Convenient Syntheses of 3′-Amino-2′,3′-dideoxynucleosides, Their 5′-Monophosphates, and 3′-Aminoterminal Oligodeoxynucleotide Primers

Abstract

5'-Protected 3'-amino-2',3'-dideoxynucleosides containing any of the four canonical nucleobases (A/C/G/T) were prepared via azides in five to six steps, starting from deoxynucleosides. For pyrimidines, the synthetic route involved nucleophilic opening of anhydronucleosides. For purines, an in situ oxidation/reduction sequence, followed by a Mitsunobu reaction with diphenyl-2-pyridylphosphine and sodium azide, provided the 3'-azidonucleosides in high yield and purity. For solid-phase synthesis of aminoterminal oligonucleotides, aminonucleosides were linked to controlled pore glass through a novel hexafluoroglutaric acid linker. These supports gave 3'-aminoterminal primers in high yield and purity via conventional DNA chain assembly and one-step deprotection/release with aqueous ammonia. Primers thus prepared were successfully tested in enzyme-free chemical primer extension, an inexpensive methodology for genotyping and labeling. Protected 5'-monophosphates of 3'-amino-2',3'-dideoxynucleosides were also prepared, providing starting materials for the preparation of labeled or photolably protected monomers for chemical primer extension.

Synthesis of Disaccharide Nucleosides by the O‐Glycosylation of Natural Nucleosides with Thioglycoside Donors

“…The glycosylation of the 3'-OH-free adenosine derivatives 29 [33] with 12 gave b-30 exclusively in 52 % yield (Scheme 4 a). In addition, the reactions of 31 [34] and 33 [35] with 10 c afforded 32 and 34 in 46 % (a/b = 3:1) and 57 % (a/b = 2.5:1) yields, respectively (Scheme 4 b and 4 c).…”

Templating efficiency of naked DNA

“…We measured rates of incorporation and fidelity on DNA templates possessing any of the 64 possible combinations of templating base and neighboring residues. Our study was made feasible by the availability of chemically activated monomers (26), amino primers (27) (Fig. 1, 3), and mass spectrometrically monitored assays for quantitative monitoring (28), unbiased by the effect of labels (29).…”

Synthesis of 3′‐BODIPY‐Labeled Active Esters of Nucleotides and a Chemical Primer Extension Assay on Beads

“…The absorption and fluorescence spectra of monomers 21 and 22 are shown in Figure 2. In our initial work, the second monomer to be employed in chemical primer extension assays (compound 22) was synthesized starting from N-benzoyl-protected 3Ј-amino-2Ј3Ј-dideoxycytidine-5Ј-monophosphate [14] 17 (Scheme 3) and commercial BODIPY ester 15. N-Hydroxysuccinimide ester 15 was coupled to 3Ј-amine 17 in aqueous DMF in the presence of N,N-diisopropylethylamine (DIEA), resulting in the protected, labeled cytidine monophosphate 19 that was deprotected with aqueous ammonia to give 20.…”

“…Here we report fluorescent oxyazabenzotriazole esters of acylated 3Ј-amino-2Ј,3Ј-dideoxynucleoside-5Ј-monophosphates [14] with more favorable properties, together with primer extension reactions that can be performed directly on beads with subsequent washing and optical read-out.…”