| In this thesis, results from studies related to the molecular recognition of oligonucleotides are reported. The thesis has three parts. The first focuses on the synthesis of small combinatorial libraries of cationic porphyrins that have the potential of binding to nucleic acids. The second part expands on the motif of generating small combinatorial porphyrin libraries, but focuses on new methodologies, namely stepwise one-pot procedures for mixed coupling reactions and reactivity tests generating equireactive building block mixtures. Equireactive mixtures were then used to prepare combinatorial libraries of porphyrins, peptides and oligonucleotides. The third part of the thesis takes the theme of multicomponent systems and molecular recognition of nucleic acids one step further and focuses on a new methodology for generating oligonucleotide arrays or model DNA chips.; In the first part of the thesis, porphyrin libraries were prepared starting from three different porphyrins scaffolds: meso-tetraanilinoporphyrin, an aminoacylated derivative of this scaffold with terminal aspartic acid residues, and coproporphyrin I. These were coupled with protected derivatives of L-arginine and L-lysine and the resulting porphyrins were fully deprotected. Interestingly, the cationic porphyrins thus prepared behaved more like denatured proteins than small molecules, in that they showed a strong tendency to aggregate and adsorb on surfaces. This complicated attempts to perform selection experiments with oligonucleotide targets.; In the second part of the thesis, tetraanilinoporphyrins were converted to less amphiphilic libraries by reacting them with non-cationic carboxylic acid building blocks. Initially, relative reactivities of pairs of activated carboxylic acids were determined and two component building block mixtures generated six component porphyrin libraries via an amide-forming one-pot reaction. A reactivity test was then developed further to allow measuring of the absolute reactivity of new carboxylic acid building blocks. Using MALDI-TOF mass spectrometry as the analyzing technique, relative reactivities could be measured rapidly. Mixed couplings with reactivity-adjusted mixtures generated porphyrin-, peptide- and oligonucleotide libraries with up to 20 components, allowing, in principle, for an efficient search of structure space for new DNA- and RNA-binding ligands.; In the third part of this thesis, a method for preparing small DNA-arrays on aldehyde-bearing glass slides is presented. The immobilization of the DNA involves reductive amination and employs oligonucleotides with 3′ -terminal lysine residues, obtained in high yield from solid phase syntheses. Spot patterns are produced by protecting selected areas of the aldehyde-bearing slides with wax, coating the free surface with a methyl triethylene glycol derivative, and removing the wax with dichloromethane. The DNA arrays give better signal-to-noise ratios in hybridization experiments than slides without passified background. A model chip with the 5′-modified sequence, chl-T*GGTTGACTGCGAT-DP-Lys, where chl stands for a cholic acid residue, DP for a dimethylhydroxypropionic acid residue, and T* for a 5′ -amino-5′deoxythymidine residue, was prepared and was shown to bind its target strand with higher affinity than its unmodified counterpart TGGTTGACTGCGAT-DP-Lys. The methodology for preparing background-passified oligonucleotide arrays is now being used to develop ‘high fidelity DNA chips’ whose mismatch discrimination is better than that of conventional chips. |
