Exploring Peptides as Template Scaffolds for Directed Olefin Metathesis Oligomerization

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DNA-templated dimerization of hairpin polyamides

Double-helical DNA accelerates the rate of ligation of two six-ring hairpin polyamides which bind adjacent sites in the minor groove via a 1,3-dipolar cycloaddition to form a tandem dimer. The rate of the templated reaction is dependent on DNA sequence as well as on the distance between the hairpin-binding sites. The tandem dimer product of the DNA-templated reaction has improved binding properties with respect to the smaller hairpin fragments. Since cell and nuclear uptake of DNA-binding polyamides will likely be dependent on size, this is a minimum first step toward the design of self-assembling small gene-regulating fragments to produce molecules of increasing complexity with more specific genomic targeting capabilities

A Chemoenzymatic Approach toward the Rapid and Sensitive Detection of O-GlcNAc Posttranslational Modifications

We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of β-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications

Template-Directed Olefin Cross Metathesis

A template containing two secondary dialkylammonium ion recognition sites for encirclement by olefin-bearing dibenzo[24]crown-8 derivatives has been used to promote olefin cross metatheses with ruthenium-alkylidene catalysts. For monoolefin monomers, the rates of metatheses and yields of the dimers are both amplified in the presence of the template. Likewise, for a diolefin monomer, the yield of the dimer is enhanced in the presence of the template under conditions where higher oligomers are not formed

Enhancing the cellular uptake of Py–Im polyamides through next-generation aryl turns

Pyrrole–imidazole (Py–Im) hairpin polyamides are a class of programmable, sequence-specific DNA binding oligomers capable of disrupting protein–DNA interactions and modulating gene expression in living cells. Methods to control the cellular uptake and nuclear localization of these compounds are essential to their application as molecular probes or therapeutic agents. Here, we explore modifications of the hairpin γ-aminobutyric acid turn unit as a means to enhance cellular uptake and biological activity. Remarkably, introduction of a simple aryl group at the turn potentiates the biological effects of a polyamide targeting the sequence 5′-WGWWCW-3′ (W = A/T) by up to two orders of magnitude. Confocal microscopy and quantitative flow cytometry analysis suggest this enhanced potency is due to increased nuclear uptake. Finally, we explore the generality of this approach and find that aryl-turn modifications enhance the uptake of all polyamides tested, while having a variable effect on the upper limit of polyamide nuclear accumulation. Overall this provides a step forward for controlling the intracellular concentration of Py–Im polyamides that will prove valuable for future applications in which biological potency is essential

DNA-Templated Dimerizations of Minor Groove-Binding Polyamides

Polyamides have emerged as a class of small molecules capable of binding the minor groove of DNA with high affinity and sequence specificity that have potential applications in molecular biology and human medicine. In efforts towards the use of polyamides in living cells, we report research directed towards DNA-templated formations of polyamide dimers. We find that formation of polyamide dimers, linked both turn-to-turn and turn-to-tail, can be templated via a 1,3-dipolar cycloaddition using a targeted sequence of DNA. The dimer products formed in situ may prove to have interesting biological effects. Also reported in this thesis are several uses of polyamides as molecular tools. We find that polyamide-biotin conjugates are able to selectively bind and capture targeted pieces of DNA via streptavidin-coated magnetic beads, effectively enriching mixtures of DNA fragments in the fragment of interest. Such molecules may find utility in the identification of DNA-protein complexes. In a second utility we report the use of polyamide-maleimide and chlorambucil conjugates to impart sequence specificity on nonspecific DNA enzymes for crystallographic studies.</p

Regulation of gene expression by synthetic DNA-binding ligands

During the past 20 years, polyamides have evolved from the natural product distamycin to a new class of programmable heterocyclic oligomers that bind a broad repertoire of DNA sequences with high affinity and specificity. This chapter details recent advances in this field of research, focusing on molecular recognition of DNA, and biological applications such as modulating gene expression by small molecules. Work presented here represents efforts towards the modulation of specific cellular function by small molecules in an addressable fashion within the context of live cells

Regulation of gene expression by synthetic DNA-binding ligands

During the past 20 years, polyamides have evolved from the natural product distamycin to a new class of programmable heterocyclic oligomers that bind a broad repertoire of DNA sequences with high affinity and specificity. This chapter details recent advances in this field of research, focusing on molecular recognition of DNA, and biological applications such as modulating gene expression by small molecules. Work presented here represents efforts towards the modulation of specific cellular function by small molecules in an addressable fashion within the context of live cells