The beta-lactam synthon method is an established technique utilizing beta-lactams in natural product synthesis. Through application of our previously described novel beta-lactam preparation, we describe the C3 functionalization and application of previously described and novel β-lactams towards the synthesis of pactamycin--a densely functionalized cyclopentane natural product--and non-natural analogs of salinosporamide A--a proteasome inhibitor currently in clinical trials for treatment of multiple myeloma.

Additionally, we present a methodology for the discovery of geometrically diverse, membrane permeable cyclic peptide scaffolds based on the synthesis and permeability screening of a combinatorial library, followed by deconvolution of membrane-permeable scaffolds to identify cyclic peptides with good to excellent passive cell permeabilities. We use a combination of experimental and computational approaches to investigate structure-permeability relationships in one of these scaffolds, and uncover structural and conformational factors that govern passive membrane diffusion in a related set of cyclic peptide diastereomers. Further, we investigate the dependency of permeability on side chain identity of one of these scaffolds through single-point diversifications to show the adaptability of these scaffolds towards development of permeability-biased libraries suitable for bioactivity screens. Overall, our results demonstrate that many novel, cell permeable scaffolds exist beyond those found in extant natural products, and that such scaffolds can be rapidly identified using a combination of synthesis and deconvolution which can, in principle, be applied to any type of macrocyclic template.

Finally, we report the application of this "split-pool" synthetic library approach towards the extension of known physical models of passive membrane permeation--in particular, the barrier domain model--to larger molecular weight chemical space. We show that the membrane permeability of macrocycles is governed by lipophilicity and solubility and that the window between the two narrows with increasing molecular size. We recognize this brings into question again the origin of intrinsic permeability of natural membrane-permeable macrocycles such as cyclosporin A.

Cyclic peptides are structurally complex molecules with the potential to access challenging biological targets. Although many cyclic peptides exhibit poor pharmacokinetic properties, a few have structural features that allow them to passively permeate cell membranes and achieve high oral bioavailability. It is well established that intramolecular hydrogen bonds and N-methylation play important roles in the passive permeability of cyclic peptides, but other structural features have been explored less extensively. In order to design synthetic peptides with better pharmacokinetic and physicochemical properties a deeper understanding of the relationship between structure and function is necessary. Cyclic peptide natural products contain a variety of non-proteinogenic structural elements such as D-amino acids, γ-amino acids, β-hydroxlation, and an enrichment in β-branched amino acids; all of which may impart to the peptide scaffolds favorable physicochemical properties. To determine the effects of these non-proteinogenic structural motifs on the passive permeability of peptidic macrocycles, a series of 14 cyclic hexapeptide stereoisomers containing γ-amino acids was synthesized and assessed for permeability, solubility, lipophilicity, and oral bioavailability. Using a combination of NMR and computational modeling, the solution structures of 5 macrocycles were determined. The compounds with more intramolecular hydrogen bonds tended to be more permeable, and one compound was found to be 21% orally bioavailable in rat. The relative effects of steric occlusion by β-branched residues, N-methylation, and aliphatic carbon count on cyclic peptide permeability were explored using multiple scaffolds. In a series of 17 sanguinamide A analogs, steric occlusion by β-branched residues was determined to have a minimal effect on passive permeability compared to that of N-methylation and scaffold lipophilicity, which were used to increase the permeability of the scaffold 15-fold. One analog was found to exhibit both high passive permeability and solubility. This behavior was attributed to the solvent-dependent backbone flexibility observed in NMR and molecular dynamics experiments. Finally, the bioactivity and permeability of a set of trimethylated cyclic hexapeptides was explored. Two new passively permeable macrocycles with different pattens of N-methylation and stereochemistry were developed and a side chain variant disrupted cell growth in several organisms.