q7. What is the effect of the incorporation of monomers on thermal stability?

Thermal denaturation experiments revealed a significant decreasing effect on duplex stability of these monomers which may at least in part be explained by interference of 30-O-benzyl group on the hydration of the phosphorus backbone. 

Q8. What was the reaction reaction used for the synthesis of the phosphoramidite?

For the synthesis of PMO-DNA chimeras containing monomer 7, the borane phosphonate intermediate was converted, through oxidative substitution,20 to the N,N-dimethylamino phosphorodiamidate (see Fig. S2, ESIy). 

Q9. What is the name of the morpholino monomers?

19Because of the current interest in PMOs, the authors decided to explore other morpholino-based monomers including the 20-amino-LNA nucleotide (5, Fig. 1), a derivative of LNA (locked nucleic acid) (4, Fig. 1) having a 20N-40C methylene bridge. 

Q10. What is the function of the 20 amino acid in oligonucleotides?

This site in oligonucleotides therefore provides a convenient handle when Nacylated and N-alkylated21 for appending amino acids residues,22 fluorescence probes,23–26 nucleobases27 and a piperizino group28while preserving the LNA-type high-affinity hybridization with complementary DNA and RNA strands. 

Q11. What is the structure of the morpholino analogue?

A new synthon, 30-O-benzyl-20-amino-LNA-T phosphorodiamidite, was used to prepare an alternative morpholino analogue having a 20-50 linkage (6 and 7, Fig. 1) and the 30-hydroxyl protected through a benzyl group. 

Q12. How many coupling rounds did the authors need?

Based upon these results, the authors conclude that the optimal conditions were 0.10 M tetrazole for each of two coupling rounds of 900 s each giving 80% total stepwise coupling yield.