Design and stereoselective synthesis of four peptide nucleic acid monomers with cyclic structures in backbone

Four isomers of the monomer of peptide nucleic acid (PNA) were derived from (2S,4R)‐4‐hydroxyproline; they had different stereochemistries at the C² and C⁴ positions in the pyrrolidine ring. These different backbone conformations corresponding to four different stereochemistries were realized through a combination of inversions at the C² and the C⁴ positions in pyrrolidine ring. The obtained backbone frameworks were reacted with N‐benzoyl thymine to give the corresponding PNA monomers. Spectroscopic comparison of the resultant monomers confirmed their stereochemistries. J. Heterocyclic Chem., (2011).     

Online Capillary Electrophoresis Reaction for Interaction Study of Amino Acid Modified Peptide Nucleic Acid and Proteins

Peptide nucleic acid (PNA) is a nucleic acid analog which consists of purines, pyrimidines bases, and a neutrally charged peptide backbone. The PNA has the potential as a very useful biological probe for protein analysis since it has more in vivo biological stability as compared to DNA- or RNA-based aptamers. Usually, the addition of amino acids or peptide to the PNA backbone is used to improve its water-solubility and cell-permeability, but these modifications may affect the interaction between PNA and proteins. To date, the investigation of the interaction between PNA and proteins is rare, and there is no reported study about the effects of modifications. In this work, we designed two types of amino acid modified PNAs, (Lys)₂-PNA and (Glu)₂-PNA, which kept the same base sequence with 15-mer thrombin aptamer and had two basic lysine and two acidic glutamic acid residues on N-terminal of the peptide backbone, respectively. To rapidly assess the binding affinity and specificity of modified PNA and proteins, the online CE reaction method was developed to analyze the interactions of (Lys)₂-PNA/(Glu)₂-PNA and three proteins: thrombin (THB), single-strand DNA-binding protein (SSB) and human serum albumin (HSA). Meanwhile, the interactions of (Lys)₂-PNA/(Glu)₂-PNA and thrombin were compared with that of the corresponding complementary base sequence (Lys)₂-cPNA/(Glu)₂-cPNA and thrombin. The online CE reaction results showed that the interaction of (Lys)₂-PNA and (Glu)₂-PNA with three proteins was in the order of THB > SSB > HSA. However, (Lys)₂-PNA and (Lys)₂-cPNA showed similar binding affinity with thrombin; while the binding affinity of (Glu)₂-PNA with thrombin was stronger than that of (Glu)₂-cPNA with thrombin. Moreover, the binding constant K_b of (Glu)₂-PNA and three proteins was determined by affinity capillary electrophoresis (ACE). The online CE reaction eliminates the requirement of incubation, and thus it is fast in detection, and easy to operate with minimum cost. The method is particularly suitable for the interaction studies of expensive modified PNAs and proteins, and can assist the design of PNA probe that binds to proteins.     

Backbone extended pyrrolidine PNA (bepPNA): a chiral PNA for selective RNA recognition

Synthesis of cationic, chiral PNA analogues with an extra atom in the backbone (bepPNA) is reported. The (2S,4S) geometry of the pyrrolidine ring, and an additional carbon atom in the backbone of homopyrimidine-bepPNAs resulted in the optimization of the inter-nucleobase distance, such that selective binding to complementary RNA over DNA was observed in the triplex mode. It was evident from circular dichroism studies that oligomers with mixed aminoethylglycyl-bep (aeg-bep) repeating units, and also bepPNA with homogeneous backbone attained structures quite different from those of aegPNA₂:RNA/DNA complexes. The bepPNA, when incorporated in a duplex forming mixed purine-pyrimidine sequence, also showed a preference for binding complementary RNA over DNA.     

(2S,5R/2R,5S)-Aminoethylpipecolyl aepip-aegPNA chimera: synthesis and duplex/triplex stability

This article reports the design and facile synthesis of novel chiral six-membered PNA analogues (2S,5R/2R,5S)-1-(N-Boc-aminoethyl)-5-(thymin-1-yl)pipecolic acid, aepipPNA IV that upon incorporation into standard aegPNA sequences effected stabilization of complexes with complementary target DNA. Substitution of aegPNA unit by the designed monomer at the C-terminus was more effective than substitution at N-terminus. The stabilizing behaviour improved with degree of substitution and was found to be dependent on their relative positions in the sequence. The six-membered piperidine ring in the design may freeze the rigid chair conformations and the relative stereochemistry of the substituents may in effect direct the complex formation with DNA/RNA by sequence-specific nucleobase recognition. In the present aepipPNA analogues, the l-trans stereochemical disposition of the substituents seems to lead to the favorable pre-organization of the PNA oligomers for complex formation with DNA. The results reported here further expand the repertoire of cyclic PNA analogues.     

Synthesis of photolabile o-nitroveratryloxycarbonyl (NVOC) protected peptide nucleic acid monomers

The chemical synthesis of peptide nucleic acid (PNA) monomers was accomplished using various combinations of the o-nitroveratryloxycarbonyl (NVOC) group (N-aminoethylglycine backbone) and base labile acyl-type nucleobase protecting groups (anisoyl for adenine and cytosine; isobutyryl for guanine), thus offering a photolithographic solid-phase PNA synthetic strategy compatible with photolithographic oligonucleotide synthesis conditions and allowing the in situ synthesis of PNA microarrays in an essentially neutral medium, by avoiding the use of the commonly used deprotection reagents such as trifluoroacetic acid or piperidine. Convenient methods were also explored to prepare 1-(carboxymethyl)-4-N-(4-methoxybenzoyl)cytosine and 9-(carboxymethyl)-2-N-(isobutyryl)guanine with good yields.     

Influence of the Type of Junction in DNA-3′-Peptide Nucleic Acid (PNA) Chimeras on Their Binding Affinity to DNA and RNA

The automated on-line synthesis of DNA-3′-PNA (PNA=Polyamide Nucleic Acids) chimeras 1 – 3 is described, in which the 3′-terminal part of the oligonucleotide is linked to the aminoterminal part of the PNA either via a N-(2-mercaptoethyl)- (X=S), a N-(2-hydroxyethyl)- (X=O), or a N-(2-aminoethyl)- (X=NH) N-[(thymin-1-yl)acetyl]glycine unit. Furthermore, the DNA-3′-PNA chimera 4 without a nucleobase at the linking unit was prepared. The binding affinities of all chimeras were directly compared by determining their T_m values in the duplex with complementary DNA, RNA, or DNA containing a mismatch or abasic site opposite to the linker unit. We found that all investigated chimeras with a nucleobase at the junction form more stable duplexes with complementary DNA and RNA than the corresponding unmodified DNA. The influence of X on duplex stabilization was determined to be in the order O>S≈NH, rendering the phosphodiester bridge the most favored linkage at the DNA/PNA junction. The observed strong duplex-destabilizing effects, when base mismatches or non-basic sites were introduced opposite to the nucleobase at the DNA/PNA junction, suggest that the base at the linking unit contributes significantly to duplex stabilization.     

Synthesis and characterization of (R)-miniPEG-containing chiral γ-peptide nucleic acids using the Fmoc strategy

Diethylene glycol (miniPEG)-containing chiral γPNA is considered to be one of the best PNA derivatives. Its preparation is mainly based on the Boc strategy for solid phase peptide synthesis (SPPS), requiring the repeated use of trifluoroacetic acid TFA, which is not suitable for the in situ synthesis of PNA arrays and some other applications under mild conditions. Herein, Fmoc/Cbz orthogonal protected miniPEG-containing chiral γPNA monomers were synthesized, and a 15mer γPNA was prepared using the Fmoc strategy under mild conditions.     

Synthesis of hydrazino-peptide nucleic acid monomers and dimers as new PNA backbone building blocks

We describe the synthesis of new hydrazinoPNA (hydPNA) monomers and new hydPNA-containing dimers. For the hydPNA monomers, the primary terminal amino group of the aminoethylglycine unit of classical aegPNA is replaced by a hydrazine moiety. An appropriate choice of two orthogonal protecting groups on the two hydrazine nitrogen atoms makes it possible to drive their coupling with other monomers selectively on one or the other nitrogen atom, thus obtaining two different types of PNA dimers. These dimers represent new building blocks that can be used to generate novel PNA oligomers.     

Synthesis of pyrrolidine analogues of solamin

A convergent synthesis of pyrrolidine analogues of solamin, which possessed a pyrrolidine in place of the tetrahydrofuran ring, was presented in a facile route from 2,5-trans-bis(methoxycarbonyl)pyrrolidine. The stereochemistry of pyrrolidine core unit was determined by ¹H NMR spectroscopic analysis.     

Lipophilic peptide nucleic acids containing a 1,3-diyne function: synthesis, characterization and production of derived polydiacetylene liposomes

Adenine-, cytosine- and thymine-containing peptide nucleic acid (PNA) monomers have been synthesized in which either diacetylenic or stearoyl moieties are attached to the N-or C-terminus; the diacetylenic group is embedded within a long hydrocarbon chain. A range of analogous lipophilic functionalized PNA oligomers have been prepared using either solid phase synthesis or a post-synthetic solution phase procedure following cleavage of the PNA oligomer from the solid support. Selected functionalized PNA monomers and oligomers have been incorporated into liposomal polydiacetylenes and characterized by UV-vis absorption spectroscopy. Preliminary investigations show that blue PDA-liposomes containing thymine-based PNAs can be formed and that production of liposomes with other PNA systems are viable.     

A lanthanide induced shift (LIS) investigation of the conformation of cyclohexanones in solution

The solution conformations of cyclohexanone1 and 4-t-butyl cyclohexanone2 have been obtained by the use of the LIS given by Yb(fod)₃. A starting geometry for the substrates was obtained by molecular mechanics calculations. The use of a two-site model for lanthanide-substrate complexing, together with iteration on the¹H and¹³C induced shifts allowed the angle of pucker of the cyclohexanone rings to be determined. In contrast, a one-site model gave no acceptable solutions. The cyclohexanone ring is somewhat flatter at the carbonyl end than cyclohexane, the angle of pucker (α) being reduced from 51° to 49° i.e. the dihedral angle (ω₁₂) is reduced trom 56 to 51°. In 4-t-butyl-cyclohexanone the angle of pucker at the carbonyl end is further reduced. The solution conformation of1 agrees closely with that deduced by MM calculations; interestingly, the conformation of2 is essentially identical with the geometry found in the crystal.     

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.     

Electrochemiluminescent monomers for solid support syntheses of Ru(II)-PNA bioconjugates: multimodal biosensing tools with enhanced duplex stability

The feasibility of devising a solid support mediated approach to multimodal Ru(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru(II)-PNA-like monomers, [Ru(bpy)(2)(Cpp-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(Cpp-L-PNA-OH)](2+) (M2), and [Ru(dppz)(2)(Cpp-L-PNA-OH)](2+) (M3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, Cpp-L-PNA-OH = [2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru(II)-PNA oligomers and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, M1 was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH(2) (PNA1) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH(2) (PNA4) to give PNA2 (H-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)) and PNA3 (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)), respectively. The two Ru(II)-PNA oligomers, PNA2 and PNA3, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (DNA: 5'-T-T-T-T-T-T-T-A-T-T-G-C-T-T-T-3') showed no major variations, suggesting that the electronic properties of the Ru(II) complexes are largely unaffected by hybridization. The thermal stability of the PNA·DNA duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (T(m)) was almost 8 °C higher for PNA2·DNA duplex, and 4 °C for PNA3·DNA duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, PNA2, PNA3, PNA2·DNA and PNA3·DNA displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools.     

Effect of backbone flexibility on charge transfer rates in peptide nucleic acid duplexes

Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.     

Synthesis and oligomerization of Fmoc/Boc-protected PNA monomers of 2,6-diaminopurine, 2-aminopurine and thymine

A Boc-protecting group strategy for Fmoc-based PNA (peptide nucleic acid) oligomerization has been developed for thymine, 2,6-diaminopurine (DAP) and 2-aminopurine (2AP). The monomers may be used interchangeably with standard Fmoc PNA monomers. The DAP monomer was incorporated into a PNA and was found to selectively bind to T (ΔT(m)≥ +6 °C) in a complementary DNA strand. The 2AP monomer showed excellent discrimination of T (ΔT(m)≥ +12 °C) over the other nucleobases. 2AP also acted as a fluorescent probe of the PNA:DNA duplexes and displayed fluorescence quenching dependent on the opposite base.     

Substituted 2-azabicyclo[2.1.1]hexanes as constrained proline analogues: implications for collagen stability

Among the proteinogenic amino acids, only proline is a secondary amine and only proline has a saturated ring. Electronegative substituents on C-4 (that is, C(gamma)) have a substantial effect on the trans/cis ratio of the prolyl peptide bond and the pucker of the pyrrolidine ring. 2-Azabicyclo[2.1.1]hexane is, in essence, a proline analogue with two C(gamma) atoms, one in each of the two prevalent ring puckers of proline. Here, 2-azabicyclo[2.1.1]hexane analogues of 2S-proline, (2S,4S)-4-hydroxyproline, and (2S,4S)-4-fluoroproline residues were synthesized, and their trans/cis ratios were shown to be invariant in a particular solvent. Thus, the substitution of a proline residue on C-4 affects the trans/cis ratio by altering the pucker of its pyrrolidine ring. This finding has implications for the conformation of collagen, which has an abundance of 2S-proline and (2S,4R)-4-hydroxyproline residues, and can be stabilized by (2S,4R)-4-fluoroproline and (2S,4S)-4-fluoroproline residues.     

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     

Diastereoselective total synthesis of (+)-morusimic acid B, an amino acid from Morus alba

An efficient, flexible and diastereoselective synthesis of the naturally occurring pyrrolidine amino acid, (+)-morusimic acid B, has been accomplished. Starting from chiral, optically active (+)-(3S)-hydroxy butyric acid methyl ester the key steps of our synthesis are diastereoselective α-alkylation of its dianion to introduce the main part of the side chain, Curtius rearrangement of the hydrazide derivative to a 2-oxazolidinone followed by N→π-cyclization with mercury(II) acetate to generate the cis-2,5-disubstituted pyrrolidine ring. The remote C-3 stereocentre is established after chain elongation with the dianion of methyl acetoacetate and asymmetric hydrogenation of the resulting β-oxoester with Noyori's Ru(II)-(R)-BINAP catalyst.     

The Implications of (2S,4S)‐Hydroxyproline 4‐O‐Glycosylation for Prolyl Amide Isomerization

The conformations of peptides and proteins are often influenced by glycans O‐linked to serine (Ser) or threonine (Thr). (2S,4R)‐4‐Hydroxyproline (Hyp), together with L ‐proline (Pro), are interesting targets for O‐glycosylation because they have a unique influence on peptide and protein conformation. In previous work we found that glycosylation of Hyp does not affect the N‐terminal amide trans/cis ratios (K_trans/cis) or the rates of amide isomerization in model amides. The stereoisomer of Hyp—(2S,4S)‐4‐hydroxyproline (hyp)—is rarely found in nature, and has a different influence both on the conformation of the pyrrolidine ring and on K_trans/cis. Glycans attached to hyp would be expected to be projected from the opposite face of the prolyl side chain relative to Hyp; the impact this would have on K_trans/cis was unknown. Measurements of ³J coupling constants indicate that the glycan has little impact on the C^γ‐endo conformation produced by hyp. As a result, it was found that the D ‐galactose residue extending from a C^γ‐endo pucker affects both K_trans/cis and the rate of isomerization, which is not found to occur when it is projected from a C^γ‐exo pucker; this reflects the different environments delineated by the proline side chain. The enthalpic contributions to the stabilization of the trans amide isomer may be due to disruption of intramolecular interactions present in hyp; the change in enthalpy is balanced by a decrease in entropy incurred upon glycosylation. Because the different stereoisomers—Hyp and hyp—project the O‐linked carbohydrates in opposite spatial orientations, these glycosylated amino acids may be useful for understanding of how the projection of a glycan from the peptide or protein backbone exerts its influence.