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The synthesis and a preliminary evaluation of the pairing properties of ribo-cyclohexanyl nucleic acids (r-CNA) is herein reported. Incorporation of a single r-CNA nucleotide into natural duplexes did not enhance their stability, while a very high pairing selectivity for RNA was found. As deduced by comparative analysis of Tm and NMR data, a relationship between pairing selectivity and conformational preferences of the "sugar" moiety of r-CNA (and more generally of six-membered nucleic acids) was suggested.

Introduction: Recently, we reported the synthesis and characterization of a new mini-library of tetramolecular G quadruplexes based on the d(TGGGAG) sequence, which was previously shown to target the HIV-1 glycoprotein gp120. Oligodeoxynucleotides (ODNs) carrying aryl groups at the 5’-end through a phosphodiester bond were endowed with prominent anti-HIV activity. Biophysical studies on these aptamers demonstrated that there is no relationship between thermal stability of G4 structures and their anti-HIV activity. To identify the properties that make these tetramolecular G-quadruplexes good anti-HIV aptamers, we studied by ESI-MS the stoichiometry and the self-assembly kinetics of G-quadruplex structures based on the most active 5’-end modified d(TGGGAG) aptamers [5]. ESI-MS results showed that the conjugation of d(TGGGAG) at the 5’-end does not necessarily increase the folding rate of the G-quadruplex structure. Unexpectedly, the folding kinetics of the inactive G4 (unmodified sequence) was similar to that of the 5’-end modified sequences. In addition to the slow G4-kinetics revealed by ESI-MS studies, PAGE experiments on the modified d(TGGGAG) highlighted the strong effect of ODN concentration (single stranded) on the G4-folding kinetics. Results and Discussion: In order to clarify the role of G-quadruplex structures in the anti-HIV activity on the basis of the above kinetic studies, we investigated the anti-HIV activity of ODNs at different concentrations of G-quadruplex structure. All ODNs, as well as the unmodified sequence, were evaluated against a variety of HIV-1 viruses and resistant strains. SPR experiments were also carried out to evaluate their binding to the HIV envelope gp120 and gp41 proteins. Almost all compounds showed a good anti-HIV activity, suggesting the absence of a straight correlation between the amount of available G-quadruplex and anti-HIV activity of ODNs. Surprisingly, we found a strong anti-HIV activity of the unmodified sequence d(TGGGAG), in contrast to what has been extensively reported by Hotoda and others, who showed a lack of anti-HIV activity of the d(TGGGAG) sequence without 5’-end modifications. Also, by SPR experiments no straight correlation between gp120 binding and anti HIV activity was observed. Conclusions: The anti-HIV activity results highlight the absence of a straight correlation between the formation of G-quadruplex structures and the antiviral potential of ODNs, suggesting instead that the G4 is not the specie providing anti-HIV activity. In addition, anti-HIV data do not strictly correlate with the SPR results, suggesting that gp120 and gp41 is not the primary target of these aptamers.

The synthesis and a preliminary investigation of the base pairing properties of (6′ → 4′)-linked 1′,5′-anhydro-l-ribo-hexitol nucleic acids (α-l-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the “all equatorial” pyranyl sugar backbone, α-l-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-l-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids.

The analysis of the physicochemical properties of sugarmodified nucleic acids is currently at the core of intense multidisciplinary investigations including chemistry, biology, biotechnology, and medicine. On one side, synthetic polymers acting as RNA/DNA mimics have extensively been devised for applications in therapy, diagnostics, and synthetic biology. On the other side, the construction of alternative pairing systems has been explored either to consider their use as orthogonal nucleic acid candidates or with the aim to potentially yield insights into the chemical evolution criteria ultimately leading to the current genetic system. In all cases, structural changes of natural (deoxy)ribose cores have been established to determine profound consequences in the pairing potential of the resulting artificial nucleic acids. In some noteworthy examples, oligonucleotide systems endowed with six-membered sugars in the backbone have been observed to hold the singular property (unique of its kind) of pairing with homochiral complements having opposite sense of chirality. Relevant to etiology-oriented investigations on nucleic acid structure, these findings could suggest the existence of a relationship between nature of the sugar backbone and chiral-selection properties of nucleic acids, thereby providing insights to enrich our understanding of the structural prerequisites for base pairing. From a comparative analysis of the pairing behavior of six-membered nucleic acids we perceived that, despite the large structural differences, oligonucleotide systems capable of isoand heterochiral hybridization (Figure 1) shared preorganized carbohydrate conformations with equatorially-oriented nucleobases. This observation took us to wonder if such an arrangement of the aglycon moiety, especially whereas inducing strong backbone-base inclination or even enabling formation of quasilinear oligomeric structures, could lead sugar chirality not to be crucial in hybridization processes. In view of systematic investigations aimed at addressing this question, we herein considered the chiral selection properties of the well-known pairing system composed of (6’!4’)-linked b-erythro-hexopyranosyl nucleotides (b-homoDNA; Figure 1). Based on above assumptions and early experimental data, we reasoned that the strongly inclined complexes provided by the “allequatorial” pyranose backbone of b-homoDNA could make the latter an interesting candidate displaying potential for heterochiral hybridization. An investigation into the enantioselectivity of the hybridization processes of b-homoDNA required access to oligomeric sequences in both enantiomeric forms (b-dand b-lhomoDNA). From a synthetic standpoint, while access to d-hexopyranosyl nucleosides was easily obtained by a carbohydrate-based route, the synthesis of the corresponding lenantiomers under the same reaction conditions was hampered by the limited commercial availability of almost all lhexoses. In an alternative path, our long studied de novo approach to l-monosaccharides and other structurallyrelated compounds was recently exploited for the preparation of the l-nucleosides 2a,b (T and A acting as model nucleobases) from the homologating agent 1 (Scheme 1). Figure 1. Sugar-modified nucleic acids displaying pairing aptitude for homochiral complements of opposite chirality.

Oligonucleotides composed of 1',5'-anhydro-arabino-hexitol nucleosides belonging to the L series ( L -HNA) were prepared and preliminarily studied as a novel potential base-pairing system. Synthesis of enantiopure L -hexitol nucleotide monomers equipped with a 2'-(N 6 -benzoyladenin-9-yl) or a 2'-(thymin-1-yl) moiety was carried out by a de novo approach based on a domino reaction as key step. The L oligonucleotide analogues were evaluated in duplex formation with natural complements as well as with unnatural sugar-modified oligonucleotides. In many cases stable homo- and hetero-chiral associations were found. Besides T m measurements, detection of hetero-chiral complexes was unambiguously confirmed by LC-MS studies. Interestingly, circular dichroism measurements of the most stable duplexes suggested that L -HNA form left-handed helices with both D and L oligonucleotides.

A stereoselective and scalable route to 1,5-anhydrohexitol nucleoside analogues belonging to l -series as building blocks for l -HNA oligonucleotide synthesis has been efficiently tuned. Key to the successful outcome of our approach is the development of a DDQ-mediated domino reaction, which leads to the formation of an unsaturated 1,6-anhydrosugar derivative. Sugar elaborations and base insertion then enable to synthesize six-membered nucleosides.