Andrei Guzaev

A NEW APPROACH FOR CHEMICAL PHOSPHORYLATION OF OLIGONUCLEOTIDES AT THE 5'-TERMINUS

Abstract A new efficient method for chemical 5′-phosphorylation of synthetic oligonucleotides is described. Accordingly, 2-cyanoethyl 3-(4,4′-dimethoxytrityloxy)-2,2-di(efhoxycarbonyl)propyl-1 N,N-diisopropyl phosphoramidite (1) was introduced as the 5-terminal building block during the normal chain assembly. Conventional ammonolysis gave rise to an oligomer protected at 5′-phosphate with a dimethoxytritylated tether. At this step, the oligonucleotide may be easily separated from truncated impurities by RP HPLC. Successive detritylation and brief treatment with aqueous ammonia gave the oligonucleotide 5′-monophosphate. Alternatively, the yield of the last coupling may be quantified by detritylation of the oligonucleotide still anchored to the solid support. Usual deprotection then leads directly to the 5′-phosphorylated oligomer.

Novel solid supports for the preparation of 3′-derivatized oligonucleotides: Introduction of 3′-alkylphosphate tether groups bearing amino, carboxy, carboxamido, and mercapto functionalities

Abstract Syntheses of 5 non-nucleosidic solid supports (1–5) that enable preparation of oligonucleotides bearing a carboxy,-amino,-carboxamido,- or mercaptoalkyl spacer arm at their 3′-terminus are described. They all contain an ester bond of moderate susceptibility toward nucleophiles. Upon the completion of oligonucleotide chain assembly, this bond may be cleaved by a variety of nucleophiles. These release the oligonucleotide from the support and simultaneously introduce the desired functionality. Differences in the reactivity between the supports prepared are discussed.

A novel solid support for synthesis of 3′-phosphorylated chimeric oligonucleotides containing internucleosidic methyl phosphotriester and methylphosphonate linkages

A novel solid phase synthesis of 3′-phosphorylated oligonucleotides is described. The chain assembly is carried out by phosphoramidite strategy on solid support 2, which allows a mild and fast release of the oligonucleotide in solution. The applicability of the method is demonstrated by preparation of 3′-phosphorylated chimeric oligonucleotides containing methyl phosphotriester and methyl phosphonate internucleosidic linkages.

Synthesis of 3'-functionalized oligonucleotides on a single solid support

Abstract Oligodeoxyribonucleotides tethered with an amino-, carboxy,- amidocarbonyl-, or mercaptoalkyl spacer arm at 3′-terminus were obtained by assembling the chains on a modified aminoalkyl-CPG ( 1 ) and releasing them with appropriate reagents.

SOLID SUPPORT SYNTHESIS OF ESTER LINKED HYDROPHOBIC CONJUGATES OF OLIGONUCLEOTIDES

Novel non-nucleosidic phosphoramidite building block 2 was employed for multiple modification of oligonucleotides with hydrophobic octyl groups. Hydrophobie sites are attached via potentially biodegradable ester bonds that are demonstrated to withstand the conditions of DNA deprotection. The chimeric oligonucleotides are capable of forming triple helix complexes that are stabilixed by forming a hydrophobic

Synthesis of 3′-O-(ω-aminoalkoxymethyl)thymidine 5′-triphosphates, terminators of DNA synthesis that enable 3′-labelling

Treatment of 5′-O-benzoylthymidine 1 with a mixture of acetic anhydride, acetic acid and dimethyl sulfoxide yielded 5′-O-benzoyl-3′-O-methylthiomethylthymidine 2, which was converted via the 3′O-bromomethyl derivative into 3′-O-(ω-aminoalkoxymethyl)thymidines 7 bearing a 6, 8 or 10 methylene groups long hydrocarbon chain, and finally to their 5′-triphosphates 10. The latter compounds were shown to be terminators of DNA synthesis catalysed by thermostable Tet/z DNA-polymerase, and may be labelled at the aliphatic amino group with fluorescent probes.

The effect of the 3′-terminal monophosphate group on the metal-ion-promoted hydrolysis of the phosphodiester bonds of short oligonucleotides

The effect of 3′-terminal monophosphate group on the metal-ion-promoted hydrolysis of the phosphodiester bonds of oligonucleotides has been studied. For this purpose, the rate constants for the hydrolysis of the following oligomers in the presence of Zn2+ and its 1,5,9-triazacyclododecane chelate, Zn2+[12]aneN3, have been determined: (i) ApUpUp(2′) and ApUpUp(3′), (ii) Up(Tp)nT (n= 0–4 and 7), (iii) Up(Tp)nTp (n= 0–4 and 7). The results obtained are used to propose a mechanism for the Zn2+ and Zn2+[12]aneN3 promoted hydrolysis of polynucleotides.

Acceleration of the Zn2+-promoted phosphodiester hydrolysis of oligonucleotides by the 3′-terminal monophosphate group: intrastrand participation over several nucleoside units

The Zn2+-promoted hydrolysis of the 5′-terminal ribonucleoside phosphodiester bond in chimeric ribo/deoxyribo oligonucleotide 3′-monophosphates, Up(Tp)4 and Up(Tp)9, and their dephosphorylated analogue, Up(Tp)3T, has been studied at various metal ion and substrate concentrations, and in the presence and absence of deoxyribooligonucleotide 3′-monophosphates, (Tp)n, containing no cleavable ribonucleoside phosphodiester bond. The results strongly suggest that the rate-accelerating effect of the 3′-terminal monophosphate group on the phosphodiester hydrolysis is of intramolecular origin: the Zn2+ ion bridges the favoured site of coordination, i.e. the terminal monophosphate group, and the cleaving phosphodiester bond. The 3′-monophosphate group also causes the reaction order in [Zn2 +] to deviate from unity, the values obtained with Up(Tp)3T, Up(Tp)4 and Up(Tp)9 being 1.1, 1.4 and 1.7, respectively. Possibly, the intramolecular participation of the 3′-monophosphate bound Zn2+ ion is facilitated by another Zn2+ ion that stabilizes the folded conformation of the oligonucleotide chain in the reactive Zn2 +/substrate macrochelate.

Novel non-nucleosidic phosphoramidite building blocks for versatile functionalization of oligonucleotides at primary hydroxy groups

Synthesis of two non-nucleosidic phosphoramidite building blocks, 1a, b, that enable attachment of various tether groups to oligonucleotides at their 5′-terminus (or 1′-OH of 3′-deoxypsiconucleoside units) is described. Introduction of these linkers during the oligonucleotide assembly on a solid support, and their subsequent derivatization upon deprotection, afforded amino-, carboxy-, and sulfanyl-alkyl-tethered oligonucleotides.

A General Approach for the Hydroxy Group Functionalization of Synthetic Oligonucleotides

Abstract Generally applicable non-nucleosidic solid supports and phosphoramidite building blocks that enable attachment of various tethers to the hydroxy groups of oligonucleotides were prepared. The key feature of the structure of these reagents is an ester bond of moderate reactivity which allows postsynthetic introduction of tethers by reaction with substituted alkylamines.