ABSTRACT
Thymine intermediate 17 has been synthesized on a multigram scale (50 g, 70 mmol) from starting sugar 1 in 15 steps in an overall yield of 73%, with only 5 purification steps. The key thymine intermediate 18 was obtained from 17 in a single step in 96% yield, whereas the key 5-methylcytosine intermediate 20 was obtained from 17 in 2 steps in 58% yield. This highly efficient large scale route necessitates only 2 and 3 novel steps to obtain N2'-functionalized thymine and 5-methylcytosine amino-LNA phosphoramidites from these key intermediates, respectively.
Subject(s)
5-Methylcytosine/chemical synthesis , Nucleosides/chemical synthesis , Organophosphorus Compounds/chemistry , Thymidine Monophosphate/analogs & derivatives , Thymine/chemical synthesis , 5-Methylcytosine/chemistry , Molecular Structure , Nucleosides/chemistry , Thymidine Monophosphate/chemical synthesis , Thymidine Monophosphate/chemistry , Thymine/chemistryABSTRACT
The triphosphate of the thymine derivative of 2'-amino-LNA (2'-amino-LNA-TTP) was synthesised and found to be a good substrate for Phusion® HF DNA polymerase, allowing enzymatic synthesis of modified DNA encoded by an unmodified template. To complement this, 2'-amino-LNA-T phosphoramidites were incorporated into DNA oligonucleotides which were used as templates for enzymatic synthesis of unmodified DNA using either KOD, KOD XL or Phusion polymerases. 2'-Amino-LNA-T in the template and 2'-amino-LNA-TTP as a substrate both decreased reaction rate and yield compared to unmodified DNA, especially for sequences with multiple 2'-amino-LNA-T nucleotides.
Subject(s)
Biopolymers/metabolism , Enzymes/metabolism , Oligonucleotides/metabolism , Base Sequence , DNA PrimersABSTRACT
Hereby we report an efficient synthesis of LNA thymine and LNA 5-methylcytosine 5'-phosphoramidites, allowing incorporation of LNA thymine and LNA 5-methylcytosine into oligonucleotides synthesized in the 5'â3' direction. Key steps include regioselective enzymatic benzoylation of the 5'-hydroxy group of unprotected LNA thymine, and subsequent 4,4'-dimethoxytritylation of the 3'-hydroxy group of the O5'-benzoylated LNA thymine nucleoside.
Subject(s)
5-Methylcytosine/chemistry , Oligonucleotides/chemistry , Organophosphorus Compounds/chemistry , Thymine/chemistry , 5-Methylcytosine/metabolism , Candida/enzymology , Fungal Proteins , Lipase/metabolism , Molecular Structure , Oligonucleotides/metabolism , Organophosphorus Compounds/metabolism , Stereoisomerism , Thymine/metabolismABSTRACT
Thymin-1-yl-acetic acid and adenin-9-yl-acetic acid have been coupled to the N2'-atom of a 2'-amino-LNA thymine nucleoside, and these "double-headed" LNA monomers have been incorporated into oligodeoxyribonucleotides via their corresponding phosphoramidite derivatives. Oligonucleotides containing these modified nucleotides show in most cases increased thermal stability when forming duplexes with complementary DNA, even allowing multiple incorporations. Incorporation of "double-headed" LNA monomers in both strands also led to stable duplexes, however, no indication of Watson-Crick base pairing between the extra nucleobases could be found.
Subject(s)
Oligonucleotides/chemistry , Base Pairing , DNA/chemistry , Models, Molecular , Molecular Structure , Nucleic Acid Denaturation , Transition TemperatureABSTRACT
A synthetic route towards novel nucleosides with 1,4- dioxane as the sugar moiety has been developed. The dioxane moiety features a second anomeric center, which has been phosphitylated giving a diastereomeric mixture of the corresponding phosphoramidites.