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A single trimethylated species is obtained in an on-resin N-methylation reaction of a cyclic hexapeptide. This regioselectivity is driven by conformation and the presence of intramolecular hydrogen bonds, and is correlated with membrane permeability of the peptides. Backbone N-methylation is common among peptide natural products and has a substantial impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was dependent on backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N -methyl variants were corroborated by computational studies on a 1,024-member virtual library of N -methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (molecular mass = 755 Da) with three N -methyl groups, showed an oral bioavailability of 28% in rat.

A modified PAMPA (parallel artificial membrane permeability assay) is proposed for evaluating the passive cuticular membrane permeability of potassium in several foliar nutrient formulations. The modified PAMPA can measure the passive permeability of ionic nutrients under fully hydrated conditions through an artificial membrane designed to more closely resemble a plant cuticle, rather than the traditional phospholipid animal model. Foliar nutrient formulations, which in some cases contain a complex organic matter component, may be evaluated with the modified PAMPA in order to develop better structure activity relationships that can help guide rational formulation development. In addition, mechanistic insights may also be uncovered with the simplified system.

Background: Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1–84) and N-terminally truncated PTH (PTH7–84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1–84] and 2 N-terminal variants, aa7–84 and aa34–84. Methods: Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. Results: Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28–84, aa48–84, aa34–77, aa37–77, and aa38–77. Quantitative SRM assays were developed for PTH1–84, PTH7–84, and variant aa34–84. Peptides exhibited linear responses (R2 = 0.90–0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16–31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%–12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%–9%. Conclusions: Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

The focus of Chapter 1 is the passive membrane permeability of cyclic hexapeptide diastereomers. A series of nine cyclic peptide diastereomers based on the sequence cyclo[Leu-Leu-Leu-Leu-Pro-Tyr] were synthesized. After synthesis and purification, each compound was run in the parallel artificial membrane permeability assay (PAMPA). Results show that the cyclic diastereomers have very different Log Pe permeability values. NMR-derived structures and deuterium exchange data for two of the cyclic diastereomers were obtained in CDCl3. Chloroform was chosen as solvent due its low dielectric, which best mimics the membrane environment. Results from the two peptides show that passive membrane permeability can be correlated to intramolecular hydrogen bonding. In Chapter 2 a series of cyclic hexa and heptapeptides were synthesized and run in PAMPA. Based on computational results from collaboration with Dr. Matthew P. Jacobson at UCSF, it was found that the ΔGl parameter was the best predictor of permeability for these compounds. The number of intramolecular hydrogen bonds in the low dielectric conformation (LDC) did not correlate as well as the ΔGl parameter, which was based on a new model of permeability. This new model accounts for the conformational switch from exposed NH's in water, to an intramolecularly hydrogen bonded conformation which is lipid soluble. The effect of specific N-methylation on permeability is studied in Chapter 3. The goal was to target exposed NH protons of the low dielectric conformation in order to improve permeability. Optimizing the reaction for cyclic peptides bound to a resin potentially allows for the creation of large libraries of permeable cyclic peptides. Results of PAMPA show that N-methylation does not always improve the permeability of cyclic peptides. In cases where permeability decreases, the limitations placed onto the conformational space of the peptide backbone by the N-methyl group may be preventing the conformational switch to the LDC. Throughout all three chapters, a concerted effort was made to relate cyclic peptide LDC structures to permeability values obtained with PAMPA.