Diversity-oriented synthesis encoded by deoxyoligonucleotides.

Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Chalcogen OCF3 Isosteres Modulate Drug Properties without Introducing Inherent Liabilities.

The synthesis of SCF3 as well as SeCF3 isosteres of two OCF3 -containing drugs was achieved through visible light and copper-catalyzed processes. Herein, we show that chalcogen replacement modulates physicochemical and ADME properties without introducing intrinsic liabilities. The SCF3 and SeCF3 groups are more lipophilic than their oxygen counterpart; however, microsomal stability is unchanged, indicating that these molecular changes may be beneficial for in vivo half-life. Enabled by modern synthetic methods, we present the chalcogen-CF3 groups as potential key players for future fluorinated pharmaceuticals.

Direct Chemical Biotinylation of RNA 5'-Ends Using a Diazo Reagent.

Introduction of chemical labels into biomolecules is of utmost importance in chemical biology research. However, methods for selective chemical labeling of in vitro transcribed RNA are scarce. Herein, we describe experimental details for direct labeling of the 5'-phosphate of RNA using a diazo biotin-reagent, as exemplified on a 110 nucleotide RNA obtained via in vitro transcription. The method exploits the fact that, under neutral buffer conditions (~pH 6.8), the 5'-phosphate carries the only mildly acidic proton in the RNA molecule, which allows for selective functionalization at that site using diazo reagents.

Covalent Chemical 5'-Functionalization of RNA with Diazo Reagents.

Functionalization of RNA at the 5'-terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5'-nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5'-phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5'-end of RNA.

Tuning the moenomycin pharmacophore to enable discovery of bacterial cell wall synthesis inhibitors.

New antibiotic drugs need to be identified to address rapidly developing resistance of bacterial pathogens to common antibiotics. The natural antibiotic moenomycin A is the prototype for compounds that bind to bacterial peptidoglycan glycosyltransferases (PGTs) and inhibit cell wall biosynthesis, but it cannot be used as a drug. Here we report the chemoenzymatic synthesis of a fluorescently labeled, truncated analogue of moenomycin based on the minimal pharmacophore. This probe, which has optimized enzyme binding properties compared to moenomycin, was designed to identify low-micromolar inhibitors that bind to conserved features in PGT active sites. We demonstrate its use in displacement assays using PGTs from S. aureus, E. faecalis, and E. coli. 110,000 compounds were screened against S. aureus SgtB, and we identified a non-carbohydrate based compound that binds to all PGTs tested. We also show that the compound inhibits in vitro formation of peptidoglycan chains by several different PGTs. Thus, this assay enables the identification of small molecules that target PGT active sites, and may provide lead compounds for development of new antibiotics.

Cyclohexyne cycloinsertion in the divergent synthesis of guanacastepenes.

The guanacastepenes are a family of 15 diterpenes that share a common 5-6-7 tricyclic core, which is decorated with quaternary centers, unsaturation, hydroxyl and carbonyl groups. Some of these natural products show interesting antimicrobial potency. Their collective structural and biological features have stirred up vibrant activity among organic chemists. Herein, we disclose an account of our studies toward the synthesis of a number of guanacastepenes. The synthetic strategy relies on the use of cyclohexyne in a cycloinsertion reaction to rapidly construct the guanacastepene core. Isolation of a cyclobutenol as intermediate in the cyclohexyne cycloinsertion provided us with the possibility to study further the reactivity of this metastable compound, and we uncovered novel rearrangements and ring-opening reactions. Stereoselective, late-stage oxidative diversification of the carbon scaffold allowed the synthesis of guanacastepenes N and O and paved the way for the synthesis of guanacastepenes H and D.

Arynes and cyclohexyne in natural product synthesis.

This Minireview highlights recent advances in the field of aryne and cyclohexyne chemistry that have allowed the extraordinary reactivity of these entities to be harnessed during the course of natural product syntheses. The syntheses presented rely on the use of these reactive species in chemoselective transformations and follow unprecedented synthetic strategies that are inspiring for the practitioners of synthetic organic chemistry.

Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics.

We present a flexible, modular route to GlcNAc-MurNAc-oligosaccharides that can be readily converted into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetrasaccharide substrate lipid IV (3), but not the disaccharide substrate lipid II (2), significantly increases the concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial cell wall synthesis with the long-term goal of identifying novel antibiotics.

Synthesis and pharmacological evaluation of fluorescent and photoactivatable analogues of antiplasmodial naphthylisoquinolines.

The naphthylisoquinoline (NIQ) alkaloids from tropical Ancistrocladaceae and Dioncophyllaceae plants show high antiplasmodial activities in vitro and in vivo, even against chloroquine-resistant strains of the malaria pathogen. For the directed optimization of these activities, an investigation of the mode of action seems most rewarding. We have therefore embarked on the identification of the respective target protein in Plasmodium falciparum. For this purpose, we have developed a flexible pathway for the synthesis of a chemically divergent series of photoactive and fluorescent derivatives of such alkaloids and succeeded in preparing the first functionalized NIQ derivatives, 10, 12, and 35, suited for fluorescence and photoaffinity labeling experiments. Pharmacological investigations ensured that the modified alkaloid derivatives retained their antiplasmodial activity. The work may pave the way for a further improvement of the activity of these natural products and will thus increase their pharmacological potential as a valuable lead structure against the widespread tropical disease malaria.