## 4 Glycol and Butyl Nucleic Acids (GNA and BuNA)
Cleavage from the solid support and deprotection of the exocyclic amino protecting groups was carried out in 15-20 min at 55 \({}^{\circ}\)C using a 1:1 mixture of 40% aqueous methylamine and 25% aqueous ammonium hydroxide. The RP-HPLC analysis confirmed a much higher quality of the crude oligonucleotide product compared to the previous method. Further studies showed that even a single GNA nucleotide incorporated in the middle of the DNA oligomer strongly destabilized the corresponding (GNA-DNA)/DNA duplex [37]. For example, for 5\({}^{\prime}\)-CACATTTA(\(S\)-T\({}^{\text{GNA}}\))TGTT and its (\(R\)-T\({}^{\text{GNA}}\)) counterpart mixed with 3\({}^{\prime}\)-GTGATAACAACAT-5\({}^{\prime}\), the Tm values were 13 and 7 \({}^{\circ}\)C lower, respectively. Three glycol nucleotides in the 15-mer DNA strand further decrease the stability of the corresponding (GNA-DNA)/DNA duplex to Tm = 29 \({}^{\circ}\)C. It was also found that (\(S\))-GNA, but not (\(R\))-GNA, hybridize well with RNA strands containing only A:T base pairs. Surprisingly, G:C base pairs lead to strong destabilization of these ((\(S\))-GNA-RNA)/RNA duplexes. To characterize the reasons for the high stability of certain GNA duplexes, the values of \(\Delta\)G (Gibbs free energy), \(\Delta\)H (change in enthalpy) and \(\Delta\)S (change in entropy) for the formation of three duplexes were determined from melting experiments [38]. The calculated thermodynamic parameters indicate that the entropic toll for GNA is much lower than that for DNA, so there must be a strong conformational preorganization of the GNA strands. There were also other results indicating particularly favorable stacking interactions in GNA duplexes. However, whereas in B-DNA the base-base stacking interactions predominantly operate in an intra-strand fashion, in GNA duplexes, these energetically beneficial contacts are between the bases of the opposite strands. These conclusions were strongly supported by the results of X-ray analysis of the (\(S\))-GNA duplex formed by the self-complementary 3\({}^{\prime}\)-CGHATHCG-2\({}^{\prime}\), where the H nucleotide contains the artificial hydroxypyridone nucleobase, which forms a highly stable H:H pair in the presence of copper(II) ions. These two Cu\({}^{2+}\)-binding pockets allowed the introduction of two heavy atoms per duplex to help for phasing the crystallographic data [39]. The analysis confirmed the extensive inter-strand base-base stacking interactions. The right-handed (\(S\))-GNA double helix differs markedly from the canonical A- and B-form nucleic acid helices. A large helical pitch of 60 A (34 A for DNA and RNA) with 16 residues per turn (10 and 12 for DNA and RNA, respectively) and a large helical rise (3.8 A vs. 3.4 A and 2.9 A) were also observed. The base pairs are displaced from the helix axis (x-displacement) by 5.1 to 8.6 A. The authors describe the GNA helix structure as \"a helical ribbon loosely wrapped around the helix axis\". However, X-ray analysis of the 3\({}^{\prime}\)-G(\({}^{\text{SB}}\)C)GCGC oligomer yielded markedly different results. Here, all nucleobases form standard Watson-Crick hydrogen-bonding patterns and the 5-bromocytosine nucleotide had virtually no distorting effect. In this duplex, a helical pitch of 26 A and 10 base pairs per turn were observed [40]. The differences have been explained by the fact that in the metallo-GNA duplex structure, all standard nucleotides adopt a _gauche_ conformation with respect to the torsional angles between C2\({}^{\prime}\)-O and C3\({}^{\prime}\)-O, whereas the hydroxypyridone glycol nucleotides adopt _anti_ conformations. These two forms of backbone conformation, an elongated M-type (containing metallo-base pairs) and the condensed N-type (containing brominated base pairs), were further analyzed using molecular dynamics (MD) simulations over a period of 20 ns [41].