cis-Cyclopentyl PNA (cpPNA) as constrained chiral PNA analogues: stereochemical dependence of DNA/RNA hybridization

DNA/RNA hybridization studies of PNA-T oligomers with cis-(1S,2R/1R,2S)-cyclopentyl units in the backbone show stereochemistry dependent binding with RNA/DNA discrimination.     

Organoboranes: XXVIII. Convenient procedures for the synthesis of borinane

Convenient syntheses of the six-ring boracyclane, borinane, have been developed. Hydroboration of 1,4-pentadiene with two molar equivalents of 9-borabicyclo-[3.3.1]nonane (9-BBN) in a suitable solvent, followed by reaction of the resulting trialkylborane with one molar equivalent of the borane-tetrahydrofuran complex (BH₃·THF) or the borane-dimethyl sulfide complex (BH₃·SMe₂, BMS), leads to the cyclization of the pentadiene moiety, forming borinane with the regeneration of two molar equivalents of 9-BBN. Complete separation of the two dialkylboranes by fractional crystallization from solvents such as THF, 1,2-dimethoxyethane (DME), 1,3-dioxolane, n-hexane, n-pentane and hexane plus dioxolane was unsuccessful. Treating the reaction mixture in hexane with the requisite amount of triethylamine (Et₃N) led to selective complexation with borinane. By cooling the reaction mixture to ?78°C, it was now possible to crystallize out the uncomplexed 9-BBN almost quantitatively (98%). Alternatively, borinane could be precipitated selectively from the hexane reaction mixture as its bis-adduct with either N, N, N′, N′-tetramethylethylenediamine (TMED) or 1,4-diazabicyclo[2.2.2]octane (DABCO). Free borinane was readily liberated from its amine adducts by treatment with boron trifluoride etherate (BF₃·OEt₂). Finally, the two dialkylboranes were readily separated by fractional distillation, using a specially designed reaction setup. Distillation at about 70–80°C and 0.01 mm pressure led to the clean distillation of borinane, leaving the 9-BBN, as a residue, readily recycled in subsequent preparations. Alternatively, once free borinane has been obtained, it can be used to hydroborate 1,4-pentadiene in place of 9-BBN. Treatment of the product with BH₃·THF or BH₃·SMe₂ converts two moles of borinane into three, without the formation of a by-product.     

Chimeric (aeg-pyrrolidine)PNAs: synthesis and stereo-discriminative duplex binding with DNA/RNA

The design and facile conversion of naturally occurring 4-hydroxyproline to all four diastereomers of thymine pyrrolidine PNA monomer, (2R,4S)-adenine, -guanine and -cytosine monomers and their incorporation into duplex forming PNA oligomers is reported. The interesting results of the hybridization studies with complementary DNA/RNA sequences in either parallel or antiparallel orientation reveal the stereochemistry-dependent DNA vs. RNA discriminations and parallel/antiparallel orientation selectivity.     

Pyrrolidine nucleic acids: DNA/PNA oligomers with 2-hydroxy/aminomethyl- 4-(thymin-1-yl)pyrrolidine-N-acetic acid

Synthesis of pyrrolidine-based chiral positively charged DNA analogues is reported. The synthesis of (2S,4S) and (2R,4R) thymin-1-ylpyrrolidine-N-acetic acid, its site specific incorporation in PNA:DNA chimera and PNA, and the study of their binding properties with complementary DNA/RNA sequences is presented.     

Chimeric peptide nucleic acids incorporating (2S,5R)-aminoethyl pipecolyl units: synthesis and DNA binding studies

The design and facile synthesis of a novel chiral six-membered PNA analogue (2S,5R )-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic acid, aepipPNA, that upon incorporation into PNA sequences effected stabilization of complexes with target complementary DNA. This is the first example where a six membered-PNA is shown to be capable of forming stable complexes with DNA and further expands the repertoire of cyclic PNA analogues.     

A hydroponic rice seedling culture model system for investigating proteome of salt stress in rice leaf

By using an in vivo hydroponic rice seedling culture system, we investigated the physiological and biochemical responses of a model rice japonica cultivar Nipponbare to salt stress using proteomics and classical biochemical methods. Yoshida's nutrient solution (YS) was used to grow rice seedlings. YS-grown 18-day-old seedlings manifested highly stable and reproducible symptoms, prominently the wilting and browning of the 3rd leaf, reduced photosynthetic activity, inhibition in overall seedling growth, and failure to develop new (5th) leaf, when subjected to salt stress by transferring them to YS containing 130 mM NaCl for 4 days. As leaf response to salt stress is least investigated in rice by proteomics, we used the 3rd leaf as source material. A comparison of 2-DE protein profiles between the untreated control and salt-stressed 3rd leaves revealed 55 differentially expressed CBB-stained spots, where 47 spots were increased over the control. Of these changed spots, the identity of 33 protein spots (27 increased and 5 decreased) was determined by nESI-LC-MS/MS. Most of these identified proteins belonged to major metabolic processes like photosynthetic carbon dioxide assimilation and photorespiration, suggesting a good correlation between salt stress-responsive proteins and leaf morphology. Moreover, 2-DE immunoblot and enzymatic activity analyses of 3rd leaves revealed remarkable changes in the key marker enzymes associated with oxidative damage to salt stress: ascorbate peroxidase and lipid peroxidation were induced, and catalase was suppressed. These results demonstrate that hydroponic culture system is best suited for proteomics of salt stress in rice seedling.     

(1S,2R/1R,2S)-cis-cyclopentyl PNAs (cpPNAs) as constrained PNA analogues: synthesis and evaluation of aeg-cpPNA chimera and stereopreferences in hybridization with DNA/RNA

Conformationally constrained chiral PNA analogues were designed on the basis of stereospecific imposition of a 1,2-cis-cyclopentyl moiety on an aminoethyl segment of aegPNA. It is known that the cyclopentane ring is a relatively flexible system in which the characteristic puckering dictates the pseudoaxial/pseudoequatorial dispositions of substituents. Hence, favorable torsional adjustments are possible to attain the necessary hybridization-competent conformations when the moiety is imposed on the conventional PNA backbone. The synthesis of the enantiomerically pure 1,2-cis-cyclopentyl PNA monomers (10a and 10b) was achieved by stereoselective enzymatic hydrolysis of a key intermediate ester 2. The chiral (1S,2R/1R,2S)-aminocyclopentylglycyl thymine monomers were incorporated into PNA oligomers at defined positions and through the entire sequence. Hybridization studies with complementary DNA and RNA sequences using UV-Tm measurements indicate that aeg-cpPNA chimera form thermally more stable complexes than aegPNA with stereochemistry-dependent selective binding of cDNA/RNA. Differential gel shift retardation was observed on hybridization of aeg-cpPNAs with complementary DNA.