Practical and innate carbon-hydrogen functionalization of heterocycles.

Nitrogen-rich heterocyclic compounds have had a profound effect on human health because these chemical motifs are found in a large number of drugs used to combat a broad range of diseases and pathophysiological conditions. Advances in transition-metal-mediated cross-coupling have simplified the synthesis of such molecules; however, C-H functionalization of medicinally important heterocycles that does not rely on pre-functionalized starting materials is an underdeveloped area. Unfortunately, the innate properties of heterocycles that make them so desirable for biological applications--such as aqueous solubility and their ability to act as ligands--render them challenging substrates for direct chemical functionalization. Here we report that zinc sulphinate salts can be used to transfer alkyl radicals to heterocycles, allowing for the mild (moderate temperature, 50 °C or less), direct and operationally simple formation of medicinally relevant C-C bonds while reacting in a complementary fashion to other innate C-H functionalization methods (Minisci, borono-Minisci, electrophilic aromatic substitution, transition-metal-mediated C-H insertion and C-H deprotonation). We prepared a toolkit of these reagents and studied their reactivity across a wide range of heterocycles (natural products, drugs and building blocks) without recourse to protecting-group chemistry. The reagents can even be used in tandem fashion in a single pot in the presence of water and air.

Oxidative activation of dihydropyridine amides to reactive acyl donors.

Amides of 1,4-dihydropyridine (DHP) are activated by oxidation for acyl transfer to amines, alcohols and thiols. In the reduced form the DHP amide is stable towards reaction with amines at room temperature. However, upon oxidation with DDQ the acyl donor is activated via a proposed pyridinium intermediate. The activated intermediate reacts with various nucleophiles to give amides, esters, and thio-esters in moderate to high yields.

Synthesis of homochiral tris-indanyl molecular rods.

Homochiral tris-indanyl molecular rods designed for supramolecular surface self-assembly were synthesized. The chiral indanol moiety was constructed via a Ti-mediated alkyne trimerization. Further manipulations resulted in a homochiral indanol monomer. This was employed as the precursor for successive Sonogashira and Ohira-Bestman reactions towards the homochiral tris-indanyl molecular rods. The molecular rods will be applied for scanning tunnelling microscopy studies of their surface self-assembly and chirality.

Synthesis of dopamine and serotonin derivatives for immobilization on a solid support.

The two important neurotransmitters dopamine and serotonin are synthesized with short PEG tethers and immobilized on a magnetic solid support. The tether is attached to the aromatic moiety of the neurotransmitters to conserve their original functional groups. This approach causes minimal alteration of the original structure with the aim of optimizing the immobilized neurotransmitters for aptamer selection by SELEX. For the dopamine derivative, the tether is attached to the aromatic core of a dopamine precursor by the Sonogashira reaction. For serotonin, a link to the indole core is introduced by a Claisen rearrangement from the allylated phenol moiety of serotonin. The tethers are azide-functionalized, which enables coupling to alkyne-modified magnetic beads. The coupling to the magnetic beads is quantified by UV spectroscopy using Fmoc-monitoring of the immobilized dopamine and serotonin derivatives.

Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

Characterization of Escherichia coli RNase H Discrimination of DNA Phosphorothioate Stereoisomers.

Phosphorothioate (PS) modification of antisense oligonucleotides (ASOs) is a critical factor enabling their therapeutic use. Standard chemical synthesis incorporates this group in a stereorandom manner; however, significant effort was made over the years to establish and characterize the impact of chiral control. In this work, we present our in-depth characterization of interactions between Escherichia coli RNase H and RNA-DNA heteroduplexes carrying chirally defined PS groups. First, using a massive parallel assay, we showed that at least a single Rp-PS group is necessary for efficient RNase H-mediated cleavage. We followed by demonstrating that this group needs to be aligned to the phosphate-binding pocket of RNase H, and that chiral status of other PS groups in close proximity to RNase H does not affect cleavage efficiency. We have shown that RNase H's PS chiral preference can be utilized to guide cleavage to a specific chemical bond. Finally, we present a strategy for ASO optimization by mapping preferred RNase H cleavage sites of a non-thioated compound, followed by introduction of Rp-PS in a strategic position. This results in a cleaner cleavage profile and higher knockdown activity compared with a compound carrying an Sp-PS at the same location.

Refining LNA safety profile by controlling phosphorothioate stereochemistry.

Drug discovery with phosphorothioate oligonucleotides is an area of intensive research. In this study we have controlled the stereochemistry of the phosphorothioate backbone of LNA oligonucleotides to investigate the differences in safety profile, target mRNA knock down, and cellular uptake in vitro. The study reveals that controlling only four stereocenters in an isomeric phosphorothioate mixture can improve the therapeutic index significantly by improving safety without compromising activity.

Dimethyl fumarate is an allosteric covalent inhibitor of the p90 ribosomal S6 kinases.

Dimethyl fumarate (DMF) has been applied for decades in the treatment of psoriasis and now also multiple sclerosis. However, the mechanism of action has remained obscure and involves high dose over long time of this small, reactive compound implicating many potential targets. Based on a 1.9 Å resolution crystal structure of the C-terminal kinase domain of the mouse p90 Ribosomal S6 Kinase 2 (RSK2) inhibited by DMF we describe a central binding site in RSKs and the closely related Mitogen and Stress-activated Kinases (MSKs). DMF reacts covalently as a Michael acceptor to a conserved cysteine residue in the αF-helix of RSK/MSKs. Binding of DMF prevents the activation loop of the kinase from engaging substrate, and stabilizes an auto-inhibitory αL-helix, thus pointing to an effective, allosteric mechanism of kinase inhibition. The biochemical and cell biological characteristics of DMF inhibition of RSK/MSKs are consistent with the clinical protocols of DMF treatment.

Locked nucleic acid: modality, diversity, and drug discovery.

Over the past 20 years, the field of RNA-targeted therapeutics has advanced based on discoveries of modified oligonucleotide chemistries, and an ever-increasing understanding of how to apply cellular assays to identify oligonucleotides with improved pharmacological properties in vivo. Locked nucleic acid (LNA), which exhibits high binding affinity and potency, is widely used for this purpose. Our understanding of RNA biology has also expanded tremendously, resulting in new approaches to engage RNA as a therapeutic target. Recent observations indicate that each oligonucleotide is a unique entity, and small structural differences between oligonucleotides can often lead to substantial differences in their pharmacological properties. Here, we outline new principles for drug discovery exploiting oligonucleotide diversity to identify rare molecules with unique pharmacological properties.

ß-Olefination of 2-alkynoates leading to trisubstituted 1,3-dienes.

A phosphine-mediated olefination of 2-alkynoates with aldehydes forming 1,3-dienes with high E-selectivity and up to 88% yield is described. Reaction conditions are optimized and reactions are demonstrated for various aryl, alkyl, and alkenyl aldehydes and for ethyl 2-alkynoates with different substituents in the δ-position. Proof of concept is shown for the generation of a ß,γ-unsaturated lactone by intramolecular olefination, and furthermore the use of the generated 1,3-dienes in the Diels-Alder reaction has been demonstrated.