UC Irvine

In my thesis I aim to explore the use of small molecules in studying peptides. All chapters utilize small molecules as either probes for peptide assembly, or as building blocks for synthesizing biologically relevant peptides.

In Chapter I, I discuss the design, synthesis, and studies of – crystal violet diazirine (CrVD) – a photoaffinity probe for Aβ-derived peptides — crystal violet diazirine (CrVD). Aβ oligomers are central to the pathogenesis of Alzheimer’s disease, however they are difficult to study due to their solubility, metastability, and heterogeneity. The development of probes that can covalently label and capture these elusive species are paramount to our understanding of them. Previously, crystal violet (CrV) was shown to interact with timer peptides derived from Aβ. Here, I describe the design of CrVD which is isosteric to CrV but contains a photoaffinity labeling group — trifluoromethyl diazirine in lieu of one of the N,N-dimethylamino groups. In this chapter I describe the computational studies that aided in the CrVD, the synthesis of CrVD in seven steps, and show that CrVD labels trimers derived from Aβ, and it also labels full-length Aβ monomers and oligomers. In Chapter II, I build upon the utility of photoaffinity labeling groups by designing and synthesizing a peptide that contains a noncanonical amino acid – photomethionine in lieu of the native methionine residue. Peptide 10 was previously shown to form toxic oligomers but none of these oligomers have ever been captured to be studied by LC-MS. I designed peptide 11 which is an isostere of peptide 10 that contains photomethionine (an alkyl diazirine containing methionine analogue). I discuss the design and synthesis of peptide 11, as well as explore new interesting assemblies that were observed. Peptide 11 forms non-covalent hexamers and covalent dimers by SDS–PAGE and assembles into a hexamer by X-ray crystallography. LC-MS studies of photolyzed peptide 11 reveal that different assemblies formed in solution as opposed to in the crystal form. Solution-based photolysis almost exclusively contains dimers of peptide 11, but the photolyzed crystals contain dimers as well as trimers that were not previously observed.

In Chapter III, I discuss and expand on the studies of Novo29 (Clovibactin), a new antibiotic peptide natural product that is active against Gram positive bacteria without inducing antibiotic resistance. Until the publication of the corresponding article that this chapter builds upon the stereochemistry of a noncanonical amino acid in Novo29 (hydroxyasparagine) was unknown. In this chapter I describe the synthesis of two building blocks Fmoc-(2R,3R)-hydroxyasparagine-OH and Fmoc-(2R,3S)-hydroxyasparagine-OH that were then used in solid-phase peptide synthesis to prepare Novo29 and epi-Novo29. Correlation with an authentic sample of Novo29 through 1H NMR spectroscopy, LC-MS, and in vitro antibiotic activity established that Novo29 contains (2R,3R)-hydroxyasparagine. X-ray crystallography reveals that epi-Novo29 adopts an amphiphilic conformation, with a hydrophobic surface and a hydrophilic surface. Four sets of epi-Novo29 molecules pack in the crystal lattice to form a hydrophobic core. I then used the crystal structure of epi-Novo29 to create a computational model of Novo29. I conclude the chapter by exploring the interesting properties of assembly of Novo29 observed in the NMR and LC-MS studies. Assembly is crucial to the activity of the related antibiotic teixobactin and could hence play an important role in the mechanism of action of Novo29.

In Chapter IV, I try to address the finding that Novo29 hydrolyzes in neutral or basic pH. This issue is currently preventing Novo29 from being further pursued as a preclinical candidate as hydrolysis leads to an inactive linear version of Novo29. To try and prevent this hydrolysis I propose the synthesis of aza-Novo29 (aza-Clovibactin) which contains an amide bond in the place of a hydrolytically labile ester bond. I explore numerous approaches toward the synthesis of aza-Novo29, however unfortunately none of the approaches thus far have been successful. This chapter serves as the groundwork for future graduate students working on the synthesis and studies of aza-Novo29.