Development of methods for the synthesis of large combinatorial libraries of macrocyclic compounds

Macrocycles have raised much interest in the pharmaceutical industry due to their ability to bind challenging targets while often still being able to cross membranes to reach intracellular proteins. However, the development of macrocyclic ligands to new disease targets is hindered by the limited availability of large, structurally diverse macrocyclic compound libraries suitable for high-throughput screening. To address this gap, the overall goal of my thesis was to develop methods and tools to synthesize large libraries of structurally diverse macrocyclic compounds.In my first project, I have investigated the efficiency of a wide range of commercially available bis-electrophilic reagents to cyclize short peptides via two thiol groups. Such reagents are of interest as they allow the synthesis of m × n macrocyclic compounds if "m" short di-thiol peptides are combinatorially reacted with "n" bis-electrophilic reagents. I have assessed the reaction efficiency of 46 different bis-electrophilic reagents undergoing either SN2, Michael addition and epoxide opening reactions. Of these reagents, 35 cyclized peptides with around 50% or greater yield, a bar considered as sufficient for synthesizing and screening combinatorially cyclized peptides as crude products.In my second project, I have developed high-density immobilized tris-(2-carboxyethyl)phosphine (TCEP) beads based on silica support for efficient disulfide bond reduction of di-thiol peptides and subsequent cyclization by bis-electrophile reagents. The immobilization of TCEP on beads allows its efficient removal, which is necessary as it would react with bis-electrophilic cyclization reagents. The generation of "high-density" TCEP beads was required as commercially provided agarose TCEP beads have a rather low reducing capacity, making them unsuitable for disulfide reduction at double-digit millimolar peptide concentrations. I developed high-density TCEP beads offering an 8-fold higher reducing capacity (129 ± 16 µmol/mL) compared to commercial agarose TCEP beads.In my third project, I have modified a commercial 96-well parallel peptide synthesizer so that it can synthesize peptides in 384-well plates. This was achieved by developing hardware parts to hold 384-well plates, a multichannel dispenser unit with 16 channels, and a reagents rack that could hold more than a hundred different Fmoc amino acids. This allowed the synthesis of peptides in four 384-well reactor plates and, thus 1,536 peptides in one run. Moreover, I have designed and developed practical tools for rapid and homogenous resin loading to 96- and 384-well plates. The new synthesizer eliminated a major bottleneck of the group's macrocycle library synthesis platform.In my last project, I have tested if microvalves can be used to precisely and rapidly transfer reagents and solvents for parallel solid-phase peptide synthesis. This was of interest as syringe-based dispensing, used in commercial parallel peptide synthesizers, is limited in precision and speed, in particular for the synthesis in 384-well plates. I was able to apply solenoid microvalves for dispensing organic solvents required for peptide synthesis such as DMF or DCM and highly corrosive TFA. I further showed that short peptides can by synthesized by microvalve dispensing in 384-well plates and even in 1,536-well plates, demonstrating the potential of microvalve dispensing in increasing the throughput and miniaturization of parallel peptide synthesis.