Triplex-forming peptide nucleic acids as emerging ligands to modulate structure and function of complex RNAs.

Over the last three decades, our view of RNA has changed from a simple intermediate supporting protein synthesis to a major regulator of biological processes. In the expanding area of RNA research, peptide nucleic acid (PNA) is emerging as a promising ligand for triple-helical recognition of complex RNAs. As discussed in this feature article, the key advantages of PNAs are high sequence specificity and affinity for RNA (>10 fold higher than for DNA) that are difficult to achieve with small molecule ligands. Emerging studies demonstrate that triple-helical binding of PNAs can modulate biological function and control dynamic conformational equilibria of complex folded RNAs. These results suggest that PNA has a unique potential as a research tool and therapeutic compound targeting RNA. The remaining problems hampering advances in these directions are limitations of sequences that can be recognized by Hoogsteen triplexes (typically purine rich tracts), poor cellular uptake and bioavailability of PNA, and potential off-target effects in biological systems. Recent exciting studies are discussed that illustrate how synthetic nucleic acid chemistry provides innovative solutions for these problems.

Improved Triplex-Forming Isoorotamide PNA Nucleobases for A-U Recognition of RNA Duplexes.

Four new isoorotamide (Io)-containing PNA nucleobases have been designed for A-U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A-U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A-binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A-binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.

Extended Peptide Nucleic Acid Nucleobases Based on Isoorotic Acid for the Recognition of A-U Base Pairs in Double-Stranded RNA.

Peptide nucleic acids (PNA) with extended isoorotamide containing nucleobases (Io ) were designed for binding A-U base pairs in double-stranded RNA. Isothermal titration calorimetry and UV thermal melting experiments revealed improved affinity for A-U using the Io scaffold in PNA. PNAs having four sequential Io extended nucleobases maintained high binding affinity.

Nucleobase-Modified Triplex-Forming Peptide Nucleic Acids for Sequence-Specific Recognition of Double-Stranded RNA.

Because of the important roles noncoding RNAs play in gene expression, their sequence-specific recognition is important for both fundamental science and the pharmaceutical industry. However, most noncoding RNAs fold in complex helical structures that are challenging problems for molecular recognition. Herein, we describe a method for sequence-specific recognition of double-stranded RNA using peptide nucleic acids (PNAs) that form triple helices in the major grove of RNA under physiologically relevant conditions. We also outline methods for solid-phase conjugation of PNA with cell-penetrating peptides and fluorescent dyes. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

Synthesis and RNA-Binding Properties of Extended Nucleobases for Triplex-Forming Peptide Nucleic Acids.

Triple-helix formation, using Hoogsteen hydrogen bonding of triplex-forming oligonucleotides, represents an attractive method for sequence-specific recognition of double-stranded nucleic acids. However, practical applications using triple-helix-forming oligonucleotides and their analogues are limited to long homopurine sequences. The key problem for recognition of pyrimidines is that they present only one hydrogen-bond acceptor or donor group in the major groove. Herein, we report our first attempt to overcome this problem by using peptide nucleic acids (PNAs) modified with extended nucleobases that form three hydrogen bonds along the entire Hoogsteen edge of the Watson-Crick base pair. New nucleobase triples (five) were designed, and their hydrogen bonding feasibility was confirmed by ab initio calculations. PNA monomers carrying the modified nucleobases were synthesized and incorporated in short model PNA sequences. Isothermal titration calorimetry showed that these nucleobases had a modest binding affinity for their double-stranded RNA (dsRNA) targets. Finally, molecular modeling of the modified triples in PNA-dsRNA helix suggested that the modest binding affinity was caused by subtle structural deviations from ideal hydrogen-bonding arrangements or disrupted π-stacking of the extended nucleobase scaffolds.

Synthetic, Structural, and RNA Binding Studies on 2-Aminopyridine-Modified Triplex-Forming Peptide Nucleic Acids.

The development of new RNA-binding ligands is attracting increasing interest in fundamental science and the pharmaceutical industry. The goal of this study was to improve the RNA binding properties of triplex-forming peptide nucleic acids (PNAs) by further increasing the pKa of 2-aminopyridine (M). Protonation of M was the key for enabling triplex formation at physiological pH in earlier studies. Substitution on M by an electron-donating 4-methoxy substituent resulted in slight destabilization of the PNA-dsRNA triplex, contrary to the expected stabilization due to more favorable protonation. To explain this unexpected result, the first NMR structural studies were performed on an M-modified PNA-dsRNA triplex which, combined with computational modeling identified unfavorable steric and electrostatic repulsion between the 4-methoxy group of M and the oxygen of the carbonyl group connecting the adjacent nucleobase to PNA backbone. The structural studies also provided insights into hydrogen-bonding interactions that might be responsible for the high affinity and unusual RNA-binding preference of PNAs.

The Chemistry of Hapalindoles, Fischerindoles, Ambiguines, and Welwitindolinones.

Isolated from diverse cyanobacteria, the hapalindole family of indole alkaloids comprises over 80 interrelated natural products. The family is further divided into several subclasses including the hapalindoles, fischerindoles, ambiguines, and welwitindolinones, each sharing one or more of the many common structural motifs, including an indole or oxindole core, a highly functionalized cyclohexane unit fused to the indole at C-3, and the uncommon isonitrile or isothiocyanate functionalities. Reported here is a comprehensive review covering the isolation of all currently known hapalindole-type alkaloids, the biological activity for several members of the family, and the proposed biosyntheses that give rise to the intricate structural diversity within the family. Finally, the major thrust of this chapter is a comprehensive outline of decades of cutting edge synthetic chemistry toward these complex alkaloids. To date, only a small fraction of the hapalindole-type alkaloids have yielded to chemical synthesis, yet the strategies and methodologies developed showcase the state of the art in synthetic chemistry and provide access to new compounds that can be evaluated for their biological activities.

The intramolecular allenolate Rauhut-Currier reaction.

An intramolecular Rauhut-Currier reaction utilizing alkynoates as the initial conjugate acceptor affords densely functionalized 5- and 6-membered rings from ynoate-enoate, ynoate-enenitrile, and alkynyl sulfone-enenitrile substrates. Trialkylphosphines catalyze the reaction, and TMSCN serves as a pronucelophile to effect turnover of the catalyst and the formation of a second C-C bond. Because of the highly electrophilic alkyne acceptor, this reaction yields products that cannot be easily accessed from the traditional Rauhut-Currier reaction.

Directed oxidative cyclizations to C2- or C4-positions of indole: efficient construction of the bicyclo[4.3.1]decane core of welwitindolinones.

Regiocontrolled oxidative cyclizations of 3-substituted indoles are described herein. Specifically, it is shown that the installation of a chloride at C2 alters the inherent propensity for cyclization at the 2-position of indole so as to favor the 4-position, enabling the construction of the unique framework found in most welwitindolinone alkaloids. The chloride functions here as more than a blocking group, as it also provides ready access to the corresponding oxindole.

Lessons Learned while Traversing the Welwitindolinone Alkaloids Obstacle Course.

We recount several unexpected results observed in the course of our work toward the synthesis of welwitindolinone alkaloids. The surprising results provide an opportunity to refine one's understanding of the interplay between chemical structure and reactivity.

Catalytic parallel kinetic resolution under homogeneous conditions.

Two complementary chiral catalysts, the phosphine 8d and the DMAP-derived ent-23b, are used simultaneously to selectively activate a mixture of two different achiral anhydrides as acyl donors under homogeneous conditions. The resulting activated intermediates 25 and 26 react with the racemic benzylic alcohol 5 to form enantioenriched esters (R)-24 and (S)-17 by fully catalytic parallel kinetic resolution (PKR). The aroyl ester (R)-24 is obtained with near-ideal enantioselectivity for the PKR process, but (S)-17 is contaminated by ca. 8% of the minor enantiomer (R)-17 resulting from a second pathway via formation of mixed anhydride 27 and its activation by 8d.

Synthesis and reactivity of new chiral bicyclic phospholanes as acyl-transfer catalysts.

[structure: see text] Synthesis of chiral phosphines 1a, 14a, and 18a as nucleophilic catalysts for anhydride activation and kinetic resolution of alcohols is described. Radical cyclization of alkenylphosphines produced the phosphabicyclooctane (PBO) core of catalysts 1a and 14a, while 18a was made by quenching a metallocycle precursor with dichlorophenylphosphine. Catalysts 1a and 14a are less reactive, while 18a is comparable to the most reactive catalysts in the PBO family. The preferred ground-state geometries of phosphine-boranes were identified using computational methods, and were correlated with the catalytic reactivity of the corresponding free phosphines.

Rapid synthesis of the N-methylwelwitindolinone skeleton.

An efficient, convergent synthesis of the core bicyclo[4.3.1]decane ring system of welwitindolinones is described. Key steps in the synthesis include an intramolecular palladium-catalyzed enolate arylation reaction to create the desired bicyclic skeleton and a Curtius rearrangement to install the bridgehead isocyanate unit. [reaction: see text]

Enantioselective acylation using a second-generation P-aryl-2-phosphabicyclo[3.3.0]octane catalyst.

The synthesis of P-aryl-2-phosphabicylco[3.3.0]octane x HBF4 salts 3a and 3c is described. Incorporation of the P-3,5-di-tert-butyl-4-methoxyphenyl group in 3c allows use of a less expensive aryl bromide starting material. Deprotonation of the air-stable salts in situ with triethylamine releases the corresponding phosphines 1a and 1c for use in the kinetic resolution of representative secondary alcohols. The method is convenient for small-scale experiments and affords enantioselectivities s close to the values obtained using the free phosphines 1a and 1b in cases where s is ca. 40 or lower.

Substituent Effect on Intramolecular Hydrogen Bonding in beta-Amino Acid-Containing Polyamides.

An alkyl substitution in the beta-amino acid-containing polyamides 1, 2, and 3 leads to an increase in the population of intramolecularly hydrogen bonded conformations. However, the most stable conformation is not always the same one when the substituent is changed from a methyl group to an isopropyl group. For the beta-amino acid derivatives of succinic acid, a methyl group enhances the formation of a head-to-tail type of folding pattern through an 11-membered ring (2b), but an isopropyl group appears to decrease the enhancement. For beta-amino acid derivatives of glutaric acid, an isopropyl group promotes the formation of a bifurcated conformation (3c). The thermodynamic parameters for the equilibrium between non-hydrogen-bonded states and the head-to-tail type of folding of 2a and 2b are obtained by a van't Hoff analysis of the variable-temperature NMR data, which gives a DeltaH of -1.0 kcal/mol and a DeltaS of -4.7 eu for triamide 2a and a DeltaH of -1.0 kcal/mol and a DeltaS of -3.7 e.u. for triamide 2b, each with a correlation coefficient of better than 0.99. The increased proportion of the intramolecular amide-amide hydrogen bond observed for triamide 2b is entirely due to entropic effects according to this analysis.