Solidphase synthesis of DNA oligonucleotides

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Solid-phase synthesis of DNA (oligonucleotides)
Dimer
Repeat cycles
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Linkage to the support
Good leaving group
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Types of phosphates
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Phosphotriester chemistry
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P(III) chemistry (developed by Marvin Caruthers)
Phosphite triester
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Phosphite triester chemistry (phosphoramidite)
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Dimethoxytrityl (DMT) for 5-OH group
Stable dimethoxytrityl cation Orange colour,
useful to monitor compeleteness of coupling
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Phosphate protection
Cyanoethyl
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Base protecting groups - remove in conc. ammonia
T needs no protection
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Step 7 1) acid 2) conc ammonia followed by HPLC
purification
Step 1 Detritylation
Step 5 Oxidation
Step 6 (same as Step 1)
Step 3 coupling
Step 2 activation
Step 4 Capping
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RNA synthesis
t-butyldimethyl silyl (TBDMS)
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Oligonucleotide Analogues Many hundreds of
possible backbone variants have been
synthesized 1) Methylphosphonate
Phosphate has stereogenic centre (Rp and Sp
isomers) More lipophilic, no negative charge
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2) Phosphorothioate
Sulphurization in place of oxidation
Beaucage Reagent
Possible to use elemental S
Phosphorothioates are used in antisense oligonucle
otides to hinder nuclease degradation
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3) Peptide Nucleic Acids
Recognizes RNA stretches to form duplexes
(sequence-specific)
Opens DNA to form triplexes (homopolymers)
PNADNAPNA
Synthesized similarly to solid-phase peptide
synthesis