## 2 Flexible Nucleic Acids (FNA)  
Flexible Nucleic Acids (FNA, **1**, Chart 1, shown are dinucleotides) were one of the first proposed acyclic nucleotides for an evolutionary precursor to DNA and RNA, and were the first synthesized acyclic analogs of DNA. Since FNA are deprived of the 2'-carbon atom, that is otherwise part of the relatively rigid D-ribose ring present in the nucleosides of DNA or RNA series, their structural flexibility is virtually unrestricted. Only the anionic phosphate moieties provide some repulsive forces of the electrostatic nature, which work against the stacking interactions caused by the nucleobases. This first synthesis of the FNA-related acyclic nucleoside was reported by [PERSON] and [PERSON] [3]. The multistep synthesis (Scheme 1) began with a 1-O-benzyl derivative of glycerol. While glycerol itself is an achiral molecule, any derivatization at either of the two primary hydroxyl groups leads to a chiral compound producing L-FNA or D-FNA depending on the chiral glycerol derivative used in the synthesis. The Swiss researchers used (S)-1-O-benzyl glycerol (**2**), but due to the O3\(\longrightarrow\)O2 migration of the pivaloyl protecting group during the chloromethylation of **2**, a mixture of two regioisomers (3 + 4, in the ratio 2:1) was formed. Substitution of thymine for the chlorine atoms in **3** and **4**, followed by removal of the benzyl group, dimethoxytriylation and removal of the pivaloyl group, afforded the desired compound **5** and its regioisomer. They were separated chromographically and **5** was routinely converted to the phosphoramidite monomer **6**, which was used in the solid phase synthesis of oligomers of the general sequences S\({}^{7}\)-d(CTTTTTTG)-3'and 5\({}^{\prime}\)-d(CAAATAAAG)-3\({}^{\prime}\). These oligomers consisted mainly of DNA, with the FNA unit at position 4 or two FNA units at positions 4,6 or 4,5. A 13-mer 5\({}^{\prime}\)-d(CttttttttG)-3\({}^{\prime}\) (\"t\" stands for the FNA-T unit) was also obtained. Interestingly, in the synthesis of these oligomers, the coupling yields were approximately 90% for 6 and >99% for standard DNA phosphoramidite monomers. The DMT-tagged oligonucleotide products were isolated by RP-HPLC and then detritylated. The resultant oligomers were hydrolyzed with spleen phosphodiesterase, snake venom phosphodiesterase (svPDE) and dephosphorylated with bacterial alkaline phosphatase, giving the mixtures containing the expected nucleosides in the expected ratios. It was found that the oligomers containing 1 or 2 FNA-T units destabilized the (FNA-DNA)/DNA duplexes by about 15 \({}^{\circ}\)C per modification, compared to Tm = 40 \({}^{\circ}\)C found for the natural DNA. The almost completely modified oligomer d(CttttttttG) did not form a duplex with the complementary 5\({}^{\prime}\)-d(GAAAAAAAAAAC)-3\({}^{\prime}\). Destabilization was attributed to an increase in entropy cost compared to the perfect duplex formed by natural nucleic acids. Pure oligothymidylate and oligoadenylate FNA and their alternating copolymers (12-19 mers) were synthesized by [PERSON] and collaborators [4]. Interestingly, using the same phosphoramidite method, the repetitive yield of +98% was observed, while subsequent thermal dissociation studies showed that (FNA-A)\({}_{12}\) hybridized effectively with dodecathymidylate (dT)\({}_{12}\) and the complex exhibited sharp melting with Tm = 24 \({}^{\circ}\)C.  
Rather unexpectedly, although DNA/DNA duplexes adopt a B-type conformation, the circular dichroism (CD) spectrum suggested that the FNA/DNA duplex exists as an A-type helix, as is the case for RNA/RNA or RNA/DNA duplexes. Some differences were observed during svPDE-assisted hydrolysis, because while t\({}_{1/2}\) = 38 s was found for T\({}_{12}\), for the oligomers FNA-A\({}_{12}\) and alternate FNA-(A(A-T)\({}_{9}\)), the values were 110 h and about 12 h, respectively.