Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides

The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.     

Synthesis and properties of nucleic acid analogues consisting of a benzene-phosphate backbone

[Chemical reaction: See text] The synthesis and properties of a nucleic acid analogue consisting of a benzene-phosphate backbone are described. The building blocks of the nucleic acid analogue are composed of bis(hydroxymethyl)benzene residues connected to nucleobases via the biaryl-like axis. Stabilities of the duplexes were studied by thermal denaturation. It was found that the thermal stabilities of the duplexes composed of the benzene-phosphate backbone are highly dependent on their sequences. The duplexes with the benzene-phosphate backbone comprised of the mixed sequences were thermally less stable than the natural DNA duplexes, whereas that composed of the homopyrimidine and homopurine sequences was thermally and thermodynamically more stable than the corresponding natural DNA duplex. It was suggested that the analogues more efficiently stabilize the duplexes in a B-form duplex rather than in an A-form duplex. Thus, the duplexes consisting of the benzene-phosphate backbone, especially composed of the homopyrimidine and homopurine sequences, may offer a novel structural motif useful for developing novel materials applicable in the fields of bio- and nanotechnologies.     

Synthesis of 2,4,5-triaminocyclohexanecarboxylic acid as a novel 2-deoxystreptamine mimic

Since aminoglycosides have been known to exert their antibacterial activities by binding to various RNA targets, 2-deoxystreptamine served as a particularly important model in understanding RNA-small molecule interaction. Herein, 2,4,5-triaminocyclohexanecarboxylic acid was prepared as a novel 2-deoxystreptamine (2-DOS) mimic, which replaces highly flexible glycosidic bonds of aminoglycosides with amide bonds and may improve binding selectivity toward RNA targets through increased rigidity and additional hydrogen-bonding capability. This unnatural g-amino acid can also be used as a potential component for synthetic foldamers.     

Site-specific cleavage of RNA by a metal-free artificial nuclease attached to antisense oligonucleotides

RNA cleaving tris(2-aminobenzimidazoles) have been attached to DNA oligonucleotides via disulfide or amide bonds. The resulting conjugates are effective organocatalytic nucleases showing substrate and site selectivity as well as saturation kinetics. The benzimidazole conjugates also degrade enantiomeric RNA. This observation rules out contamination effects as an alternative explanation of RNA degradation. The pH dependency shows that the catalyst is most active in the deprotonated state. Typical half-lifes of RNA substrates are in the range of 12-17 h. Thus, conjugates of tris(2-aminobenzimidazoles) can compete with the majority of metal-dependent artificial nucleases.     

Synthesis and photovoltaic properties of copolymers with a fluoro quinoxaline unit

Two novel accepter units, namely, difluoroquinoxaline and monofluoroquinoxaline, were prepared and used for the synthesis of the conjugated polymers containing electron donor–acceptor pairs for use in organic photovoltaics. The introduction of a fluorine atom into the quinoxaline moiety resulted in polymers with lowered highest occupied molecular orbital (HOMO) energy levels; this increased the open circuit voltage of the devices based on the synthesized polymers. The conjugated polymers containing difluoroquinoxaline and monofluoroquinoxaline, namely, thiophene and benzodithiophene, were synthesized using the Stille polymerization reaction to produce PEHBQxF2, PEHBQxF1, PEHBDTQxF2, and PEHBDTQxF1. The HOMO energy levels of PEHBQxF2, PEHBQxF1, PEHBDTQxF2, and PEHBDTQxF1 were determined to be −5.66, −5.52, −5.54, and −5.39 eV, respectively. The device with PEHBDTQxF2/PC₇₁BM (1:2, w/w) and containing diiodooctane (3 vol %) exhibited the best photovoltaic performance, with its V_OC being 0.79 V, J_SC being 10.44 mA/cm², FF being 68%, and PCE being 5.58%. © 2018 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 821–830     

Synthesis and peroxidase-like mimic study in H2O2 detection of a stable polyoxometalate-pillared coordination polymer

Abstract

A stable hamburger-like polyoxometalate (POM)-pillared coordination polymer (CP) [Ag₁₅(trz)₁₀][SiW₁₂O₄₀] (Ag₁₅SiW₁₂) (trz = 1,2,3-triazole) has been synthesized and characterized. Single crystal X-ray diffraction (SCXRD) analysis indicates that SiW₁₂ polyanions are sandwiched between adjacent Ag-trz sheets with two distinct types of pores, giving a three-dimensional POM-pillared sheet framework. Inspired by the reported horseradish peroxidase (HRP) and other enzyme mimics, the peroxidase-like activity of Ag₁₅SiW₁₂ was investigated to catalyze oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂. Compared with other POM-involved enzyme mimics, Ag₁₅SiW₁₂ exhibits excellent peroxidase-like activity. Detection of H₂O₂ was also studied in buffer solution, and the result shows broader application range (1–100?μM), faster response time (1?min) and lower detection limit (0.27?μM), which may be ascribed to the distinct structure and maximized synergistic effect of individual components.

     

Bridged nucleic acid conjugates at 6'-thiol: synthesis, hybridization properties and nuclease resistances

The bridged nucleic acid (BNA) containing a thiol at the 6'-position in the bridged structure was synthesized from the disulfide-type BNA and conjugated with various functional molecules via the thioether or the disulfide linkage post-synthetically and efficiently in solution phase. The disulfide-linked conjugate was cleaved under reductive conditions derived from glutathione and an oligonucleotide bearing a free thiol was released smoothly. Conjugated functional molecules had great effects on duplex stability with the DNA complement. In contrast, the molecules little influenced the stability with the RNA complement. Moreover, the oligonucleotides with functional groups at the 6'-position had as high or higher resistances against 3'-exonuclease than phosphorothioate oligonucleotide (S-oligo).     

Synthesis and physical properties of laterally fluoro substituted ferroelectric liquid crystals with a fluoro substituted chiral terminal chain

A new series of ferroelectric liquid crystals (FLCs) has been designed for active matrix displays based upon the chiral smectic C phase. The FLCs have been derived from optically active fluorinated alkanols and a laterally fluoro substituted biphenylyl-1,3-dioxan core. Their physical properties such as spontaneous polarization, current response time, and tilt angle have been determined. The FLC derived from 2-fluoro-octanol showed a very short current response time of 10μs at T _Sm*-N* - T= 10°C, while another FLC with the same core derived from 5-fluoro-octanol gave a value of 150μs.     

Synthesis and ferroelectric properties of laterally substituted fluoro liquid crystals derived from 4-mercaptobenzoic acid

Several new ferroelectric liquid crystals with thiobenzoate mesogenic cores have been synthesized and the influence of a single fluorine atom ortho to the alkyloxy tail on the mesomorphic properties of some two and three benzene ring core compounds has been studied. The two benzene ring derivatives do not display the ferroelectric smectic C phase but the three phenyl ring compounds exhibit a wide temperature range S_c* phase. The transition temperatures and enthalpies of transition for these compounds have been determined; the spontaneous polarization, response time and tilt angle have also been measured and are discussed as a function of the structure near to the chiral centre.     

Synthesis and conformational analysis of a ribo-type cyclohexenyl nucleoside

A straightforward approach to a novel class of ribo-type cyclohexenyl nucleosides is described. An electron-demand Diels-Alder reaction forms the key-step of the chosen synthetic pathway. Although the difference is small, conformational analysis using NMR shows that this nucleoside analogue adopts preferentially an 2H3 conformation (S-type), while the "deoxy" cyclohexenyl analogue has a preference for a C3' endo conformation (N-type). Analyses of the conformational equilibrium reveal that, in the given experimental conditions, the difference between adenosine and its cyclohexenyl congener resides in their different DeltaG values; furthermore, in adenosine, the conformational preference is of enthalpic origin, whereas in the cyclohexenyl congener, the conformational preference is of entropic origin.     

Synthesis and oligodeoxynucleotide binding properties of pyrrolidinyl peptide nucleic acids bearing prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones

A series of novel conformationally rigid pyrrolidinyl peptide nucleic acids (PNA) based on d-prolyl-2-aminocyclopentanecarboxylic acid (ACPC) backbones has been synthesized. Investigation of the binding properties of four stereoisomeric PNAs possessing different stereochemistry at the ACPC part with DNA revealed that a precise stereochemistry of the backbone is very important in determining the binding properties. Only the PNA containing (1S,2S)-ACPC can form a very stable 1:1 complex with the complementary DNA in a sequence-specific manner.     

Synthesis and properties of a bridged nucleic acid with a perhydro-1,2-oxazin-3-one ring

A novel derivative of 2',4'-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with an N-O linkage in the six-membered bridged structure is the unique structural feature of the novel 2',4'-bridged nucleic acid analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl function, which would improve the properties of the modification. The synthesized monomer was incorporated into oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be higher than that of the corresponding natural and 2',4'-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA modified oligonucleotide to 3'-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover, ring-opening of only one modification at the 3'-end of the oligonucleotides was observed, even if two or three HxNA modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in antisense technology.     

Synthesis of N-(fluoro phenyl) maleamic acids and N-(fluoro phenyl) maleimides

A series of seven N-(fluoro phenyl) maleamic acids and their N-(fluoro phenyl) maleimides were prepared by the reaction of fluoro phenyl amines ( o-, m-, and p-fluoro, 2.4-, 2.5-difluoro, 2.3.5.6-tetrafluoro and 2.3.4.5.6.-pentafluoro anilines) with maleic anhydride according to substantial modifications made to the reaction conditions used by Searle for the preparation of normal N-aryl maleimides.     

2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH

Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.     

Synthesis and properties of oligonucleotides that contain a triazole‐linked nucleic acid dimer

New chemically modified oligonucleotides at the site of the backbone are needed to improve the properties of oligonucleotides. A practical synthesis for a triazole‐linked nucleoside dimer based on a PNA‐like structure has been developed. This involves synthesizing two uracil‐based monomers that contain either an azide or an alkyne functionality, followed by copper‐catalyzed 1,3‐dipolar cycloaddition. This dimer was incorporated within an oligonucleotide via phosphoramidite chemistry and UV‐monitored thermal denaturation data illustrates slight destabilization relative to its target complementary sequence. This chemically modified dimer will allow for a future investigation of its properties within DNA and RNA‐based applications. J. Heterocyclic Chem., (2011).     

Synthesis and properties of a stable 6-stannapentafulvene

The first donor-free 6-stannapentafulvene stable at ambient temperature was synthesized and isolated, exhibiting the shortest tin-carbon bond length among those previously reported.     

Synthesis of a New Bivalent Hirudin Analog (hirufos), which includes a stable 4′-phosphono-L-phenylalanine mimic of (L-tyrosine O4-sulfate)-63

The synthesis on solid phase of a new derivative of the anticoagulant protein hirudin is described (see Scheme and Fig.1, I ). The henicosapeptide is a bivalent conjugate of the C-terminus of hirudin and of the active-site-binding tetrapeptide D -Phe-Pro-Arg-Pro linked via a tetraglycine spacer. The peptide, for which the name hirufos was coined, incorporates a stable phosphono derivative of L -phenylalanine which, combined with the other structural modifications, leads to a potent anticoagulant agent. Synthesis was readily achieved by the (9H-fluoren-9-yl)-methoxycarbonyl (Fmoc) strategy followed by acidolytic cleavage from the resin and deprotection, including the liberation of the crucial phosphonic group on L -phenylalanine.     

Backbone modifications of aromatic peptide nucleic acid (APNA ) monomers and their hybridization properties with DNA and RNA

Aromatic peptide nucleic acid (APNA) monomers containing N-(2-aminobenzyl)-glycine, N-(2-aminobenzyl)-(R)- or -(S)-alanine, and N-(2-aminobenzyl)-beta-alanine moieties as part of their backbone were synthesized. These novel analogues were incorporated as a single "point mutation" in PNA hexamers, and their physicochemical properties were investigated by UV thermal denaturation and CD experiments. Destabilization in triplex formation between the PNA-APNA chimeras and complementary DNA or RNA oligomers was observed, as compared to the PNA control. The APNA monomer composed of the N-(2-aminobenzyl)-glycine backbone led to the smallest decrease in the thermal stability of the triplexes formed with DNA and RNA, while maintaining selectivity for base-pairing recognition. Since the PNA-APNA chimeras are more lipophilic than the corresponding PNA homopolymers, these oligomers may also exhibit better cell membrane permeability properties.     

Synthesis and RNA binding selectivity of oligonucleotides modified with five-atom thioacetamido nucleic acid backbone structures

Convenient chemical synthesis and incorporation of dithymidine and thymidine-cytidine dimer blocks connected with a five-atom amide linker N3'-CO-CH2-S-CH2 into oligonucleotides (ONs) are reported. The UV-Tm experiments for binding affinities of these mixed backbone ONs with complementary DNA and RNA sequences revealed important results such as significantly higher RNA-binding selectivity as compared with complementary DNA. NMR studies of the dimer blocks suggested a marginal increase in the N-type sugar conformations over that of the native DNA.