(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.     

Cyclohexanyl peptide nucleic acids (chPNAs) for preferential RNA binding: effective tuning of dihedral angle beta in PNAs for DNA/RNA discrimination

[structures: see text] A serious drawback of peptide nucleic acids (PNAs) from an application perspective that has not been adequately dealt with is nondiscrimination of identical DNA and RNA sequences. An analysis of the available X-ray and NMR solution structures of PNA complexes with DNA and RNA suggested that it might be possible to rationally impart DNA/RNA duplex binding selectivity by tuning the dihedral angle beta of the flexible ethylenediamine part of the PNA backbone (II) via suitable chemical modifications. Cyclohexanyl PNAs (chPNAs) with beta approximately = 65 degrees were designed on the basis of this rationale. The chPNAs introduced remarkable differences in duplex stabilities among their DNA and RNA complexes, with melting temperatures (deltaTm(RNA-DNA) = +16-50 degrees C) depending on the number of modifications and the stereochemistry. This is a highly significant, exceptional binding selectivity of a mix sequence of PNA to RNA over the same DNA sequence as that seen to date. In contrast, cyclopentanyl PNAs (cpPNAs) with beta approximately = 25 degrees hybridize to DNA/RNA strongly without discrimination because of the ring puckering of the cyclopentane ring. The high affinity of chPNAs to bind to RNA without losing base specificity will have immediate implications in designing improved PNAs for therapeutic and diagnostic applications.     

Cyclopropane PNA: observable triplex melting in a PNA constrained with a 3-membered ring

The first peptide nucleic acid (PNA) with a cyclopropane in the backbone has been synthesized, and the effects of the ring on DNA/RNA binding properties of the PNA have been examined. Well-defined triplex to duplex melting transitions of PNA₂ DNA complexes is clearly observed by variable temperature UV absorbance with the cyclopropane-constrained PNA.     

trans-5-aminopipecolyl-aegPNA chimera: design, synthesis, and study of binding preferences with DNA/RNA in duplex/triplex mode

The design and synthesis of novel chiral PNA monomer based on trans-5-aminopipecolic acid is reported. The trans diequatorial disposition of the 1,4 ring substituents in six-membered 5-aminopipecolic acid derivative could be favorable over trans 1,3 axial-equatorial disposition in 4-aminopipecolic acid of PNA. Studies on the synthesis of trans-4/5-aminopipecolyl PNA-eagPNA chimeras and their binding preferences to DNA/RNA in duplex/triplex modes are described.     

Synthesis and evaluation of (1S,2R/1R,2S)-aminocyclohexylglycyl PNAs as conformationally preorganized PNA analogues for DNA/RNA recognition

Conformationally constrained cis-aminocyclohexylglycyl PNAs have been designed on the basis of stereospecific imposition of 1,2-cis-cyclohexyl moieties on the aminoethyl segment of aminoethylglycyl PNA (aegPNA). The introduction of the cis-cyclohexyl ring may allow the restriction of the torsion angle beta in the ethylenediamine segment to 60-70 degrees that is prevalent in PNA(2):DNA and PNA:RNA complexes. The synthesis of the optically pure monomers (10a and 10b) is achieved by stereoselective enzymatic hydrolysis of an intermediate ester 2. The chiral PNA oligomers were synthesized with (1S,2R/1R,2S)-aminocyclohexylglycyl thymine monomers in the center and N-terminus of aegPNA. Differential gel shift retardation with one or more units of modified monomer units was observed as a result of hybridization of PNA sequences with complementary DNA sequences. Hybridization studies with complementary DNA and RNA sequences using UV-T(m) measurements indicate that PNA with (1S,2R)-cyclohexyl stereochemistry enhances selective binding with RNA over DNA as compared to control aegPNA and PNA with the other (1R,2S) isomer.     

Base dependent pyrrolidine ring pucker in aep-PNA monomers: NMR and PSEUROT analysis

The aep-PNA is a chiral and cyclic PNA analogue, which has a stronger and base dependent binding affinity with complementary DNA. To understand the base dependent properties at monomer level, the structural studies of aep-PNA-(T/C/A) monomers have been carried out focussing on the conformational analysis of pyrrolidine ring pucker in aep-PNA by ¹H NMR and the coupling constant data fitted into PSEUROT software. The results indicate that the type of pyrrolidine pucker depends on the electronic nature of substituent, implying the effect of pyrimidine or purine substituents in determining the ring pucker in monomers. This may consequently influence the aep-PNA oligomer conformation. Since pyrrolidine nucleic acids have emerged as an important class of PNA analogues, present results may have importance for their future development.     

Backbone-extended pyrrolidine peptide nucleic acids (bepPNA): design, synthesis and DNA/RNA binding studies

One-carbon extended conformationally constrained pyrrolidine PNA monomer (bepPNA) has been synthesized, incorporated into PNA sequences at predefined positions, and showed selective RNA binding properties.     

Synthesis and supramolecular assemblies of bipolar archaeal glycolipid analogues containing a cis-1,3-disubstituted cyclopentane ring

Unsymmetrical archaeal tetraether glycolipid analogues 1-2 incorporating a 1,3-disubstituted cyclopentane ring into the bridging chain have been synthesized. The cyclopentane has been introduced with a totally controlled cis configuration, either into the middle of the aliphatic chain or at three methylene groups from the glycerol unit linked to the bulkier disaccharide residue. Freeze-fracture and cryotransmission electron microscopy experiments clearly demonstrated unprecedented glycolipid supramolecular organizations involving two-by-two monolayer associations coupled with interconnection and fusion phenomena. Furthermore, a significant difference in the hydration properties and in the lyotropic liquid crystalline behavior of bipolar lipids 1-2 was found depending on the position of the cyclopentane residue.     

PNA-DNA duplexes, triplexes, and quadruplexes are stabilized with trans-cyclopentane units

Peptide nucleic acids (PNAs) are non-natural nucleic acid mimics that bind to complementary DNA and RNA with high affinity and selectivity. PNA can bind to nucleic acids in a number of different ways. Currently, the formation of PNA-oligonucleotide duplex, triplex, and quadruplex structures have been reported. PNAs have been used in numerous biomedicial applications, but there are few strategies to predictably improve the binding properties of PNAs by backbone modification. We have been studying the benefits of incorporating (S,S)-trans-cyclopentane diamine units (tcyp) into the PNA backbone. In this Communication, we report the improvement in stability associated with tcyp incorporation into PNA-DNA duplexes, triplexes, and quadruplexes. The broad utility of this modification across multiple types of PNA structures is unique and should prove useful in the development of applications that rely on PNA.     

Enhanced stability of G-quadruplexes from conformationally constrained aep-PNA backbone

Nucleic (DNA) acids having contiguous stretch of G sequence form quadruplex structure, which is very critical to control cell division. Recently the existence of G-quadruplex in RNA is also reported in presence of monovalent metal ion. PNA is a promising DNA analogue which binds strongly to DNA to form PNA:DNA duplex or PNA(2):DNA triplex. PNA also forms quadruplexes such G-quadruplex and i-motif in G and C-rich sequences respectively. aep-PNA containing a prolyl ring is one of several PNA analogues that provide rigidity and chirality in backbone and has binding affinity to natural DNA which is higher than that of PNA. Here we examine the ability of aep-PNA-G to form a quadruplex by UV, CD and mass spectroscopic techniques.     

Biosynthetic studies on the cyclopentane ring formation of allosamizoline, an aminocyclitol component of the chitinase inhibitor allosamidin

Allosamizoline (1) is an aminocyclitol component of allosamidin, a Streptomyces metabolite, and has a cyclopentane ring originated from D-glucosamine. Biosynthesis of the cyclopentane ring was studied by feeding experiments with a variety of deuterium-labeled glucosamine and glucose. In the feeding experiments with [3-(2)H]- and [4-(2)H]-D-glucosamine and [1-(2)H]-D-glucose, deuterium was incorporated into C-3, C-4, and C-1 of 1, respectively. On the other hand, feeding experiments with [5-(2)H]- and [6,6-(2)H(2)]-D-glucosamine showed that deuterium on C-5 and one of the two deuterium atoms on C-6 of glucosamine were lost during the cyclopentane ring formation of 1. In the feeding experiments with (6R)- and (6S)-[6-(2)H(1)]-D-glucose, the (6R)-deuterium of glucose was incorporated into the proS position on C-6 of 1, but the (6S)-deuterium of glucose was not incorporated into 1. These results suggested that an intermediate with a 6-aldehyde group is involved in the biosynthesis of the cyclopentane ring moiety of 1 and overall inversion of stereochemistry of the C-6 methylene group occurred by stereospecific oxidation and reduction on C-6 during the formation of 1. The 6-aldehyde intermediate may play a key role in the biosynthetic step(s) of cyclization to form the cyclopentane ring and/or deoxygenation at C-5.     

Saturated hydrocarbon-benzene complexes: theoretical study of cooperative CH/pi interactions

High-level ab initio calculations at the CCSD(T)/aug-cc-pVTZ//MP2/aug(d,p)-6-311G(d,p) level were employed to investigate the cooperative CH/pi effects between the pi face of benzene and several modeled saturated hydrocarbons, propane, isobutane, cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclopentane, cyclooctane, and bicyclo[2.2.2]octane. In all cases, multiple C-H groups (2-4) are found to interact with the pi face of benzene, with one C-H group pointing close to the center of the benzene ring. The geometries of these complexes are governed predominantly by electrostatic interaction between the interacting systems. The calculated interaction energies (10-14 kJ mol(-1)) are 2-3 times larger than that of the prototypical methane-benzene complex. The trends of geometries, interaction energies, binding properties, as well as electron-density topological properties were analyzed. The calculated interaction energies correlate well with the polarizabilities of the hydrocarbons. AIM analysis confirms the hydrogen-bonded nature of the CH/pi interactions. Significant changes in proton chemical shift and stretching frequency (blue shift) are predicted for the ring C-H bond in these complexes.     

Quantum chemical studies of peptide nucleic acid monomers and role of cyclohexyl modification on backbone flexibility

Peptide nucleic acids (PNA) bind sequence specifically to DNA/RNA and are of major interest for all fields of molecular biology and could form the basis for gene‐targeted drugs. Modifications are introduced in PNA to overcome problems associated with orientational selectivity in binding, to restrict conformational flexibility of backbone, and to discriminate binding for either DNA or RNA. The addition of geometrical isomers (1R,2S and 1S,2R) of cyclohexyl ring in the backbone of PNA could bring rigidification to PNA backbone and may impart specificity toward RNA. Therefore, quantum chemical studies are aimed to explore the conformational space, to find out preferred stable conformations of PNA and modified (1R,2S and 1S,2R) cyclohexyl PNA monomer. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications

The ability of peptide nucleic acids (PNA) to form specific higher-order (i.e., three- and four-stranded) complexes with DNA makes it an ideal structural probe for designing strand-specific dsDNA biosensors. Higher-order complexes are formed between a dye-labeled charge-neutral PNA probe and complementary dsDNA. Addition of a light-harvesting cationic conjugated polymer (CCP) yields supramolecular structures held together by electrostatic forces that incorporate the CCP and the dye-labeled PNA/DNA complexes. Optimization of optical properties allows for excitation of the CCP and subsequent fluorescence resonance energy transfer (FRET) to the PNA-bound dye. In the case of noncomplementary dsDNA, complexation between the probe and target does not occur, and dye emission is weak. The binding between PNA and noncomplementary and complementary dsDNA was examined by several methods. Gel electrophoresis confirms specificity of binding and the formation of higher-order complexes. Nano-electrospray mass spectrometry gives insight into the stoichiometric composition, including PNA/DNA, PNA(2)/DNA, PNA/DNA(2), and PNA(2)/DNA(2) complexes. Finally, structural characteristics and binding-site specificity were examined using ion mobility mass spectrometry in conjunction with molecular dynamics. These results give possible conformations for each of the higher-order complexes formed and show exclusive binding of PNA to the complementary stretch of DNA for all PNA/DNA complexes. Overall, the capability and specificity of binding indicates that the CCP/PNA assay is a feasible detection method for dsDNA and eliminates the need for thermal denaturing steps typically required for DNA hybridization probe assays.     

Reversibly controllable guest binding in precisely defined cavities: selectivity, induced fit, and switching in novel resorcin[4]arene-based container molecules

Two molecular baskets are presented, which were constructed based on a resorcin[4]arene platform. The molecules completely surround suitable guests, such as cyclo- or oxacycloalkanes, and bind them with high strength. The thermodynamic parameters for inclusion complexation were determined as well as the influence of encapsulation on the ring inversion barrier of bound cyclohexane. Two-dimensional NMR spectroscopy clearly shows the existence of a directed attractive interaction between oxacyclohexane and one of the hosts, which constrains the rotation of the bound molecule. Both containers exhibit remarkable binding selectivity as a consequence of their precisely defined structures. They both differentiate between homologous cycloalkanes, and whereas cyclohexane binds best within the larger of the two interior cavities, cyclopentane fits best in the smaller one. The selectivity is governed by ideal filling of space. We have conducted molecular dynamics experiments to understand the thermal fluctuations in the cavity sizes when a guest is bound. The simulations show that within a very narrow range the hosts adapt their binding site to different guests in order to optimize the fraction of occupied space. Once a binding geometry is established, it is characterized by a very low degree of flexibility. The guest-hosting properties of both molecules can be suspended by an external stimulus: addition of acid induces an opening of portals in the structures and immediately releases all bound cargo. Neutralization of the solution completely restores the initial state.     

Ring Contraction Through Epoxide Rearrangement: A Formal Synthesis of Capsorubin

An eight-step synthesis of the cyclopentane keto-alcohol 2, which has previously been converted in one step into the carotenoid pigment capsorubin (1), is described. The key step in our synthesis is a stereospecific epoxide rearrangement with ring contraction, thus producing the cyclopentane ring from an epoxide of a cyclohexene.     

Nagelamide J, a novel dimeric bromopyrrole alkaloid from a sponge Agelas species

A novel dimeric bromopyrrole alkaloid, nagelamide J (1), with antimicrobial activity has been isolated from an Okinawan marine sponge Agelas species, and the structure and stereochemistry were elucidated from spectroscopic data. Nagelamide J (1) is the first bromopyrrole alkaloid possessing a cyclopentane ring fused to an amino imidazole ring.     

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.     

Stabilization of zwitterions in solution: GABA analogues

The solution-phase structures of a number of conformationally restricted gamma-aminobutyric acid (GABA) analogues are investigated at the MP2/6-31+G* level of theory, using both explicit water molecules and the conductor-like screening solvation model (COSMO) to model solvation. GABA analogues constrained in a cis conformation by either a double bond or cyclopropane ring have the potential to attain either folded, intramolecularly hydrogen-bonded, or partially folded conformations in solution. Systems constrained in a cis conformation by a cyclopentane or cyclopentene ring are more conformationally restricted and exist only in a folded, intramolecularly hydrogen-bonded form. GABA analogues constrained in a trans conformation by either a double bond or cyclopropane ring have the potential to adopt either partially folded or fully extended conformations in solution. Due to a lack of conformational flexibility, analogues that are constrained in a trans conformation by a cyclopentane or cyclopentene ring attain only partially folded conformations. Like GABA, conformationally flexible GABA analogues possess a large number of stable rotamers, and may exist in any or all of these conformations in aqueous solution. The structures of these analogues provide an essential foundation for subsequent structure-activity analysis of ligand binding at GABA receptors and transporters. This work is therefore expected to facilitate the design and development of new biologically active GABA analogues to treat GABA-related neurological disorders.     

An unusual samarium diiodide mediated reductive ring contraction of a tricyclic oxazine to a highly-functionalized cyclopentane and cyclobutane

[reaction--see text] Samarium diiodide mediated reductive ring contraction of a substituted tricyclo[2.2.2]oxazine at 25 degrees C leads to a mixture of cyclopentane and cyclobutane rearrangement products with complete diastereoselectivity in each case. At -78 degrees C, the anticipated amidocyclohexanol reduction product is obtained exclusively, while the cyclopentane is the sole product at reflux in THF.