We are investigating, developing and applying Knoevenagel adducts as a platform for multifunctionalization and complex molecular synthesis. Initial results revealed that Knoevenagel adducts can act as trimethylenemethane (TMM) dipole surrogates where intermolecular α,γ-difunctionalization is possible. Along with that, a route to 1-aryl tetralin lignans by Knoevenagel adducts via deconjugative α-alkylation/intramolecular 6-endo-trig Heck cyclization was developed. This demonstrated the intramolecular version of α,γ-difunctionalization of Knoevenagel adducts. Recent advancement on α,γ-difunctionalization of Knoevenagel adducts include a simple, tunable synthetic plan to convene an array of galiellactone and analogues, utilizing deconjugative α-alkylation/decarboxylative-Saegusa oxidation methodology.

G-quadruplex (GQ) has been developed extensively over the past decades. It has been reported that GQs could be used in many directions of chemical, material and biological researches, including supramolecular hydrogel, molecular switch and ionophores. To form a controllable G-quadruplex, one general consideration of self-assembly process is the competition between ΔH formed by hydrogen bonding and the ΔS generated from formation of highly organized structure. It is reasonable to assume that structurally more rigid monomer will lead to the formation of more stable G-quartet due to the reduction of conformation flexibility. One general approach to achieve structurally rigid G-monomer is the modification of C-8 position by fixing the sugar syn/anti conformation. However, according to literature, the studies of C-8-modified G-quadruplexes are rare, mainly due to the challenges associated with the substrate synthesis.

Taking advantage of the electron deficiency of 1,2,3-triazole and electron rich guanosine, we designed a triazole substituted guanosine and successfully achieved high fluorescent intensity which can be used in the application of molecular switch. Herein, we report the new synthesis of 8-aryl guanosine and fluorescent active 8-triazole guanosine and their selfassembly property in solid state and in solution. Through cation templation (Mⁿ⁺= Na⁺, K⁺, Ba²⁺, Pb²⁺, Sr²⁺, La³⁺), discrete self-assembled G-quartet structures were formed. Both structurally novel and functional enriched G-quartets are achieved using this new system. Potential applications in molecular sensing and biological target recognition are expected with this new system.

The NIH Molecular Libraries Program (MLP) was founded to translate the discoveries of the Human Genome Project into therapeutics through a network of high-throughput screening (HTS) centers. A decade of discovery produced hundreds of probes — highly selective small molecules that modulate cellular function — but centralized compound screening bears the same cost and infrastructure burdens of millennial DNA sequencing centers, which has limited access to the technology and, more significantly, the rate of small molecule discovery. We are building a next-generation distributable drug discovery platform analogous to next-generation DNA sequencing. We have developed DNA-encoded solid-phase synthesis strategies to produce ultra-miniaturized compound libraries where each microscopic bead displays many copies of a small molecule library member and a corresponding amplifiable DNA that that encodes the structure. In parallel, we engineered microfluidic instrumentation for miniaturizing automated screening. The integrated microfluidic compound screening circuit loads individual compound library beads into picoliter-scale droplets of assay reagent. Compound, which is attached to the bead via photolabile linker, is released into the droplet in a UV dose-dependent fashion (0.01–10 µM compound) using an integrated UV fiber optic, the dosed droplets are then incubated, evaluated for activity using laser-induced confocal fluorescence detection, and sorted for PCR amplification and high-throughput sequencing. To demonstrate the feasibility of the platform, we synthesized a modest (~50k compounds) DNA-encoded combinatorial protease inhibitor library and developed droplet-scale biochemical assays of HIV-1 protease, ZIKV NS2B-NS3 protease, and cathepsin D. Library screening of HIV-1 protease is under way. Not only are the molecular libraries and screening technology deployable in any laboratory setting, but dose-response screening will generate whole-library structure activity relationship profiles. The unprecedented molecular detail of these data will yield portfolios of new leads and replenish the pipeline of therapeutics, especially those targeting rapidly-evolving bacterial and viral pathogens.

Structurally-complex terpenoid natural products (NPs), are used in the treatment of various diseases including cancer. NP drugs and leads are procured most commonly by isolation and semisynthesis from the natural source. Terpenoid total-synthesis is an alternative strategy to access terpenoid derived drug candidates. Although significant progress has been made in chemical synthesis, there is immediate need improve synthetic efficiency and devise routes that produce medicinally relevant NPs and NP-analogs rapidly. We are attempting to develop a operationally simple, unified route to various "terpenoid building blocks" for processing into terpenoid natural products and analogs for application in the drug discovery process. Our progress toward frondosin-, guaianolide-, tigliane-, and dolastane-terpenoid NP families is presented.

The macrocyclic tetrapeptides CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]) and its D-Trp isomer both bind to kappa opioid receptors (KOR) and exhibit KOR antagonist activity. The change in the tryptophan stereochemistry, however, changes the in vivo opioid activity profile; while the D-Trp isomer exhibits KOR antagonism with minimal agonist activity, CJ-15,208 containing L-Trp exhibits mixed agonist/KOR antagonist activity in a mouse antinociceptive assay (Ross et al., 2012). Therefore we explored the effects of changing the stereochemistry of the phenylalanine residues on opioid activity. The peptides were synthesized by a combination of solid phase synthesis of the linear precursors followed by cyclization of the peptides in dilute solutions using optimized reaction conditions. All of the new stereoisomers examined exhibited antinociceptive (agonist) activity in vivo in the mouse 55 ^oC warm-water tail-withdrawal assay, with multiple opioid receptors contributing to the antinociceptive activity. However, unlike the lead peptides most of the stereoisomers did not exhibit KOR antagonism. Several of the stereoisomers were also active after oral administration and some exhibited promising results in tests for liabilities associated with standard narcotic analgesics (sedation, tolerance). Thus these peptides represent new lead compounds for the potential development of novel analgesics. Research supported by NIDA grants R01 DA18832 and R01 DA023924.

Noncanonical amino acids (ncAAs) are highly valuable building blocks for the preparation of active pharmaceutical ingredients. De novo chemical synthesis of ncAAs, however, is still a tedious endeavor and often requires non-trivial methods to provide high enantioenrichment for the desired products.  A biocatalytic, direct functionalization of existing amino acids offers an alternative strategy that could potentially streamline synthetic approaches towards this class of molecules. This talk will describe our efforts in characterizing a highly proficient leucine hydroxylase with relaxed substrate specificity, allowing for the regio- and enantioselective hydroxylation of a range of aliphatic amino acids. In addition, applications of this transformation in the synthesis of bioactive peptide building blocks and an alkaloid natural product will be discussed.

Many surface proteins are anchored to the cell membrane through glycosylphosphatidylinositols (GPIs), a class of complex glycolipids. GPI-anchored proteins have their polypeptide C-termini linked to a fixed position of the highly conserved core structure of GPI anchors. GPI-anchored proteins play a pivotal role in various biological and pathological processes.  To study these events, it is necessary to have access to GPI-anchored peptides and proteins, as well as other GPI conjugates, in homogeneous and structurally defined forms.  The aims of this project are to develop methods for the synthesis of these important biomolecules and use synthetic GPI conjugates to investigate GPI biology.  In this regard, we have established several methods for GPI-anchored peptide, glycopeptide, and protein synthesis.  These methods include chemical synthesis based upon regioselective chemical ligation of peptides or glycopeptides with GPI anchors and chemoenzymatic synthesis that used enzymes, such as bacterial sortase, to couple synthetic peptides and glycopeptides or recombinant proteins with GPI anchors. The synthesized GPIs and GPI conjugates have been employed to explore problems such as bacterial toxin-GPI interactions, GPI organization on the cell surface, GPI-anchored proteomics, etc.