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.     

A new route to (2S,4R)- and (2R,4S)-4-hydroxypipecolic acid

Both enantiomers of cis-4-hydroxypipecolic acid have been prepared by asymmetric synthesis using (S)- or (R)-glycidol as the chiral source, and involving a stereoselective hydrogenation of a six-membered cyclic imine. The latter is obtained by reduction and cyclization of a cyano β-hydroxy ketone.     

alpha-L-ribo-configured locked nucleic acid (alpha-L-LNA): synthesis and properties

The syntheses of monomeric nucleosides and 3'-O-phosphoramidite building blocks en route to alpha-L-ribo-configured locked nucleic acids (alpha-L-LNA), composed entirely of alpha-L-LNA monomers (alpha-L-ribo configuration) or of a mixture of alpha-L-LNA and DNA monomers (beta-D-ribo configuration), are described and the alpha-L-LNA oligomers are studied. Bicyclic 5-methylcytosin-1-yl and adenine-9-yl nucleoside derivatives have been prepared and the phosphoramidite approach has been used for the automated oligomerization leading to alpha-L-LNA oligomers. Binding studies revealed very efficient recognition of single-stranded DNA and RNA target oligonucleotide strands. Thus, stereoirregular alpha-L-LNA 11-mers containing a mixture of alpha-L-LNA monomers and DNA monomers ("mix-mer alpha-L-LNA") were shown to display DeltaT(m) values of +1 to +3 degrees C per modification toward DNA and +4 to +5 degrees C toward RNA when compared with the corresponding unmodified DNA x DNA and DNA x RNA reference duplexes. The corresponding DeltaT(m) values per modification for the stereoregular fully modified alpha-L-LNA were determined to be +4 degrees C (against DNA) and +5 degrees C (against RNA). 11-Mer alpha-L-LNAs (mix-mer alpha- L-LNA or fully modified alpha- L-LNA) were shown in vitro to be significantly stabilized toward 3'-exonucleolytic degradation. A duplex formed between RNA and either mix-mer alpha-L-LNA or fully modified alpha-L-LNA induced in vitro Escherichia coli RNase H-mediated cleavage, albeit very slow, of the RNA targets at high enzyme concentrations.     

Analogues of a locked nucleic acid with three-carbon 2',4'-linkages: synthesis by ring-closing metathesis and influence on nucleic acid duplex stability and structure

Two bicyclic 2'-deoxynucleoside analogues containing a saturated and an unsaturated three-carbon 2',4'-linkage, respectively, have been synthesized using a ring-closing metathesis-based linear strategy starting from uridine. Both analogues have been incorporated into oligodeoxynucleotide sequences and increased the stability of DNA:RNA hybrid duplexes (DeltaT(m) approximately 2.5-5.0 degrees C per modification) and decreased the stability of dsDNA duplexes (DeltaT(m) approximately 2.5-1.0 degrees C per modification). CD spectroscopy revealed that the bicyclic nucleosides induced formation of A-type-like duplexes albeit to a lesser degree than found for locked nucleic acid (LNA) monomers. From the CD data and UV melting analysis, we propose that the 2'-oxygen atom of the bicyclic moiety is essential for the formation of stabilized A-type-like dsDNA but not for the formation of a stabilized A-type DNA:RNA hybrid.     

Synthesis and thermal denaturation studies of novel 2'-O,3'-C-linked bicyclic oligonucleotides with a methoxy or a piperazino group facing the major groove of nucleic acid duplexes

With the aim of evaluating duplex stabilities of oligonucleotides (ONs) with major groove facing functionalities, two novel 2'-O,3'-C-linked bicyclic nucleoside phosphoramidite building blocks were synthesized by routes involving regioselective O-methylation or piperazine attachment using carbonyldiimidazole coupling chemistry. The novel monomers were incorporated into 9-mer mixed base ONs and the thermal stability toward complementary single stranded DNA and RNA was evaluated by thermal denaturation experiments. O-methylated ONs confirmed the applicability of the functionalized bicylic sugar unit for attachment of groups facing the major groove and satisfactory binding properties towards the RNA complement were observed. For the piperazino modified ONs, experiments were performed in aqueous buffers with low (40 mM) and medium (110 mM) salt concentrations, at pH 5 and pH 7. A change from a medium to a low salt concentration induced a significant relative increase in the thermal stability of modified duplexes toward both DNA and RNA complements, which suggests protonation of the piperazino group under the experimental conditions applied.     

LNA (locked nucleic acid) and analogs as triplex-forming oligonucleotides

The triplex-forming abilities of some conformationally restricted nucleotide analogs are disclosed and compared herein. 2'-Amino-LNA monomers proved to be less stabilising to triplexes than LNA monomers when incorporated into a triplex-forming third strand. N2'-functionalisation of 2'-amino-LNA monomers with a glycyl unit induced the formation of exceptionally stable triplexes. Nucleotide analogs containing a C2',C3'-oxymethylene linker (E-type furanose conformation) or a C2',C4'-propylene linker (N-type furanose conformation) had no significant effect on triplex stability proving that conformational restriction per se is insufficient to stabilise triplexes.     

An efficient route to 4-halophosphaisocoumarins via CuX2-mediated direct halocyclization of 2-(1-alkynyl)phenylphosphonic acid diesters

A series of 4-halophosphaisocoumarins were synthesized via CuX2 (X = Br, Cl) -mediated direct halocyclization of 2-(1-alkynyl)phenylphosphonic acid diesters in dichloroethane with the addition of n-Bu4NX or/and AgI in good to excellent yields. This reaction provides an efficient route to 4-halophosphaisocoumarins and represents the first example of bromo- and chlorocyclization of unsaturated phosphonic acid diesters.     

Syntheses and base-pairing properties of locked nucleic acid nucleotides containing hypoxanthine, 2,6-diaminopurine, and 2-aminopurine nucleobases

Second generation 2'-O,4'-C-methylene-linked nucleotides 1-3 containing hypoxanthine, 2,6-diaminopurine, and 2-aminopurine nucleobases were synthesized and incorporated into locked nucleic acid (LNA) oligonucleotides by means of the automated phosphoramidite method. The required phosphoramidite monomeric units were efficiently prepared via convergent synthesis. The glycosyl donor 4 was stereoselectively coupled with hypoxanthine and 6-chloro-2-aminopurine to give the 4'-C-branched nucleosides 5 and 17. The methods for conversion of 5 and 17 into phosphoramidites 11, 25, and 29 were developed and described in full details for the first time. Hybridization properties of LNA octamers containing the new LNA nucleotides were assessed against perfect and singly mismatched DNA. The binding studies revealed that all LNA octamers hybridize very efficiently to DNA following Watson-Crick base-pairing rules with increased binding affinity compared to the DNA analogues. The unique properties of the nucleotides 1-3 make them very useful for further strengthening of the LNA technology.     

A new and efficient chemoenzymatic route to both enantiomers of 4-hydroxycyclohex-2-en-1-one

[reaction: see text] A chemoenzymatic synthesis of both enantiomers of the pharmacologically interesting 4-hydroxycyclohex-2-en-1-one in three steps starting from 3-methoxycyclohex-2-en-1-one is described. Manganese(III) acetate-mediated acetoxylation followed by enzyme-mediated hydrolysis of alpha-acetoxy enone affords acetoxy enone 3 and hydroxy enone 4 with high enantiomeric excesses and in good yields. The reduction of the acetoxy and hydroxy enones furnished both enantiomers of 4-hydroxycyclohex-2-en-1-one in high enantiomeric excess.     

An efficient route to 4-(substituted benzyl)piperidines

A novel approach to 4-(substituted benzyl)piperidines has been developed. The key steps involve the cyclization of imines bearing an allylsilane in the side-chain followed by the palladium-catalyzed cross-coupling of the resulting 4-methylenepiperidine with organoboronic acids under an atmosphere of oxygen.     

A concise and efficient route to the Alzheimer's therapeutic agent (R)-arundic acid

A short and efficient procedure for the preparation of (R)-arundic acid, a therapeutic agent for the treatment of Alzheimer's disease, has been developed. Based on cheap and commercially available (1R)-(+)-camphor as the source of chiral information, (R)-arundic acid is synthesized in a four-step cyclic sequence with 55% overall yield and high optical purity, ≥98% ee. Alkyl halide and acetylene constitute the only consumable carbon sources of this method, which allows obtaining of both enantiomers and recycling of chiral auxiliary.     

Parallel nucleic acid recognition by the LNA (locked nucleic acid) stereoisomers beta-L-LNA and alpha-D-LNA; studies in the mirror image world

Two LNA (locked nucleic acid) stereoisomers (beta-L-LNA and alpha-D-LNA) are evaluated in the mirror-image world, that is by the study of two mixed sequences of LNA and alpha-L-LNA and their L-DNA and L-RNA complements. Both are found to display high-affinity RNA-recognition by the formation of duplexes with parallel strand orientation.     

Functionalized LNA (locked nucleic acid): high-affinity hybridization of oligonucleotides containing N-acylated and N-alkylated 2''-amino-LNA monomers

A molecular dynamics (MD) simulation, synthesis and very efficient hybridization for a series of N-acylated and N-alkylated derivatives of 2'-amino-LNA are reported.     

A simplified and efficient bromine-facilitated SF4-preparation method

A novel, simple method for SF₄-preparation has escaped discovery over the last 50 years of sulfur-fluorine chemistry and is described in the present study. Sulfur tetrafluoride is readily produced according to the equation:

S + (2 + x)Br₂ + 4KF → SF₄↑ + xBr₂ + 4KBr     

Alpha-LNA (locked nucleic acid with alpha-D-configuration): synthesis and selective parallel recognition of RNA

Alpha-LNA is presented as a stereoisomer of LNA (locked nucleic acid) with alpha-D-configuration. Three different approaches towards the thymine alpha-LNA monomer as well as the 5-methylcytosine alpha-LNA monomer are presented. Different alpha-LNA sequences have been synthesised and their hybridisation with complementary DNA and RNA has been evaluated by means of thermal stability experiments and circular dichroism spectroscopy. In a mixed pyrimidine sequence, alpha-LNA displays unprecedented parallel-stranded and selective RNA binding. Furthermore, a remarkable selectivity for hybridisation with RNA over DNA is indicated.     

Palladium-catalyzed [2 + 2 + 2] cocyclotrimerization of benzynes with bicyclic alkenes: an efficient route to anellated 9,10-dihydrophenanthrene derivatives and polyaromatic compounds

An efficient method for the cocyclotrimerization of bicyclic alkenes and benzynes catalyzed by palladium phosphine complexes to give the corresponding norbornane anellated 9,10-dihydrophenanthrene derivatives is described. Bicyclic alkenes 1a-i undergo [2 + 2 + 2] cocyclotrimerization with benzynes generated from precursors 2a-d [2-(trimethylsilyl)phenyl triflate (2a), 4,5-dimethyl-2-(trimethylsilyl)phenyl triflate (2b), 6-(trimethylsilyl)-2,3-dihydro-1H-5-indenyl triflate (2c), 4-methyl-2-(trimethylsilyl)phenyl triflate (2d)] in the presence of PdCl(2)(PPh(3))(2) in acetonitrile at ambient temperature to yield anellated 9,10-dihydrophenanthrene products 3a-r in moderate to excellent yields. The [2 + 2 + 2] cocyclotrimerization products from oxa- and azabicyclic alkenes can be applied for the synthesis of polyaromatics, substituted benzo[b]triphenylenes (8a-f), via a simple Lewis acid mediated deoxyaromatization in good yields. In addition the [2 + 2 + 2] products undergo retro Diels-Alder reaction readily, providing a new method for the synthesis of substituted phenanthrenes and for generating isobenzofurans. A plausible mechanism is proposed to account for the catalytic [2 + 2 + 2] cycloaddition reaction.     

A facile and general route to 4-substituted-2 butenolides

Grignard reagents react with 7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic anhydride and produce in high yields the corresponding substituted bicyclo-γ-butanolides. These lactones produce the title compounds by retro Diels-Alder reaction during distillation.