Solid-phase Synthesis of PNA Monomer by Ugi Four-component Condensation Reaction

Peptide nucleic acids (PNA) oligomers were synthesized in most cases by peptide a peptide synthesis from N-protected monomers. In this work a new method of obtaining PNA monomer by Ugi four-component condensation reaction was tested by solid-phase synthesis. The Fmoc protected PNA monomer was build up with thymin-l-yl acetic acid, 3-methylbutyl aldehyde, Fmoc protected aminoethyl isocyanide and Gly-Wang resin.     

Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution- and solid-phase synthesis of multiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labelled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.     

High-affinity peptide nucleic acid oligomers containing tricyclic cytosine analogues

[structure: see text] Peptide nucleic acid (PNA) monomers containing the tricyclic cytosine analogues phenoxazine, 9-(2-aminoethoxy)phenoxazine (G-clamp), and 9-(3-aminopropoxy)phenoxazine (propyl-G-clamp) have been synthesized. The modified nucleobases were incorporated into PNA oligomers using Boc-chemistry for solid-phase synthesis. PNAs containing single G-clamp modifications exhibit significantly enhanced affinity toward RNA and DNA targets relative to unmodified PNA while maintaining mismatch discrimination. These PNA G-clamp modifications exhibit the highest increase in affinity toward nucleic acid targets reported so far for PNA modifications.     

In situ, on-resin synthesis of 8-Br/NH2 adeninyl peptide nucleic acid (PNA) oligomers and complementation studies with DNA

A new, novel and efficient in situ synthesis of 8-aminoadeninyl PNA oligomers from corresponding 8-bromoadeninyl PNA oligomers is reported. The study of hybridisation properties of (8-Br/8-NH₂) PNA oligomers with cDNA reveals substitution-site dependent stabilization of derived triplexes and duplexes.     

Parallel synthesis of PNA-peptide conjugate libraries

An optimized semi-automatic protocol for parallel synthesis of up to 96 peptide nucleic acids (PNA) or PNA-peptide conjugates using Boc-protection strategy has been developed using a robotic system. The approach is illustrated by synthesizing PNA and PNA-peptide libraries varying between 15 and 27 amino acid units. The peptides (NLS (nuclear localization signal) or Tat-peptide) were attached to N-terminus of the PNA. The method was found to be far superior to that based on the SPOT/Fmoc protocol by which PNA oligomers are synthesized on a modified cellulose membrane. On a 0.5 micromole scale the method typically yielded 2 mg product of 90% purity by HPLC/MALDI-TOF analysis. This approach is suitable for screening of a large number of PNA and/or peptide sequences for biochemical and biological studies.     

Rapid Synthesis of Propargyl-γ-Modified Peptide Nucleic Acid Monomers for Late-Stage Functionalization of Oligomers

The exceptional hybridization properties of peptide nucleic acids (PNAs) coupled with the ease of their synthesis has made this artificial nucleic acid mimetic a desirable platform for diagnostics, therapeutics and supramolecular engineering. PNA backbone modifications have been extensively explored to finetune physicochemical properties and for conjugation of functional molecules. Here, we detail the synthesis of a universal γ-propargyl-PNA backbone from serine, and its acylation with the four DNA canonical nucleobases. The availability of serine as d or l enantiomer provide simple accesses to PNA oligomers for hybridization with natural oligonucleotides or for orthogonal hybridization circuitry. We show that late-stage conjugation enables optimization of the physicochemical properties. This approach is appealing due to its orthogonality to Fmoc-SPPS, its flexibility and ease for introducing diversity by on-resin copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). We exemplified the utility of these novel monomers with PNA based hybridization chain reactions (HCRs).     

Peptide nucleic acids (PNAs) with a functional backbone

The synthesis of 10 new T-PNA monomers derived from L-amino acids is presented. The monomers were incorporated into decameric PNA oligomers, and the hybridisation with RNA, DNA and PNA complements studied by thermal stability measurements.     

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.     

Synthesis and characterization of cationic PNA bearing 5-ω-aminopropyl-uracil

5-ω-Aminopropyl-uracil bearing PNA monomers are synthesized for solid phase oligomer synthesis using FMOC protection. Several PNA oligomers with differing amounts of aminopropyluracil modification were prepared. These oligomers were found to associate with complementary DNA oligonucleotides.     

Efficient and improved synthesis of triazole-linked DNA (DNA) oligomers

A method for the synthesis of triazole-linked DNA oligomers has been revisited to incorporate a reliable protective group and linker for solid-phase synthesis. The new solid-phase synthesis allowed the preparation of oligomers with the efficiency of elongation reaching over 90%.     

Cyanuryl peptide nucleic acid: synthesis and DNA complexation properties

The synthesis of cyanuryl PNA monomer (CyaPNA) 6 was achieved by direct N-monoalkylation of cyanuric acid with N-(2-Boc-aminoethyl)-N′-(bromoacetyl)glycyl ethyl ester 4. Compound 6 was incorporated as a T-mimic into PNA oligomers and biophysical studies on their triplexes/duplex complexes with complementary DNA oligomers indicated unusual stabilization of PNA:DNA hybrids when the cyanuryl unit was located in the middle of the PNA oligomer.     

Solid-phase synthesis of cyclic PNA and PNA-DNA chimeras

[structure: see text] A new and versatile on-line automated solid-phase approach to obtain cyclic PNA (I and III) and cyclic PNA-DNA chimeras (II) in highly pure form has been developed. Starting from a Tentagel matrix functionalized with a 3-chloro-4-hydroxyphenylacetic linker, the synthesis of representative, new cyclic molecules by standard peptide and phosphoramidite-based chemistry has been achieved.     

8-Vinylguanine nucleo amino acid: a fluorescent PNA building block

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as quadruplex forming RNA/PNA oligomers based on fluorescence intensity.     

Organic Chemistry on Solid Supports

Until recently, repetitive solid-phase synthesis procedures were used predominantly for the preparation of oligomers such as peptides, oligosaccharides, peptoids, oligocarbamates, peptide vinylogues, oligomers of pyrrolin-4-one, peptide phosphates, and peptide nucleic acids. However, the oligomers thus produced have a limited range of possible backbone structures due to the restricted number of building blocks and synthetic techniques available. Biologically active compounds of this type are generally not suitable as therapeutic agents but can serve as lead structures for optimization. “Combinatorial organic synthesis” has been developed with the aim of obtaining low molecular weight compounds by pathways other than those of oligomer synthesis. This concept was first described in 1971 by Ugi.^[56f,g,59c] Combinatorial synthesis offers new strategies for preparing diverse molecules, which can then be screened to provide lead structures. Combinatorial chemistry is compatible with both solution-phase and solid-phase synthesis. Moreover, this approach is conducive to automation, as proven by recent successes in the synthesis of peptide libraries. These developments have led to a renaissance in solid-phase organic synthesis (SPOS), which has been in use since the 1970s. Fully automated combinatorial chemistry relies not only on the testing and optimization of known chemical reactions on solid supports, but also on the development of highly efficient techniques for simultaneous multiple syntheses. Almost all of the standard reactions in organic chemistry can be carried out using suitable supports, anchors, and protecting groups with all the advantages of solid-phase synthesis, which until now have been exploited only sporadically by synthetic organic chemists. Among the reported organic reactions developed on solid supports are Diels–Alder reactions, 1,3-dipolar cycloadditions, Wittig and Wittig–Horner reactions, Michael additions, oxidations, reductions, and Pd-catalyzed C? C bond formation. In this article we present a comprehensive review of the previously published solid-phase syntheses of nonpeptidic organic compounds.     

Synthesis of a C-linked glycosylated thymine-based PNA monomer and its incorporation into a PNA oligomer

Backbone modification of peptide nucleic acids (PNAs) by glycosylation has been shown to enhance selective biodistribution and cellular targeting of PNA oligomers based on sugar and cell surface lectin interactions. Here we report the synthesis of a new backbone-glycosylated thymine-based PNA monomer (T(gal)). The sugar residue was attached to the backbone of PNA via a stable carbon-carbon linkage between the sugar and the PNA monomers. Also, incorporation of the modified monomer into a PNA decamer (H-Ala(gal)-G-G-G-T(gal)-C-A-G-C-T(gal)-T-Lys-NH2) was successfully performed. Melting temperature (UV-Tm) of the modified PNA against the complementary DNA was only slightly lower than unmodified PNA.     

Synthesis of gamma-substituted peptide nucleic acids: a new place to attach fluorophores without affecting DNA binding

Molecular beacon strategies using PNA are currently restricted to fluorophore attachment to the ends of the PNA. We report the synthesis of PNA oligomers wherein fluorophores can be attached to the PNA backbone from novel gamma-lysine PNA monomers. Oligomers incorporating the modified PNA showed comparable thermal stability to the corresponding aegPNA oligomer with DNA. When the modified PNA oligomer was annealed with complementary DNA, the fluorescence intensity increased 4-fold over the unbound PNA. [structure: see text]     

Synthesis of alpha-(2-->5)Neu5Gc oligomers

A facile synthesis of the sialic acid oligomers alpha-(2-->5)Neu5Gc (1) is presented. Monosaccharides 2-4 with suitable functionality were used as the building blocks. After selective removal of the paired carboxyl and amine protecting groups, the fully protected oligomers were assembled through consecutive coupling of the building blocks by well established peptide coupling techniques. By this approach, fully protected oligomers as large as an octasaccharide were synthesized. Deprotection of these fully protected oligomers was conducted in two steps (LiCl in refluxing pyridine and 0.1 n NaOH) to afford the desired products in high yield. Enzymatic degradation of the octamer with neuraminidase, monitored by capillary electrophoresis (CE), was also accomplished. The stepwise exo-cleavage adducts were all well separated and identified in the CE spectrum. The strategy described here for solution-phase synthesis also provides the basis for future solid-phase synthesis of poly-alpha-(2-->5)Neu5Gc.     

Synthesis of thiol-modified peptide nucleic acids designed for post-assembly conjugation reactions

Two orthogonally protected PNA monomers were prepared having the mercaptomethyl moiety attached to the PNA backbone. These building blocks were employed in solid-phase PNA synthesis and it was shown that Boc/S-p-methoxybenzyl protection scheme was only satisfactory for the introduction of N-terminal thiol modification while the Fmoc/S-butylthio protected monomer proved to be amenable to elongation. The mercaptomethyl modification did not influence the thermal stability of a PNA/RNA duplex. The feasibility of PNA-PNA native ligation was demonstrated.     

Development of an efficient and low-cost protocol for the manual PNA synthesis by Fmoc chemistry

An efficient and low-cost protocol for the manual synthesis of Peptide Nucleic Acids is reported here. The protocol relies on coupling reactions carried out with 2.5 equiv of PNA monomers activated with HOBT/HBTU, in the presence of pyridine/NMM. The protocol has been tested on four PNA oligomers with a length ranging from 9 to 12 bases and a purine content up to 67%.     

A convenient route to N-[2-(Fmoc)aminoethyl]glycine esters and PNA oligomerization using a Bis-N-Boc nucleobase protecting group strategy

A simple and practical synthesis of the benzyl, allyl, and 4-nitrobenzyl esters of N-[2-(Fmoc)aminoethyl]glycine is described starting from the known N-(2-aminoethyl)glycine. These esters are stored as stable hydrochloride salts and were used in the synthesis of peptide nucleic acid monomers possessing bis-N-Boc-protected nucleobase moieties on the exocyclic amino groups of ethyl cytosin-1-ylacetate, ethyl adenin-9-ylacetate and ethyl (O(6)-benzylguanin-9-yl)acetate. Upon ester hydrolysis, the corresponding nucleobase acetic acids were coupled to N-[2-(Fmoc)aminoethyl]glycine benzyl ester or to N-[2-(Fmoc)aminoethyl]glycine allyl ester in order to retain the O(6) benzyl ether protecting group of guanine. The Fmoc/bis-N-Boc-protected monomers were successfully used in the Fmoc-mediated solid-phase peptide synthesis of mixed sequence 10-mer PNA oligomers and are shown to be a viable alternative to the currently most widely used Fmoc/Bhoc-protected peptide nucleic acid monomers.