Title page for ETD etd-05112012-113039

Type of Document Dissertation
Author Crumpton, Jason
URN etd-05112012-113039
Title Click Chemistry on DNA and Targeting RNA structure with Peptide Boronic Acids
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
Santos, Webster L. Committee Chair
Capelluto, Daniel G. S. Committee Member
Carlier, Paul R. Committee Member
Etzkorn, Felicia A. Committee Member
  • click chemistry
  • DNA
  • matrix
  • peptide sequencing
Date of Defense 2012-04-20
Availability unrestricted
The utilization of click chemistry to perform inter- and intramolecular ligation on DNA has become ubiquitous in the literature. Advances in copper (I) stabilizing ligands that prevent DNA degradation via redox pathways have provided nucleic acid researchers access to the efficiency and quantitative nature of the click reaction. The majority of ligation procedures in the literature are performed in solution after DNA assembly and modification with alkyne reporter groups. However, without specialty alkyne reagents that can be sequentially and selectively deprotected, the solution phase method requires that the click reaction be performed on all DNA-attached alkynes simultaneously. Therefore, the variability of the azide reagent is limited to a singular R group. However, performing the click reaction on DNA during synthetic elongation (immediately after each alkyne installation) allows for the possibility of performing multiple click reactions with variable azide reagents. Unfortunately, most solid phase click procedures require long reaction times or the utilization of microwave irradiation to accelerate the reaction. The development of methods for the ligation of azides to alkynes without the use of microwave irradiation on solid phase is potentially very useful. Herein, we report a simple, efficient, and robust solid phase synthetic method for the ligation of azido-diamondoids to the alkyne-modified phosphate backbone of DNA with click chemistry using [Cu(CH3CN)4]PF6 without stabilizing ligand. Interestingly, it was found that as the size of diamondoid increased, a corresponding increase in melting temperature of hybridized duplexes was observed. The developed method has the potential to complement existing DNA ligation procedures for applications in biotechnology and diagnostics.

Interest in peptides incorporating boronic acid moieties is increasing due to their potential as therapeutics/diagnostics for a variety of diseases such as cancer. The utility of peptide boronic acids may be expanded with access to vast libraries that can be deconvoluted rapidly and economically. Unfortunately, current detection protocols using mass spectrometry are laborious and confounded by boronic acid trimerization, which requires time consuming analysis of dehydration products. These issues are exacerbated when the peptide sequence is unknown, as with de novo sequencing, and especially when multiple boronic acid moieties are present. Thus, a rapid, reliable and simple method for peptide identification is of utmost importance. Herein, we report the identification and sequencing of linear and branched peptide boronic acids containing up to five boronic acid groups by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protocols for preparation of pinacol boronic esters were adapted for efficient MALDI analysis of peptides. Additionally, a novel peptide boronic acid detection strategy was developed in which 2,5-dihydroxybenzoic acid (DHB) served as both matrix and derivatizing agent in a convenient, in situ, on-plate esterification. Finally, we demonstrate that DHB-modified peptide boronic acids from a single bead can be analyzed by MALDI-MSMS analysis, validating our approach for the identification and sequencing of branched peptide boronic acid libraries.

It is well known that RNA ligands incorporating basic and intercalating moieties display high RNA affinity. Unfortunately, these ligands are also often plagued by promiscuous binding to off-target substrates. Due to the potential utility of RNA ligands

in biology and medicine, it is imperative to elucidate RNA binders which display high specificity as well as affinity. Boronic acid peptides promise unique RNA binding motifs through the interaction between the empty p-orbital of boron and the 2’-hydroxyl group of RNA. Herein, we describe the incorporation of lysine and phenylalanine boronic acid analogues into a branched peptide combinatorial library in an effort to impart increased selectivity towards the HIV-1 Rev Response Element (RRE). We were able to easily select and deconvolute 6 resulting “hit” peptides from 65,536 unique library members by high throughput screening and de novo sequencing. Although we were unable to evaluate peptide selectivity towards RRE due to general insolubility in aqueous media, we demonstrated the efficient deconvolution of a branched peptide library that incorporates boronic acids.

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