Oligodeoxynucleotides containing fluorophore / quencher pairs

Abstract

Four phosphoramidite monomers were prepared for use in solid phase oligodeoxynucleotide synthesis.  Each monomer held the florescence quencher molecule (2-(4-dimethylamino-phenlazo)-benzoic acid (methyl red) with attachment by aminoalkyl pendant arms.  The monomer 1-α-O-[(2-(dimethylamino-phenylazo)-benzoyl)-6-aminohexyl]-5’-O-(5-bis(4-methoxyphenyl)-phenylmethyl-2-doxy-D-ribose-3’-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite) was prepared in 9 steps with an overall yield of 10% and was tested in solid phase oligodeoxynucleotide synthesis giving coupling efficiencies of 96%. The monomer 4-(di-n-butylamino-methyleneamino)-7-(O-(5-bis-(4-methoxyphenyl)-phenylmethyl)-O-[3-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite)]-2-deoxy-β-D-ribosyl)-5-(3-N-(4-dimethylamino-phenylazo-benazmido)-prop-2-ynyl)-pyrrolo[2,3-d]pyrimidine was synthesised in 14 steps with an overall yield of 0.9%.  Molecular beacon studies using oligodeoxynucleotides containing these monomers showed the required fluorogenic responses.

Two further major groove monomers were synthesised. 4-(Di-n-butylamino-methyleneamino)-7-(O-5-bis(4-methyoxyphenyl)-phenylmethyl)-2-deoxy-β-D-ribosyl-O-3-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite)-5-(6-(2-(dimethylamino-phenylazo)-benzoyl)-prop-2-ynyl-hexanamido)-pyrrolo[2,3-d]pyrimidine was synthesised in 0.73% yield over 12 steps.  7-[O-(5-Bis-(4-methoxyphenyl)-phenylmethyl-2-deoxy-β-Di-ribosyl-O-3-(2-cyanoethyl-N,N-diisopropyl-phosphoramidite))]-5-(6-(2-(dimethylamino-phenylazo)-prop-2-ynyl-hexanamido)-pyrrolo[2,3-d]pyrimidin-5-one was synthesised in 0.50% yield over 12 steps.

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