Synthesis of pyrimidine bases

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Presentation transcript:

Chemical syntheses of nucleobases, nucleosides, nucleotides a oligonucleotides

Synthesis of pyrimidine bases

Transformations of pyrimidine bases

Syntheses of purine bases

Transformations of purine bases

Synthesis of nucleosides

Synthesis of nucleosides

Modifications of Nucleosides and Nucleotides acyclic nucleoside/nucleotide analogues cyclonucleosides fused and bicyclic analogues homonucleosides modified oligomucleotides

Biological Activity of Nucleoside Analogues

Synthesis of nucleotides Enzymatic: nucleoside kinase

Synthesis of nucleotides

Synthesis of oligonucleotides Phosphodiester method Phosphotriester method H-Phosphonate method Phosphoramidite method

Phosphodiester method History…

Phosphodiester method

Phosphotriester method

Phosphotriester method

Phosphotriester method

Phosphotriester method

H-Phosphonate method

H-Phosphonate method

H-Phosphonate method

H-Phosphonate method

Phosphoramidite method

Phosphoramidite method

Phosphoramidite method

Phosphoramidite method

Protection of bases

Protection of sugar

Protection of sugar

Solid-phase oligodeoxyribonucleotides

Solid-phase oligodeoxyribonucleotides

Solid-phase oligodeoxyribonucleotides Detritylation Coupling with phosphoramidite Capping Oxidation ….. 6. Deprotection and release (aq. NH3, 50°C, 5h) 7. Purification (HPLC, GEP) Total yield Yn= Yi(n-1) 20-mer 80% -> 1.4% 90% -> 13% 99% -> 82% 99.8% -> 96%

REGULATION OF GENE EXPRESSION interaction with proteins ANTISENSE STRATEGY interaction with RNA ANTIGENE STRATEGY interaction with DNA APTAMER STRATEGY interaction with proteins

No protein synthesis Translation arest Hybrid duplex m-RNA*DNA-oligomer

Products of m-RNA cleavage DNA-oligomer RNase H Products of m-RNA cleavage Hybrid duplex m-RNA*DNA-oligomer

REQUIREMENTS FOR MODIFIED OLIGONUCLEOTIDES Resistance against nuclease cleavage High affinity towards target sequences of RNA/DNA Selectivity – discrimination between DNA and RNA Low non-specific binding and high sensitivity to mismatch pairs Activation of RNase H cleavage ability

MOST IMPORTANT MODIFICATIONS OF INTERNUCLEOTIDE LINKAGES ! !!

Peptide Nucleic Acids

Classical synthesis of genes (duplexes DNA) Synthesis of oligonucleotide fragments (20-40-mers, cohesive ends) 5’-Phospohorylation (enzymatic or chemical) Ligation – T4 DNA ligase

PCR (Polymerase Chain Reaction) Add primers complementary to flanking sequence Add all nucleoside triphosphates and thermostable DNA polymerase Heat 95°C 15s - strand separation Cool 54°C – hybridization Heat 72°C (optimal temp.) – DNA synthesis

DNA cloning