Development of the Right- and Left-Handed Gamma Peptide Nucleic Acid Building Blocks for On-Resin Chemical Functionalization.

Gamma peptide nucleic acids (PNAs) are a promising class of nucleic acid mimics that adopt either a right- or left-handed helical motif as individual strands and hybridize to DNA or RNA with high affinity and sequence specificity, or not at all, depending on the helical sense. They are attractive as antisense and antigene reagents, as well as building blocks for molecular self-assembly; however, they have not been widely adopted due to their relatively poor biophysical attributes and the challenge in chemical modifications. Here, we report the development of a set of universal monomers, four each for both the right- and left-handed conformers, that permit rapid and selective on-resin chemical functionalization and diversification. The system is modular, permitting incorporation of different chemical groups in the backbone without causing adverse effects on hybridization. The approach overcomes the need to prepare a new set of monomers each time a different chemical group is introduced in the backbone. The newly added synthetic flexibility, along with superior hybridization property, recognition orthogonality, and helical sense translational capability, significantly expands the scope of gamma PNA in biology, biotechnology, and molecular engineering.

Rapid self-assembly of γPNA nanofibers at constant temperature.

Peptide nucleic acids (PNAs) have primarily been used to achieve therapeutic gene modulation through antisense strategies since their design in the 1990s. However, the application of PNAs as a functional nanomaterial has been more recent. We recently reported that γ-modified peptide nucleic acids (γPNAs) could be used to enable formation of complex, self-assembling nanofibers in select polar aprotic organic solvent mixtures. Here we demonstrate that distinct γPNA strands, each with a high density of γ-modifications can form complex nanostructures at constant temperatures within 30 minutes. Additionally, we demonstrate DNA-assisted isothermal growth of γPNA nanofibers, thereby overcoming a key hurdle for future scale-up of applications related to nanofiber growth and micropatterning.

Synthesis of ( R)- and ( S)-Fmoc-Protected Diethylene Glycol Gamma PNA Monomers with High Optical Purity.

A robust synthetic route has been developed for preparing optically pure, Fmoc-protected diethylene glycol-containing ( R)- and ( S)-γPNA monomers. The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of epimerization in the O-alkylation and reductive amination steps. The optical purities of the final monomers were determined to be greater than 99.5% ee, as assessed by 19F-NMR and HPLC. The new synthetic methodology is well-suited for large-scale monomer production, with most synthetic steps providing excellent chemical yields without the need for chromatographic purification other than a simple workup and precipitation.

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington's Disease.

We report the development of a new class of nucleic acid ligands that is comprised of Janus bases and the MPγPNA backbone and is capable of binding rCAG repeats in a sequence-specific and selective manner via, inference, bivalent H-bonding interactions. Individually, the interactions between ligands and RNA are weak and transient. However, upon the installation of a C-terminal thioester and an N-terminal cystine and the reduction of disulfide bond, they undergo template-directed native chemical ligation to form concatenated oligomeric products that bind tightly to the RNA template. In the absence of an RNA target, they self-deactivate by undergoing an intramolecular reaction to form cyclic products, rendering them inactive for further binding. The work has implications for the design of ultrashort nucleic acid ligands for targeting rCAG-repeat expansion associated with Huntington's disease and a number of other related neuromuscular and neurodegenerative disorders.

Design of a "Mini" Nucleic Acid Probe for Cooperative Binding of an RNA-Repeated Transcript Associated with Myotonic Dystrophy Type 1.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUGexp from the wild-type transcript and disrupt the rCUGexp-MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

RNA-Templated Concatenation of Triplet Nucleic-Acid Probe.

Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. Although it is central to many biological processes, such an approach has rarely been applied to the in situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic-acid probe containing a C-terminal thioester group and an N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of an RNA target and self-deactivation in its absence. The work has implications for the development of millamolecular nucleic-acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders.

Shape selective bifacial recognition of double helical DNA.

An impressive array of antigene approaches has been developed for recognition of double helical DNA over the past three decades; however, few have exploited the 'Watson-Crick' base-pairing rules for establishing sequence-specific recognition. One approach employs peptide nucleic acid as a molecular reagent and strand invasion as a binding mode. However, even with integration of the latest conformationally-preorganized backbone design, such an approach is generally confined to sub-physiological conditions due to the lack of binding energy. Here we report the use of a class of shape-selective, bifacial nucleic acid recognition elements, namely Janus bases, for targeting double helical DNA or RNA. Binding occurs in a highly sequence-specific manner under physiologically relevant conditions. The work may provide a foundation for the design of oligonucleotides for targeting the secondary and tertiary structures of nucleic acid biopolymers.

[Au]/[Ag]-catalysed expedient synthesis of branched heneicosafuranosyl arabinogalactan motif of Mycobacterium tuberculosis cell wall.

Emergence of multidrug-resistant and extreme-drug-resistant strains of Mycobacterium tuberculosis (MTb) can cause serious socioeconomic burdens. Arabinogalactan present on the cellular envelope of MTb is unique and is required for its survival; access to arabinogalactan is essential for understanding the biosynthetic machinery that assembles it. Isolation from Nature is a herculean task and, as a result, chemical synthesis is the most sought after technique. Here we report a convergent synthesis of branched heneicosafuranosyl arabinogalactan (HAG) of MTb. Key furanosylations are performed using [Au]/[Ag] catalysts. The synthesis of HAG is achieved by the repetitive use of three reactions namely 1,2-trans furanoside synthesis by propargyl 1,2-orthoester donors, unmasking of silyl ether, and conversion of n-pentenyl furanosides into 1,2-orthoesters. Synthesis of HAG is achieved in 47 steps (with an overall yield of 0.09%) of which 21 are installation of furanosidic linkages in a stereoselective manner.

Facile synthesis of aminooxy glycosides by gold(III)-catalyzed glycosidation.

The O-glycosidation of hydroxysuccinimides and hydroxyphthalimides with a variety of aldose derived propargyl 1,2-orthoesters under the gold(III)-catalyzed glycosidation conditions is reported. A wide range of hydroxysuccinimidyl and hydroxyphthalimidyl glycosides were synthesized from corresponding glycosyl orthoesters including glucosyl, mannosyl, galactosyl, ribofuranosyl, arabinofuranosyl, lyxofuranosyl and xylofuranosyl using gold catalysis repertoire. The protocol is identified to be compatible for the synthesis of aminooxy glycosides of higher oligosaccharides as well.

Efficient synthesis of oligosaccharyl 1,2-O-orthoesters from n-pentenyl glycosides and application to the pentaarabinofuranoside of the mycobacterial cell surface.

Complex oligosaccharide syntheses employ the use of more than one glycosyl donor and hence, methods for the interconversion of glycosyl donors are highly valuable for the overall synthesis plan. Herein, n-pentenyl glycosides are efficiently converted to glycosyl 1,2-O-orthoesters in the presence of both acid and base sensitive functional groups. The identified protocol was found to be suitable for the synthesis of trisaccharyl and tetrasaccharyl 1,2-O-orthoester as well. Furthermore, an iterative synthesis of pentaarabinofuranoside present on the Mycobacterium tuberculosis cell surface was accomplished using this method.

Gold(III)-catalyzed glycosidations for 1,2-trans and 1,2-cis furanosides.

Stereoselective synthesis of furanosides is still a daunting task, unlike the pyranosides, for which several methods exist. Herein, a unified stereoselective strategy for the synthesis of 1,2-trans and 1,2-cis furanosides is revealed for seven out of eight possible isomers of pentoses. The identified protocol gives access to diastereoselective synthesis of α- and ß-araf, ribf, lyxf, and α-xylf furanosides. 1,2-trans glycosides were synthesized by the use of propargyl 1,2-orthoesters under gold-catalyzed glycosidation conditions, and subsequently, they are converted into 1,2-cis glycosides through oxidation-reduction as the key functional group transformation. All the reactions are found to be fully diastereoselective, mild, and high yielding.

Facile synthesis of ß- and α-arabinofuranosides and application to cell wall motifs of M. tuberculosis.

Propargyl 1,2-orthoesters of arabinose are exploited for the synthesis of 1,2-trans furanosides; easily accessible 1,2-trans ribofuranosides are converted to challenging 1,2-cis-arabinofuranosides by oxidoreduction. Utility of these protocols was demonstrated by the successful synthesis of major structural motifs present in the cell surface of Mycobacterium tuberculosis. Key furanosylations were carried out under gold-catalyzed glycosidation conditions.

Gold catalyzed glycosidations for the synthesis of sugar acrylate/acrylamide hybrids and their utility.

Propargyl glyco 1,2-orthoesters were exploited for the efficient synthesis of interesting glycomonomers such as glyco-acrylates and acrylamides using gold catalysts. It was observed that propargyl glyco 1,2-orthoesters with hydroxyethyl acrylates gives very good yield of the corresponding glyco-acrylates in a single step in the presence of catalytic amount of gold(III) catalyst; whereas, gold catalyzed glycosidation reaction on hydroxyethyl acrylamides was found to yield the corresponding acrylamidoyl 1,2-orthoester which was then converted to the corresponding glycol-acrylamide in the presence of catalytic amount of TMSOTf. Synthesized glyco-acrylate/acrylamide monomers are shown to undergo thiolate addition as well as free radical polymerization.

Orthogonal activation of propargyl and n-pentenyl glycosides and 1,2-orthoesters.

An orthogonal activation strategy with propargyl and n-pentenyl glycosides has been identified. According to this methodology, n-pentenyl glycosides can be selectively activated with NIS/TMSOTf in the presence of either armed or disarmed propargyl O-glycosides. In addition, we report herein that propargyl 1,2-orthoesters can be selectively activated with AuBr(3) in CH(2)Cl(2) at room temperature in the presence of n-pentenyl glycosides. Similarly, pentenyl 1,2-orthoesters can be selectively activated with NIS/Yb(OTf)(3) in the presence of propargyl glycosides.