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Funding Information: V.G. and P.V. acknowledge Academy of Finland’s Project No. 308931. J.J.V.D. and M.Ö. acknowledge Academy of Finland’s Flagship Programme under Project Nos. 318890 and 318891 (Competence Center for Materials Bioeconomy, FinnCERES). M.Y. acknowledges Academy of Finland’s Flagship Programme under Project No. 337430 (Gene, Cell and Nano Therapy Competence Cluster for the Treatment of Chronic Diseases, GeneCellNano). Publisher Copyright: © 2021 The Authors. Published by American Chemical Society. Bingel cyclopropanation between Buckminster fullerene and a heteroarmed malonate was utilized to produce a hexakis-functionalized C60 core, with azide and tetrazine units. This orthogonally bifunctional C60 scaffold can be selectively one-pot functionalized by two pericyclic click reactions, that is, inverse electron-demand Diels-Alder and azide-alkyne cycloaddition, which with appropriate ligands (monosaccharides, a peptide and oligonucleotides tested) allows one to control the assembly of heteroantennary bioconjugates.

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs. publishedVersion

Phosphoramidite building blocks of CF3-modified N4(6-amino-2-pyridinyl)deoxycytidines were synthesized, incorporated into triplex-forming 2'-deoxyoligonucleotide strands and the applicability of the probes to recognize nucleobase content in the pyrimidine-rich strand of double helical DNA targets was evaluated. As expected, the obtained 19F NMR resonances were sensitive to the base content and unique 19F NMR-spectral fingerprints could be obtained.

Triplex-forming peptide nucleic acids (TFPNAs) were targeted to double-helical regions of 19 F-labeled RNA hairpin models (a UA-rich duplex with a hexaethylene glycol (heg) loop and a microRNA model, miR-215). In addition to conventional UV- and circular dichroism (CD)-based detection, binding was monitored by 19 F NMR spectroscopy. Detailed information on the stoichiometry and transition between the triple-helical peptide nucleic acid (PNA)/RNA and (PNA)2 /RNA binding modes could be obtained. γ-(R)-Hydroxymethyl-modified thymine-1-yl- and 2-aminopyridin-3-yl-acetyl derivatives of TFPNAs were additionally synthesized, which were targeted to the same RNA models, and the effect of the γ-(R)-hydroxymethyl group on binding was studied. An appropriate pattern of γ-(R)-hydroxymethyl modifications reduced the stability of the ternary complex and preferred stoichiometric binding to the miR-215 model.

Glycosidic (&beta, 1&prime, &prime, &rarr, 6, 3&prime, and 4&prime, ) site isomers of neomycin B (i.e., neobiosamine (&beta, ) neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3&prime, 4&prime, tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4&prime, 5,3&prime, and 5,6,3&prime, tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.

Neomycin-conjugated homopyrimidine oligo 2′-deoxyribonucleotides have been synthesized on a solid phase and their potential as triplex forming oligonucleotides (TFOs) with DNA-duplexes has been studied. For the synthesis of the conjugates, C-5, C-2′ and C-4′-tethered alkyne-modified nucleoside derivatives were used as an integral part of the standard automated oligonucleotide chain elongation. An azide-derived neomycin was then conjugated to the incorporated terminal alkynes by Cu(I)-catalyzed 1,3-dipolar cycloaddition (the click chemistry). Concentrated ammonia released the desired conjugates in acceptable purity and yields. The site of conjugation was expectedly important for the Hoogsteen-face recognition: C-5-conjugation showed a notable positive effect, whereas the influence of the C-2′ and C-4′-modification remained marginal. In addition to conventional characterization methods (UV- and CD-spectroscopy), 19F NMR spectroscopy was applied for the monitoring of triplex/duplex/single strand-conversions.