Homopolymeric pyrrolidine-amide oligonucleotide mimics: Fmoc-synthesis and DNA/RNA binding properties

By chemically modifying or replacing the backbone of oligonucleotides it is possible to modulate the DNA and RNA recognition properties and fine-tune the physiochemical properties of oligomers. This is important because it challenges our understanding of natural nucleic acid structural and recognition properties and can lead to nucleic acid mimics with a wide range of applications in nucleic acid targeting, analysis or diagnostics. In this paper we describe the solid phase synthesis of pyrrolidine-amide oligonucleotide mimics (POMs) using Fmoc-peptide chemistry. This required the synthesis of adeninyl, cytosinyl, thyminyl and guaninyl pyrrolidine monomers, with Fmoc- and standard acyl-protecting groups on the exocyclic amino groups and nucleobases respectively. These monomers were used to synthesise several thyminyl and adeninyl POM pentamers, with modest coupling efficiency. The pentamers were purified by RP-HPLC, characterised by mass spectrometry and their DNA and RNA binding properties were investigated using UV thermal denaturation/renaturation experiments. This revealed that all the pentamers exhibit strong affinity for complementary nucleic acids. The further evaluation of longer mixed-sequence POMs is described in a second accompanying paper (R. J. Worthington et al., Org. Biomol. Chem., 2006, DOI: 10.1039/b613386j).     

Nucleic acid binding properties of thyminyl and adeninyl pyrrolidine-amide oligonucleotide mimics (POM)

Adeninyl POM was prepared using solid-phase peptide chemistry and shown to exhibit higher affinity for complementary DNA and RNA than the corresponding adeninyl PNA.     

Biophysical and cellular-uptake properties of mixed-sequence pyrrolidine-amide oligonucleotide mimics

Previously we introduced the positively charged pyrrolidine-amide oligonucleotide mimics (POM), which possess a pyrrolidine ring and amide linkage in place of the sugar-phosphodiester backbone of natural nucleic acids. Short POM homo-oligomers have shown promising DNA and RNA recognition properties. However, to better understand the properties of POM and to assess their potential for use as modulators of gene expression and bioanalytical or diagnostic tools, more biologically relevant, longer, mixed-sequence oligomers need to be studied. In light of this, several mixed-sequence POM oligomers were synthesised, along with fluorescently labelled POM oligomers and a POM-peptide conjugate. UV thermal denaturation showed that mixed-sequence POMs hybridise to DNA and RNA with high affinity but slow rates of association and dissociation. The sequence specificity, influence of terminal amino acids, and the effect of pH and ionic strength on the DNA and RNA hybridisation properties of POM were extensively investigated. In addition, isothermal titration calorimetry (ITC) was used to investigate the thermodynamic parameters of the binding of a POM-peptide conjugate to DNA. Cellular uptake experiments have also shown that a fluorescently labelled POM oligomer is taken up into HeLa cells. These findings demonstrate that POM has the potential for use in a variety of applications, alongside other modified nucleic acids developed to date, such as peptide nucleic acids (PNA) and phosphoramidate morpholino oligomers (PMO).     

RNA-selective cross-pairing of backbone-extended pyrrolidine-amide oligonucleotide mimics (bePOMs)

Pyrrolidine-amide oligonucleotide mimics (POMs) can cross-pair strongly with complementary parallel and antiparallel DNA and RNA targets in a sequence-specific fashion. As a result POMs have significant potential for applications including in vivo gene silencing, diagnostics and bioanalysis. To further modulate the DNA- and RNA-recognition properties and fine-tune the physiochemical properties of POMs for nucleic acid targeting, backbone-extended pyrrolidine-amide oligonucleotide mimics (bePOM I and II) were introduced. The bePOMs differ from the original POMs through the insertion of an additional methylene group into the backbone units, which increases the flexibility of the oligomers. bePOM I and II oligomers were synthesised using solid-phase peptide chemistry. Interestingly, UV thermal denaturation and circular dichroism studies reveals bePOM I and II can hybridise with complementary RNA, but not DNA.     

Stereospecific backbone methylation of pyrrolidine-amide oligonucleotide mimics (POM)

The introduction of (7'S)-methyl groups into the backbone of pyrrolidine-amide oligonucleotide mimics (POM) does not interfere with high affinity recognition of complementary nucleic acids, whereas (7'R)-methylation disrupts hybridisation significantly.     

Design, synthesis, conformational analysis and nucleic acid hybridisation properties of thymidyl pyrrolidine-amide oligonucleotide mimics (POM)

Pyrrolidine-amide oligonucleotide mimics (POM) 1 were designed to be stereochemically and conformationally similar to natural nucleic acids, but with an oppositely charged, cationic backbone. Molecular modelling reveals that the lowest energy conformation of a thymidyl-POM monomer is similar to the conformation adopted by ribonucleosides. An efficient solution phase synthesis of the thymidyl POM oligomers has been developed, using both N-alkylation and acylation coupling strategies. 1H NMR spectroscopy confirmed that the highly water soluble thymidyl-dimer, T2-POM, preferentially adopts both a configuration about the pyrrolidine N-atom and an overall conformation in D2O that are very similar to a typical C3'-endo nucleotide in RNA. In addition the nucleic acid hybridisation properties of a thymidyl-pentamer, T5-POM, with an N-terminal phthalimide group were evaluated using both UV spectroscopy and surface plasmon resonance (SPR). It was found that T5-POM exhibits very high affinity for complementary ssDNA and RNA, similar to that of a T5-PNA oligomer. SPR experiments also showed that T5-POM binds with high sequence fidelity to ssDNA under near physiological conditions. In addition, it was found possible to attenuate the binding affinity of T5-POM to ssDNA and RNA by varying both the ionic strength and pH. However, the most striking feature exhibited by T5-POM is an unprecedented kinetic binding selectivity for ssRNA over DNA.     

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.     

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.     

Serine-cis-proline and serine-trans-proline isosteres: stereoselective synthesis of (Z)- and (E)-alkene mimics by Still-Wittig and Ireland-Claisen rearrangements

Two new amide isosteres of Ser-cis-Pro and Ser-trans-Pro dipeptides were designed and stereoselectively synthesized to be incorporated into potential inhibitors of the phosphorylation-dependent peptidylprolyl isomerase Pin1, an essential regulator of the cell cycle. The cis mimic, the (Z)-alkene isomer, was formed through the use of a Still-Wittig [2,3]-sigmatropic rearrangement, while the trans mimic, the (E)-alkene, was synthesized through the use of an Ireland-Claisen [3,3]-sigmatropic rearrangement. Starting from N-Boc-Ser(OBn)-N(OMe)Me, both mimics were synthesized in Boc-protected form suitable for peptide synthesis with an overall yield of 20% in 10 steps for the cis mimic and 13% in eight steps for the trans mimic.     

Fluorous Boc ((F)Boc) carbamates: new amine protecting groups for use in fluorous synthesis

The first fluorous variants of the Boc (tert-butyloxycarbonyl) group have been prepared and tested for their suitability as nitrogen protecting groups. A group with two fluorous chains and an ethylene spacer, (RfCH2CH2)2(CH3)COC(O)-, was readily attached to a representative amine but was difficult to cleave. In contrast, groups with two fluorous chains and a propylene spacer, (RfCH2CH2CH2)2(CH3)COC(O)-, or one fluorous chain and an ethylene spacer, (RfCH2CH2)(CH3)2COC(O)-, were readily formed and cleaved. The fluorous alcohol component of the (F)Boc group can be removed by evaporation and can be recovered and reused. The utility of the new (F)Boc group (C8F17CH2CH2)(CH3)2COC(O)- was demonstrated in 16 and 96 compound library synthesis exercises. Separations can be achieved either by manual, parallel fluorous solid-phase extraction, or automated, serial fluorous chromatography. The results provide additional confirmation of the value of "light" fluorous synthesis techniques, and the new fluorous Boc groups expand the applicability of fluorous synthesis techniques to many classes of nitrogen-containing organic compounds.     

(9Z)- and (11Z)-8-methylretinals for artificial visual pigment studies: stereoselective synthesis, structure, and binding models

Artificial visual pigment formation was studied by using 8-methyl-substituted retinals in an effort to understand the effect that alkyl substitution of the chromophore side chain has on the visual cycle. The stereoselective synthesis of the 9-cis and 11-cis isomers of 8-methylretinal, as well as the 5-demethylated analogues is also described. The key bond formations consist of a thallium-accelerated Suzuki cross-coupling reaction between cyclohexenylboronic acids and dienyliodides (C6-C7), and a highly stereocontrolled Horner-Wadsworth-Emmons or Wittig condensation (C11-C12). The cyclohexenylboronic acid was prepared by trapping the precursor cyclohexenyllithium species with B(OiPr)(3) or B(OMe)(3). The cyclohexenyllithium species is itself obtained by nBuLi-induced elimination of a trisylhydrazone (Shapiro reaction), or depending upon the steric hindrance of the ring, by iodine-metal exchange. In binding experiments with the apoprotein opsin, only 9-cis-5-demethyl-8-methylretinal yielded an artificial pigment; 9-cis-8-methylretinal simply provided residual binding, while evidence of artificial pigment formation was not found for the 11-cis analogues. Molecular-mechanics-based docking simulations with the crystal structure of rhodopsin have allowed us to rationalize the lack of binding displayed by the 11-cis analogues. Our results indicate that these isomers are highly strained, especially when bound, due to steric clashes with the receptor, and that these interactions are undoubtedly alleviated when 9-cis-5-demethyl-8-methylretinal binds opsin.     

The synthesis and spectral properties of new DNA binding ligands

Two new ligands based on anthracene or carbazole planar skeletons, and a phenyloxazoline moiety linked by a vinyl bridge are synthesized as potential DNA-interacting drugs. Their spectral characteristics and DNA binding affinity are assessed.     

Base-labile tert-butoxycarbonyl (Boc) group on phenols

Phenols are deprotected with weak bases from their tert-butoxycarbonyl (Boc) derivatives. Boc deprotection with bases can avoid side reactions during the deprotection with acids. We note the lability of the Boc to bases and are able to utilize it as a new cleavage condition for synthetic studies.     

Organoruthenium(II) thiosemicarbazone complexes: synthesis, spectral characterization, DNA binding and DNA cleavage studies

A series of new ruthenium(II) complexes were synthesized with Schiff bases derived from salicylaldehyde / o-hydroxyacetophenone/ o-vanillin / 2-hydroxy-1-naphthaldehyde with thiosemicarbazide and acetyl furan. They are characterized by elemental analysis, IR, electronic, ¹H NMR, ¹³C NMR and mass spectral studies. The elemental analysis suggests the stoichiometry to be 1:1 (metal:ligand). Four of these complexes were tested for its binding with CT-DNA using absorption spectroscopic studies and two of these complexes exhibit efficient DNA cleavage activity.      

Revisit to (Z)-civetone synthesis

The synthesis of (Z)-civetone (1) is described starting from oleic acid (5) via a series of reactions, intermolecular olefin self-metathesis, bromination/dehydrobromination into acetylene, semi-hydrogenation and intramolecular Dieckmann macrocyclization.     

Heteroannelated (+)-muricatacin mimics: synthesis, antiproliferative properties and structure-activity relationships

Six new (+)-muricatacin mimics bearing a furano-furanone core have been synthesized and their in vitro antiproliferative activity was evaluated against a panel of human tumour cell lines. A straightforward total synthesis of (+)-muricatacin (1) from d-xylose is disclosed providing a sample of 1 that served as a positive control in antitumour assays. All new compounds showed diverse antiproliferative effects against human malignant cell lines, but were devoid of any significant cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). Additionally, the most of (+)-muricatacin analogues show selective cytotoxicities towards certain cancer cell lines, whereas only two of six analogues are broadly toxic against all cell lines under evaluation. A SAR study reveals the structural features that may be beneficial for the antiproliferative activity of these lactones. These include the absolute stereochemistry, introduction of a THF ring, interchange of the O₈ ether functionality and the C₈ methylene group in the side chain of muricatacin oxa analogues, as well as the one- or two-carbon homologation of the side chain in both 3 and 6.     

Solid-phase synthesis, characterization and DNA binding properties of the first chloro(polypyridyl)ruthenium conjugated peptide complex

A general method for the synthesis of chloro(polypyridyl)ruthenium conjugated peptide complexes via a solid-phase strategy is described. The method is applied to synthesize two positional isomers of the complex [Ru(terpy)(4-CO2H-4'-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6). Even though the separation of the isomers was only partially achieved chromatographically, the isomers were unambiguously assigned by NMR spectroscopy. The interactions of the complex [Ru(terpy)(4-CO2H-4'-Mebpy-Gly-L-His-L-LysCONH2)Cl](PF6) with CT-DNA and plasmid DNA, have been studied with various spectroscopic techniques, showing that (i) the complexes coordinatively bind to DNA preferring the bases guanine and cytosine over the bases thymine and adenine after hydrolysis of the coordinated chloride, (ii) electrostatic interactions between the complex cation and the polyanionic DNA chain assist this binding (iii) only in the case of one isomer the peptide does interact further with DNA as evidenced from 31P NMR spectroscopy, (iv) DNA unwinding occurs in all cases with high binding ratio (Ru/base) values (r > 0.3).     

Carbohydration of 1,4,8,11-tetraazacyclotetradecane (cyclam): synthesis and binding properties toward concanavalin A

Two novel glycocluster ligands with cyclam core bearing thiourea-linked d-glucose and 2-acetamido-2-deoxy-d-glucose at the periphery have been synthesized. The interaction with concanavalin A has been studied by isothermal titration microcalorimetry for characterizing protein-ligand interactions. The sugar-containing multivalent ligands showed higher association affinity compared to the sugar monomers, which is attributed to an entropy driven glycoside clustering effect.     

Metal(II) complexes of bioactive aminonaphthoquinone-based ligand: synthesis,characterization and BSA binding,DNA binding/cleavage,and cytotoxicity studies

An aminonaphthoquinone ligand, L, and its metal complexes of general formula [MLCl₂] {M = Co(II), Ni(II), Cu(II) and Zn(II)} have been synthesized and characterized by analytical and spectral techniques. Tetrahedral geometry has been assigned to Ni(II) and Zn(II) complexes and square planar geometry to Co(II) and Cu(II) complexes on the basis of electronic spectral and magnetic susceptibility data. The binding of complexes with bovine serum albumin (BSA) is relatively stronger than that of free ligand and alters the conformation of the protein molecule. Interaction of these complexes with CT-DNA has been investigated using UV-Vis and fluorescence quenching experiments, which show that the complexes bind strongly to DNA through intercalative mode of binding (K_app 10⁵ M^?1). Molecular docking studies reiterate the mode of binding of these compounds with DNA, proposed by spectral studies. The ligand and its complexes cleave plasmid DNA pUC18 to nicked (Form II) and linear (Form III) forms in the presence of H₂O₂ oxidant. The in vitro cytotoxicity screening shows that Cu(II) complex is more potent against MCF-7 cells and Zn(II) complex exhibits marked cytotoxicity against A-549 cells equal to that of cisplatin. Cell imaging studies suggested apoptosis mode of cell death in these two chosen cell lines.     

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.