Phosphorothioates, Essential Components of Therapeutic Oligonucleotides

Address correspondence to: Fritz Eckstein, PhD, Max-Planck-Institut für Experimentelle Medizin, Hermann-Rein-Strasse 3, Göttingen 37075, Germany, E-mail: [email protected]

Introduction

This review will focus on the impact of the phosphorothioate group in oligonucleotides, which renders them suitable for therapeutic applications. This backbone modification is present in a large proportion of the oligonucleotides in various phases of clinical trials. Additionally, the two oligonucleotides approved by the U.S. Food and Drug Administration as medicaments are fully phosphorothioated: Vitravene (Fomivirsen) for local delivery to the eye in 1998 [1,2], and Mipomersen (Kynamro) for systemic delivery in 2013 [3]. As background information, I will describe some of the chemical/biochemical properties of this particular nucleic acid modification to illustrate its differences from the phosphates. Although some of these are not directly relevant for the antisense methodology, they nevertheless help us to understand this class of compounds.

The focus of this review is on phosphorothioates containing a nonbridging sulfur. The replacement of an oxygen by a sulfur might be considered a minor one, but as will be discussed, there are considerable differences arising from the somewhat larger Van der Waals radius of 1.85 versus 1.44 Å [4] and the resultant slight lengthening of the P-S bond [5]. When phosphorothioate occurs as a diester in the internucleotidic linkage, phosphorous has four nonidentical ligands. These can be arranged either like the fingers of the right or the left hand. This is called a chiral arrangement from the Greek word for hand. Thus two compounds exist, called diastereomers, with different configurations around phosphorus.This results in the presence of two diastereomers which can be expected to interact differently with enzymes. The literature on phosphorothioate nucleic acids is extensive; thus, I apologize for having to be selective. For reviews see [6,7].

Development of Nucleoside Phosphorothioates

Early days

The study of phosphorothioate biochemistry began with the synthesis of adenosine 5[variant prime]-phophorothioate, which was unexpectedly resistant to phosphatases that of course readily degrade adenosine 5[variant prime]-phosphate (Fig. 1) [8]. This resistance could not be explained on the basis of differences between the chemical reactivity of phosphate and phosphorothioate moieties. An explanation for the mechanism underlying this difference was clarified by X-ray structural work on a phosphorothioate oligonucleotide and its resistance to...