4-Methyltrityl-Protected Pyrrole and Imidazole Building Blocks for Solid Phase Synthesis of DNA-Binding Polyamides

Abstract

DNA-binding polyamides are synthetic oligomers of pyrrole/imidazole units with high specificity and affinity for double-stranded DNA. To increase their synthetic diversity, we report a mild methodology based on 4-methyltrityl (Mtt) solid phase peptide synthesis (SPPS), whose building blocks are more accessible than the standard Fmoc and Boc SPPS ones. We demonstrate the robustness of the approach by preparing and studying a hairpin with all precursors. Importantly, our strategy is orthogonal and compatible with sensitive molecules and could be readily automated.

Controlling Nuclease Degradation of Wireframe DNA Origami with Minor Groove Binders

“…The use of MGB-alkylating agent conjugates represents one possible strategy to address this challenge via the formation of covalent bonds. For the pyrrole-imidazole polyamide class of MGBs, represented by compound 6 , recent advances in synthetic methodologies and the development of hairpin structures facilitate the tuning of sequence specificity and leverage avidity effects. ,, This might enable the rational design of MGBs tailored to the sequence space of a given DNA-VLP. At the same time, the characteristics outlined above enable the engineering of degradation time scales and thereby provide control over cargo bioavailability and release.…”

“…DNase I preferentially cleaves double-stranded DNA (dsDNA) over ssDNA, with a preference for AT tracts. , Because the endonuclease binds dsDNA via the minor groove, minor groove binders (MGBs) have been previously explored to modulate DNase I-dependent degradation of dsDNA and are commonly used in DNase I footprinting assays. , Three major classes of MGBs have been described: diamidines, benzimidazoles, and pyrrole-imidazole polyamides . MGBs typically display preferential binding to AT tracts, yet their specificity and affinity are readily tunable. − Platin-based phosphate clamps (PCs, hereafter also termed MGBs) interact with the phosphate backbone but have also been shown to restrict access to the minor groove of dsDNA and thereby modulate DNase I activity. − While binding of MGBs to DNA origami has been characterized previously, protection against endonucleases was not evaluated . Upon cellular uptake, DNA-VLPs additionally encounter the intracellular endonuclease DNase II. , Endolysosomal degradation of DNA-based materials is of particular importance for gene therapeutic delivery and has been studied by fluorescence microscopy. , In contrast to DNase I, the dsDNA binding mode of DNase II has not been elucidated, and it remains unknown whether MGBs inhibit its activity …”

Controlling wireframe DNA origami nuclease degradation with minor groove binders

“…Moreover, we have not yet explored the third canonical class of MGBs, pyrroleimidazole polyamides. For this class of compounds, recent advances in synthetic methodology and the development of hairpin structures facilitate the tuning of sequence specificity and leverage avidity effects [42][43] . This might enable the rational design of MGBs tailored to the sequence space of a given DNA nanoparticle.…”

“…Extensive medicinal chemistry efforts have resulted in to the development of FDA-approved experimental drugs or clinical candidates for the treatment of both infectious diseases and cancer, with inhibition of replication being the primary mode of action (NCT 03824795 is an ongoing phase II clinical trials) [40][41] . MGBs typically display preferential binding to AT tracts, yet their specificity and affinity are readily tunable [42][43] . A related class of compounds, platin-based phosphate clamps (PCs, hereafter also termed MGBs), interact with the phosphate backbone but have also been shown to restrict access to the minor groove of dsDNA and to thereby modulate DNAse I activity in footprinting assays [44][45][46] .…”

Papain-Mediated Conjugation of Peptide Nucleic Acids to Delivery Peptides: A Density Functional Theory/Molecular Mechanics Metadynamics Study in Aqueous and Organic Solvent

“…Nevertheless, peptide synthesis usually relies on the solid-phase synthesis (SPPS) method, which is good for small peptides but is harder to apply to longer sequences (with companies only producing peptides with up to 40 residues), since as the sequence length increases, impurities do also, making the final product more difficult to obtain and isolate . Furthermore, the method requires multiple protection and deprotection steps as well as the use of toxic chemicals. , …”