Folding and Duplex Formation in Sequence-Defined Aniline Benzaldehyde Oligoarylacetylenes.

In all known genetic polymers, molecular recognition via hydrogen bonding between complementary subunits underpins their ability to encode and transmit information, to form sequence-defined duplexes, and to fold into catalytically active forms. Reversible covalent interactions between complementary subunits provide a different way to encode information, and potentially function, in sequence-defined oligomers. Here, we examine six oligoarylacetylene trimers composed of aniline and benzaldehyde subunits. Four of these trimers self-pair to form two-rung duplex structures, and two form macrocyclic 1,3-folded structures. The equilibrium proportions of these structures can be driven to favor each of the observed structures almost entirely depending upon the concentration of trimers and an acid catalyst. Quenching the acidic trimer solutions with an organic base kinetically traps all species such that they can be isolated and characterized. Mixtures of complementary trimers form exclusively sequence-specific 3-rung duplexes. Our results suggest that reversible covalent bonds could in principle guide the formation of more complex folded conformations of longer oligomers.

Solid-Phase Synthesis of Sequence-Defined Informational Oligomers.

Genetic biopolymers utilize defined sequences and monomer-specific molecular recognition to store and transfer information. Synthetic polymers that mimic these attributes using reversible covalent chemistry for base-pairing pose unique synthetic challenges. Here, we describe a solid-phase synthesis methodology for the efficient construction of ethynyl benzene oligomers with specific sequences of aniline and benzaldehyde subunits. Handling these oligomers is complicated by the fact that they often exhibit multiple conformations because of intra- or intermolecular pairing. We describe conditions that allow the dynamic behavior of these oligomers to be controlled so that they may be manipulated and characterized without needing to mask the recognition units with protecting groups.

Transfer of Sequence Information and Replication of Diimine Duplexes.

The ability of the biopolymers RNA and DNA to store and transfer information is essential to life. Herein, we demonstrate template-directed replication in a set of dimer duplexes that use reversible covalent bonds to form base-pairing interactions. Binary sequence information was encoded as a sequence of aniline and benzaldehyde subunits linked together by a diethynyl benzene backbone. These dimers formed sequence-specific, imine-linked duplexes, which could be separated and used as templates for the synthesis of daughter duplexes with identical sequences.

Automated quantitative image analysis for ex vivo metastasis assays reveals differing lung composition requirements for metastasis suppression by KISS1.

Imaging is broadly used in biomedical research, but signal variation complicates automated analysis. Using the Pulmonary Metastasis Assay (PuMA) to study metastatic colonization by the metastasis suppressor KISS1, we cultured GFP-expressing melanoma cells in living mouse lung ex vivo for 3 weeks. Epifluorescence images of cells were used to measure growth, creating large datasets which were time consuming and challenging to quantify manually due to scattering of light from outside the focal plane. To address these challenges, we developed an automated workflow to standardize the measurement of disseminated cancer cell growth by applying statistical quality control to remove unanalyzable images followed and a filtering algorithm to quantify only in-focus cells. Using this tool, we demonstrate that expression of the metastasis suppressor KISS1 does not suppress growth of melanoma cells in the PuMA, in contrast to the robust suppression of lung metastasis observed in vivo. This result may suggest that a factor required for metastasis suppression is present in vivo but absent in the PuMA, or that KISS1 suppresses lung metastasis at a step in the metastatic cascade not tested by the PuMA. Together, these data provide a new tool for quantification of metastasis assays and further insight into the mechanism of KISS1 mediated metastasis suppression in the lung.

Gallium(III)-Promoted Halocyclizations of 1,6-Diynes.

Cyclization of 1,6-diynes promoted by stoichiometric Ga(III) halides produces vinyl halides in good to excellent yields. Under acidic conditions, initially formed iodocyclization products undergo in situ Friedel-Crafts cyclizations, giving access to iodoindenopyridines. Application of the vinyl halides in cross-coupling reactions has been explored, and mechanistic aspects of the cyclization are discussed.

Identification of a broad-spectrum inhibitor of viral RNA synthesis: validation of a prototype virus-based approach.

There are no approved therapeutics for the most deadly nonsegmented negative-strand (NNS) RNA viruses, including Ebola (EBOV). To identify chemical scaffolds for the development of broad-spectrum antivirals, we undertook a prototype-based lead identification screen. Using the prototype NNS virus, vesicular stomatitis virus (VSV), multiple inhibitory compounds were identified. Three compounds were investigated for broad-spectrum activity and inhibited EBOV infection. The most potent, CMLDBU3402, was selected for further study. CMLDBU3402 did not show significant activity against segmented negative-strand RNA viruses, suggesting proscribed broad-spectrum activity. Mechanistic analysis indicated that CMLDBU3402 blocked VSV viral RNA synthesis and inhibited EBOV RNA transcription, demonstrating a consistent mechanism of action against genetically distinct viruses. The identification of this chemical backbone as a broad-spectrum inhibitor of viral RNA synthesis offers significant potential for the development of new therapies for highly pathogenic viruses.

Phosphorylation of enkephalins: NMR and CD studies in aqueous and membrane-mimicking environments.

Phosphorylation of l-serine-containing enkephalin analogs has been explored as an alternative to glycosylation in an effort to increase blood-brain barrier permeability and CNS bioavailability of peptide pharmacophores. Two enkephalin-based peptides were modified for these studies, a set related to DTLES, a mixed µ/δ-agonist, and one related to DAMGO, a highly selective µ-agonist. Each unglycosylated peptide was compared to its phosphate, its mono-benzylphosphate ester, and its ß-d-glucoside. Binding was characterized in membrane preparations from Chinese hamster ovary cells expressing human µ, δ and κ-opiate receptors. Antinociception was measured in mice using the 55 °C tail-flick assay. To estimate bioavailability, the antinociceptive effect of each opioid agonist was evaluated after intracerebroventricular (i.c.v.) or intravenous administration (i.v.) of the peptides. Circular dichroism methods and high-field nuclear magnetic resonance were used in the presence and absence of sodium dodecylsulfate to understand how the presence of a membrane might influence the peptide conformations.