Synthesis and studies of 2´-o-alkylated oligonucleotides with enhanced stability and cellpenetrating properties

Basic structures of natural oligonucleotides (ONs) are RNA and DNA are giving rise to conformations and secondary structures inside cells and are the fundamentals for life. Many chemically modified oligonucleotides have been synthesized over the years. Studying the mechanisms on how RNA and DNA interact with different type of modified oligonucleotides will and have given new understandings of their functions. It is also important in the order of pursuing a new type of medicines. Today we can find that these molecules have been realized in certain types of medicines for hereditary diseases and as vaccines, for example.

This thesis describes synthetic strategies to be able to perform 2´-OH alkylations and which protecting groups are required to reach the target compounds. This includes the synthesis of modified nucleotide building blocks intended for oligonucleotide synthesis. The goal is to reach synthesis methods of oligonucleotides with a 2´-O-carbamoylmethyl (CM) functionality including the version of the CM where the amino group is extended with ethylene-amine giving the 2´-O-[N-(aminoethyl)-carbamoyl]methyl (AECM) modification. Thereafter, to study these molecules with respect to: • Protecting group strategy for the synthesis of the 2´-O-modified building blocks. • Chemical stability of the modification during conditions used in oligonucleotide synthesis. • Stability against degradation by nucleases (enzymes) provided by the CM and AECM modification. • Binding ability to RNA and DNA. • Study of how the 2´-O-AECM functionality affects cell uptake.

Paper I present the synthesis of a 2′-O-carbamoylmethyl (CM) containing H-phosphonate building block as well as synthesis of model dinucleotide. Paper II studies the stability of the 2´-O-carbamoylmethyl (CM) group under ammonolysis conditions used during oligonucleotide synthesis as well as enzymatic stability of the phosphate diester bond vicinal to the 2´-O-carbamoyl group. Paper III present two different classes of protection for the uridine lactam function, intended for use in synthesis of 2'-O-alkyl-uridines. These are benzoyl protections and different acetal functions. Paper IV includes strategies for the synthesis of a 2`-O-[N-(aminoethyl)-carbamoyl]methyl (AECM) modified H-Phosphonate building block and the synthesis of a model dinucleotide. In addition, studies on the chemical and enzymatic stability of this dinucleotide is reported. Paper V present the synthesis of a 2`-O-AECM modified phosphoramidite building block and the synthesis of AECM-modified oligonucleotides. These AECM-oligonucleotides are studied with respect to resistance towards enzymatic degradation and cellular uptake.

List of scientific papers

I. Synthesis of A 2′-O-(Carbomoylmethyl)Ribonucleoside H-Phosphonate Building Block and A Model Dinucleotide. Stefan Milton, Raunak, Esther Yeheskiely & Roger Strömberg. Nucleosides, Nucleotides & Nucleic Acids. 2007;26(10-12).
https://doi.org/10.1080/15257770701542900

II. Stability of a 2′-O-(Carbamoylmethyl)adenosine-Containing Dinucleotide. Stefan Milton, Charlotte Ander, Esther Yeheskiely, Roger Strömberg. EurJOC. 2012;3:539-543.
https://doi.org/10.1002/ejoc.201101264

III. Evaluation of lactam protection for synthesis of 2'-O-alkylated uridines. Stefan Milton, Roger Strömberg. Nucleosides Nucleotides Nucleic Acids. 2007;26(10-12):1491-3.
https://doi.org/10.1080/15257770701542868

IV. Synthesis and Stability of a 2′-O-[N-(Aminoethyl)carbamoyl]methyladenosine-Containing Dinucleotide. Stefan Milton, Charlotte Ander, Dmytro Honcharenko, Małgorzata Honcharenko, Esther Yeheskiely, Roger Strömberg. EurJOC. 2013;31:7184-7192.
https://doi.org/10.1002/ejoc.201300699

V. Nuclease resistant oligonucleotides with cell penetrating properties. Stefan Milton, Dmytro Honcharenko, Cristina S. J. Rocha, Pedro M. D. Moreno, C. I. Edvard Smith and Roger Strömberg. ChemCom. 2015;51(19):4044-4047.
https://doi.org/10.1039/c4cc08837a