Stability of Stereoregular Oligo(nucleoside Phosphorothioate)s in Human Plasma: Diastereoselectivity of Plasma 3‵-Exonuclease

Abstract

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3'-Exonuclease present in human plasma appeared to be RP specific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereo-controlled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [S(P)]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3'-end of PS-oligo substrate significantly inhibits the enzyme activity.

The mononucleotide-dependent, nonantisense mechanism of action of phosphodiester and phosphorothioate oligonucleotides depends upon the activity of an ecto-5′-nucleotidase

“…For purity control and hydrolysis experiments, the oligomers were 5Ј-labeled with ␥-32 P]ATP and T4 polynucleotide kinase as described previously. 2 …”

“…Although they are much more resistant to nucleolytic degradation than the PO oligos, reports indicating their stereodependent hydrolysis have been published recently. 2,3 Moreover, results published by Vaerman et al 4 indicate that the cytotoxicity of PO and PS oligos might have been, in part, caused by mononucleotides deoxyribonucleoside-5Ј-phosphates (dNMPs) or their phosphorothioate analogs, deoxyribonucleoside-5Ј-monophosphorothioates (dNMPSs) released during enzymatic degradation of the oligonucleotides. The toxicity of the dNMPs to leukemia cell lines depends on their concentration and/or the type of nucleobase.…”

The P‐Stereocontrolled Synthesis of PO/PS‐Chimeric Oligonucleotides by Incorporation of Dinucleoside Phosphorothioates Bearing an O‐4‐Nitrophenyl Phosphorothioate Protecting Group

“…Incorporation of 9b was achieved by prolonging the coupling time to 10 min, without further modifications of the standard protocol. Since incorporation of the diastereomerically pure phosphorothioate at the terminal 3Ј-position of the chimeric oligonucleotide is of special interest due to effective protection against exonuclease degradation, [7,8] we prepared a CPG support functionalized with diastereomerically pure (Ͼ96 %) dinucleoside (3Ј,5Ј)-O-arylphosphorothioate 2b linked to the support through a standard succinyl linker. [4] The use of (R P )-2b and (S P )-2b allowed the preparation of supports with a loading capacity of 28.6 and 20.1 μmol g -1 , respectively.…”

The effect of divalent cations on the catalytic activity of the human plasma 3′-exonuclease

“…1), a large number studies with stereodefined PS-oligos have been performed to determine the stereochemical course of reactions catalyzed by certain kinases (Orr et al 1978), nucleotidyl transferases (Eckstein et al 1977;Sheu and Frey 1978), polymerases (Eckstein et al 1976; Bartlett and Eckstein 1982), endonucleases (Connolly et al 1984;Grasby and Connolly 1992), and exonucleases (Eckstein et al 1979; Gupta and Benkovic 1984;Koziolkiewicz et al 2002). Among enzymes acting in exonucleolytic fashion, much attention has been paid to the 3 0 -exonuclease present in human plasma (HP) in regard to its phosphodiesterase activity toward unmodified and phosphorothioate oligonucleotides degraded successively from the 3 0 -end to the 5 0 -end with releasing of nucleoside-5 0 -phosphate and 5 0 -phosphorothioates, respectively (Eder et al 1991;Koziolkiewicz et al 1997;Gilar et al 1998). It is now accepted that the human plasma 3 0 -exonuclease possesses the [R P ]-selective nature and its digestion mechanism proceeds with retention of configuration at a phosphorus atom, that is, the reaction occurs via a two-step mechanism with participation of a covalent enzymesubstrate intermediate (Gijsbers et al 2001;Koziolkiewicz et al 2002).…”

“…Whereas the stereochemical mechanism and the substrate specificity of the 3 0 -exonuclease have been well characterized (Eder et al 1991;Koziolkiewicz et al 1997Koziolkiewicz et al , 2002Gilar et al 1998), nothing is known about its three-dimensional structure. This prompted us to carry out functional studies aiming at the determination of divalent cations which contribute to catalysis of PO-and PS-oligos in HP.…”