Acyclic Phosphono Nucleosides, Nucleotides, and Oligonucleotides: Synthesis, Antiviral Evaluation, and Genetic Selection

  The chemical diversification and enzymatic proliferation of artificial genetic polymers (xeno nucleic acids, XNAs) is an intriguing avenue of research, especially in view of potential applications in synthetic biology. One of the main goals in this area is to achieve the genetic reprogramming of novel microorganisms through the development of both chemical and biological tools. For this purpose, oligonucleotide analogues, showing good hybridization properties under physiological conditions (high affinity and high specificity) and higher stability than their natural congeners against degradation by cellular and serum nucleases, are attractive molecular targets.

  It has been reported that a number of XNAs, such as HNA, TNA, CeNA and GNA, can store genetic information and can be recognized and incorporated by engineered thermophilic polymerase mutants. One of the disadvantages of the present XNA approach is the potential instability of the phosphodiester bond in vivo. As a consequence, the information could be lost over time. The phosphonate unit is known to be a biological active mimic of the natural phosphate, while not being susceptible to enzymatic cleavage by phosphatases in vivo. In this work, we are interested in the replacement of the enzymatically labile phosphodiester bond (O-P) by a phosphonate linkage (C-P) to avoid this problem. In addition, the C-P moiety could reduce interactions with natural nucleic acids binding factors by changing the surface electrostatic potential of the relevant XNA polymer.

  Almost 20 years after the discovery of the broad antiviral activity spectrum of the first acyclic nucleoside phosphonate, several structural analogues have become important drugs in the treatment of DNA virus and retrovirus infections. Acyclic nucleoside phosphonates (ANPs) represent an important class of antiviral nucleoside derivatives. In addition, acyclic nucleosides are structurally simplified analogues of naturally occurring nucleosides due to the reduced number of stereocentres, and they should therefore serve in principle as ideal building blocks as initial proof of concept.

  On this basis, we aim at exploring chemical synthetic methods for the preparation of acyclic phosphonate oligonucleotides (aPhoNA) for further hybridization and in vivo studies, as well as screening polymerases suitable for this particular modification in order to identify potential candidates for directed enzyme evolution. In addition, novel prodrugs of ANPs were synthesized and evaluated against a range of retro- and DNA viruses.