Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.

A supercritical fluid chromatography method was developed for the detection of intramolecular hydrogen bonds in pharmaceutically relevant molecules. The identification of compounds likely to form intramolecular hydrogen bonds is an important drug design consideration given the correlation of intramolecular hydrogen bonding with increased membrane permeability. The technique described here correlates chromatographic retention with the exposed polarity of a molecule. Molecules that can form an intramolecular hydrogen bond can hide their polarity and therefore exhibit lower retention than similar compounds that cannot. By use of a pairwise analysis strategy, intramolecular hydrogen bonds are identified within a test set of compounds with diverse topologies. The chromatographic results are confirmed by NMR chemical shift and temperature coefficient studies.

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Supporting InformationGeneral. Microanalyses were performed by Galbraith Laboratories or Atlantic Microlab. The actual charges of substrates and reagents are given below. The molar amounts are calculated based on the assays of the materials. Similarly, yields are calculated based on assay corrected moles of substrates and products. Proton ( 1 H) nuclear magnetic resonance (NMR) spectra were recorded on either a Unity Inova Varian 300 MHz or Unity Inova Varian 400 MHz spectrometer. 1 H NMR descriptions are reported as: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or br (broad).Carbon ( 13 C) nuclear magnetic resonance (NMR) spectra were recorded on either a Unity Inova Varian 300 or Unity Inova Varian 400 spectrometer at 75 MHz and 100 MHz, respectively.Melting points were determined using a Laboratory Devises Mel-Temp Instrument equipped with a Fluke 51 Thermocouple. Thin-layer chromatography was performed on EM Science 0.25 nm Silica Gel 60, glass-backed plates with F 254 indicator. UV light was employed for visualization. Flash chromatography was performed on Universal Scientific 0-63 mesh Silica Gel. Liquid chromatography and mass spectrum analysis were performed on an Agilent 1100 Series LC/MSD model number G1946D equipped with an APCI ionization source and photodiiode array. Chiral HPLC method for separation of

Cyclic peptides have long tantalized drug designers with their potential ability to combine the best attributes of antibodies and small molecules. An ideal cyclic peptide drug candidate would be able to recognize a protein surface like an antibody while achieving the oral bioavailability of a small molecule. It has been hypothesized that such cyclic peptides balance permeability and solubility using their solvent-dependent conformational flexibility. Herein we report a conformational deconvolution NMR methodology that combines residual dipolar couplings, J-couplings, and intramolecular hydrogen bond analysis along with conformational analysis using molecular dynamics simulations and density functional theory calculations for studying cyclic peptide conformations in both low-dielectric solvent (chloroform) and high-dielectric solvent (DMSO) to experimentally study the solvent-dependent conformational change hypothesis. Taken together, the combined experimental and computational approaches can illuminate conformational ensembles of cyclic peptides in solution and help identify design opportunities for better permeability.