Peptide nucleic acids and their structural modifications

Peptide (polyamide) analogues of nucleic acids (PNAs) make very promising groups of natural nucleic acid (NA) ligands and show many other interesting properties. Two types of these analogues may be highlighted as particularly interesting: the first, containing a polyamide with alternating peptide/pseudopeptide bonds as its backbone, consisting of N-(aminoalkyl)amino-acid units (type I), with nucleobases attached to the backbone nitrogen with the carboxyalkyl linker; and the second, containing a backbone consisting of amino-acid residues carrying the nucleobases in their side chains (type II). So far, these two groups have been studied most intensively. The paper describes main groups of peptide nucleic acids, as well as various other amino acid-derived nucleobase monomers or their oligomers, which were either studied in order to determine their hybridisation to nucleic acids, or only discussed with respect to their potential usefulness in the oligomerisation and nucleic acids binding.     

Pyrrolidine carbamate nucleic acids: synthesis and DNA binding studies

An efficient solid phase synthesis of pyrrolidine carbamate nucleic acids is reported. The protected (2S, 4S)-4-aminopyrrolidine-2-methanol with nucleobases thymine and cytosine attached to the ring nitrogen through an acetyl linker can be activated as nitrophenyl carbonates for the synthesis of dimer, trimer and oligomers.     

Synthesis and properties of chiral peptide nucleic acids with a N-aminoethyl-D-proline backbone

A synthon of D-proline substituted at the 4-position by thymine and at N by a flexible aminoethyl linker, has been used to prepare a novel chiral peptide nucleic acid (cPNA) with (2R,4R) stereochemistry using solid phase methodology. The homothymine decamer cPNA binds to complementary polyadenylic acid to form a 2:1 hybrid with high affinity and specificity according to UV and CD studies, whereas no binding to the corresponding polydeoxyadenylic acid was observed.     

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.     

Synthesis of a novel ester analog of nucleic acids bearing a serine backbone

A novel analog of nucleic acids bearing an optically active serine ester backbone, serine-based nucleobase-linked polyester (SNE), was synthesized. Monomers containing a thymine base were synthesized from L- and D-serines. Furthermore, reaction conditions were thoroughly examined for the ester bond formation by using a new phosphonium-type condensing reagent on a solid support without racemization. The release of the dimer from the resin was also investigated using a new type of linker, which could be cleaved under neutral conditions.     

Synthesis of novel nucleoside 5'-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids

A series of novel nucleoside 5'-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative functional capabilities: an amino group for the reaction of amino-alkynyl-modified oligonucleotides with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the new linker can be used to synthesize a diversity of modified pyrimidine nucleosides.     

In vitro stability of alpha-helical peptide nucleic acids (alphaPNAs)

Alpha-helical peptide nucleic acids (alphaPNAs) are synthetic molecules that merge the alpha-helix secondary structure of peptides with the codified Watson-Crick base pairing capability of nucleic acids. It is now demonstrated that alphaPNAs made up of either L- or D-amino acids are resistant to degradation by the proteases present in human serum. The increased stability of alphaPNAs towards proteases may be attributable to the presence of unnatural nucleoamino acid residues [-NHCH(CH(2)OCH(2)B)CO-, where B=thymine or cytosine] since the replacement of these amino acids by serine yields a control peptide that does break down in human serum. The stability of alphaPNAs towards proteases makes them attractive candidates for further development as antisense agents.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a β-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.     

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.     

Ritter-based glycoconjugation of amino acids and peptides--access to novel glycoconjugates displaying a beta-amide linkage between amino acid and sugar moiety

Beta-peptidic-D-gluco-, D-galacto-, and L-fuco-configured glycosyl amino acids can be prepared from the corresponding 2-deoxy-oct-3-ulopyranosonic acids via a one-pot intramolecular Ritter reaction. Initially, a ketopyranoside-based acid condenses under Lewis acid promoted conditions with nitriles (PhCN, MeCN) and a partially protected diamino ester (Boc-DAB-O-t-Bu, Boc-Orn-O-t-Bu) to form a beta-peptidic glycosyl amino t-butylesters. The glycosyl amino t-butylesters can be converted into Fmoc-protected glycosyl amino acids that are suitably protected for solid-phase glycopeptide synthesis. Furthermore, replacement of the protected diamino ester by immobilized peptide amines permits post-synthetic N-terminal- and N(epsilon)-glycoconjugation of peptides on the solid phase.     

Monoadduct forming photochemical reagents for labeling nucleic acids for hybridization.

We describe the synthesis of three angelicin derivatives which can be used for labeling nucleic acids with biotin. These compounds were used to label nucleic acids in the presence of lysed cell constituents. The resulting labelled nucleic acids show hybridization to a genus specific probe for E. coli. The relative comparison of sensitivity indicates that a polyamine linker is better than a polyethylene oxide linker between the biotin and angelicin moieties.     

Enhancing solid phase synthesis by a noncovalent protection strategy-efficient coupling of rhodamine to resin-bound peptide nucleic acids.

Resins for solid-phase synthesis can affect coupling efficiencies by interacting with reactants. We have observed that polyethylene glycol-polystyrene (PEG-PS) solid support absorbs added activated fluorophores, preventing efficient labeling of peptide nucleic acids (PNAs). We now report that addition of an inexpensive unactivated fluorophore blocks the resin and allows efficient labeling. This protection strategy may have general benefits for peptide and combinatorial synthesis.     

Fluorescein-based amino acids for solid phase synthesis of fluorogenic protease substrates

An efficient synthesis of new type fluorescent amino acids is described. The Fmoc-protected dyes can be prepared in a four-step procedure with approximately 30% overall yield from aminofluoresceins and other inexpensive commercially available precursors. The dyes are much more photostable compared to fluorescein and exhibit constant pH-independent fluorescence that is advantageous in biological applications. The Fmoc-protected fluorescent amino acids are ready for use in solid phase peptide synthesis. As a proof of concept, a fluorogenic papain substrate was synthesized and employed for on-bead detection of the protease activity. By using a novel technique for quantitative analysis of bead fluorescence, a approximately 2.7-fold increase in mean bead brightness was measured and was attributed to substrate cleavage by papain. The new type fluorescent amino acids seem to be a promising tool for the synthesis of fluorescent peptide ligands and fluorogenic protease substrates.     

Mechanistic studies on DNA damage by minor groove binding copper-phenanthroline conjugates

Copper–phenanthroline complexes oxidatively damage and cleave nucleic acids. Copper bis-phenanthroline and copper complexes of mono- and bis-phenanthroline conjugates are used as research tools for studying nucleic acid structure and binding interactions. The mechanism of DNA oxidation and cleavage by these complexes was examined using two copper–phenanthroline conjugates of the sequence-specific binding molecule, distamycin. The complexes contained either one or two phenanthroline units that were bonded to the DNA-binding domain through a linker via the 3-position of the copper ligand. A duplex containing independently generated 2-deoxyribonolactone facilitated kinetic analysis of DNA cleavage. Oxidation rate constants were highly dependent upon the ligand environment but rate constants describing elimination of the alkali-labile 2-deoxyribonolactone intermediate were not. Rate constants describing DNA cleavage induced by each molecule were 11–54 times larger than the respective oxidation rate constants. The experiments indicate that DNA cleavage resulting from β-elimination of 2-deoxyribonolactone by copper–phenanthroline complexes is a general mechanism utilized by this family of molecules. In addition, the experiments confirm that DNA damage mediated by mono- and bis-phenanthroline copper complexes proceeds through distinct species, albeit with similar outcomes.     

Are there structural analogies between amino acids and nucleic acids?

Space-filling molecular models have been used to examine structural analogies between amino acids and nucleic acids. The three-dimensional structures of amino acidR groups appear to be stereochemically related to cavities formed by removal of single bases in double helical nucleic acids. The common L amino acids may thus be complementary to their codons.Their publications are indicated by an asterisk in the references at the end of this paper.     

Solid phase synthesis of peptide hydroxamic acids on poly(ethylene glycol)‐based support

A novel resin designed for solid‐phase synthesis of peptide hydroxamic acids (PHA) combining the trityl linker with poly(ethylene glycol)‐based support, ChemMatrix® type, is described. The synthesis of PHA can be performed according to a standard protocol, providing products in excellent purity and reasonable yields. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

RNA cleavage by 2,9-diamino-1,10-phenanthroline PNA conjugates

We report on the synthesis of 2,9-diamino-1,10-phenanthroline PNA conjugates as well as on their action in cleavage of a target RNA. Synthesis of the PNA conjugates are performed on solid support and the phenanthroline derivative is conjugated either to the amino-end or to a centrally positioned diaminopropionic acid in the PNA via a urea linker. Cleavage of the target RNA is achieved and compared to cleavage with the corresponding 2,9-dimethyl-1,10-phenanthroline and glycine conjugates.