The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

Functional Group Tolerance of a Micellar on-DNA Suzuki-Miyaura Cross-Coupling Reaction for DNA-Encoded Library Design.

DNA-encoded libraries (DELs) offer great promise for the discovery of new ligands for proteins. Many current reactions used for DEL synthesis do not proceed efficiently over a wide range of substrates. Combining a diverse array of multicomponent reactions with micellar-promoted Suzuki-Miyaura cross-coupling provides a strategy for synthesizing highly diverse DELs with exceptionally high fidelity. These results demonstrate that the micellar Suzuki-Miyaura reaction has exceptional functional group tolerance and broad applicability.

Chemically Stabilized DNA Barcodes for DNA-Encoded Chemistry.

DNA-encoded compound libraries are a widely used small molecule screening technology. One important aim in library design is the coverage of chemical space through structurally diverse molecules. Yet, the chemical reactivity of native DNA barcodes limits the toolbox of reactions for library design. Substituting the chemically vulnerable purines by 7-deazaadenine, which exhibits tautomerization stability similar to natural adenine with respect to the formation of stable Watson-Crick pairs, yielded ligation-competent, amplifiable, and readable DNA barcodes for encoded chemistry with enhanced stability against protic acid- and metal ion-promoted depurination. The barcode stability allowed for straightforward translation of 16 exemplary reactions that included isocyanide multicomponent reactions, acid-promoted Pictet-Spengler and Biginelli reactions, and metal-promoted pyrazole syntheses on controlled pore glass-coupled barcodes for diverse DEL design. The Boc protective group of reaction products offered a convenient handle for encoded compound purification.

Macrocyclic Modalities Combining Peptide Epitopes and Natural Product Fragments.

"Hot loop" protein segments have variable structure and conformation and contribute crucially to protein-protein interactions. We describe a new hot loop mimicking modality, termed PepNats, in which natural product (NP)-inspired structures are incorporated as conformation-determining and -restricting structural elements into macrocyclic hot loop-derived peptides. Macrocyclic PepNats representing hot loops of inducible nitric oxide synthase (iNOS) and human agouti-related protein (AGRP) were synthesized on solid support employing macrocyclization by imine formation and subsequent stereoselective 1,3-dipolar cycloaddition as key steps. PepNats derived from the iNOS DINNN hot loop and the AGRP RFF hot spot sequence yielded novel and potent ligands of the SPRY domain-containing SOCS box protein 2 (SPSB2) that binds to iNOS, and selective ligands for AGRP-binding melanocortin (MC) receptors. NP-inspired fragment absolute configuration determines the conformation of the peptide part responsible for binding. These results demonstrate that combination of NP-inspired scaffolds with peptidic epitopes enables identification of novel hot loop mimics with conformationally constrained and biologically relevant structure.

Translation of the copper/bipyridine-promoted Petasis reaction to solid phase-coupled DNA for encoded library synthesis.

The Petasis three-component reaction gives rise to diverse substituted α-aryl glycines from readily available amines, boronic acids and glyoxalic acid. Thus, this reaction is highly attractive for DNA-encoded small molecule screening library synthesis. The Petasis reaction is for instance promoted by a potentially DNA damaging copper(I)/bipyridine reagent system in dry organic solvents. We found that solid phase-coupled DNA strands tolerated this reagent system at elevated temperature allowing for synthesis of diverse substituted DNA-tagged α-aryl glycines from DNA-conjugated secondary amines.

TEAD-YAP Interaction Inhibitors and MDM2 Binders from DNA-Encoded Indole-Focused Ugi Peptidomimetics.

DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or "hot spot", regions of protein-protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide "hexT", encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors.

DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences.

DNA-encoded compound libraries are a highly attractive technology for the discovery of small molecule protein ligands. These compound collections consist of small molecules covalently connected to individual DNA sequences carrying readable information about the compound structure. DNA-tagging allows for efficient synthesis, handling and interrogation of vast numbers of chemically synthesized, drug-like compounds. They are screened on proteins by an efficient, generic assay based on Darwinian principles of selection. To date, selection of DNA-encoded libraries allowed for the identification of numerous bioactive compounds. Some of these compounds uncovered hitherto unknown allosteric binding sites on target proteins; several compounds proved their value as chemical biology probes unraveling complex biology; and the first examples of clinical candidates that trace their ancestry to a DNA-encoded library were reported. Thus, DNA-encoded libraries proved their value for the biomedical sciences as a generic technology for the identification of bioactive drug-like molecules numerous times. However, large scale experiments showed that even the selection of billions of compounds failed to deliver bioactive compounds for the majority of proteins in an unbiased panel of target proteins. This raises the question of compound library design.

Catalytic enantioselective 1,3-dipolar cycloadditions of azomethine ylides for biology-oriented synthesis.

Cycloaddition reactions are among the most powerful methods for the synthesis of complex compounds. In particular, the development and application of the 1,3-dipolar cycloaddition, an important member of this reaction class, has grown immensely due to its powerful ability to efficiently build various five-membered heterocycles. Azomethine ylides are commonly used as dipoles for the synthesis of the pyrrolidine scaffold, which is an important motif in natural products, pharmaceuticals, and biological probes. The reaction between azomethine ylides and cyclic dipolarophiles allows access to polycyclic products with considerable complexity. The extensive application of the 1,3-dipolar cycloaddition is based on the fact that the desired products can be obtained with high yield in a regio- and stereocontrolled manner. The most attractive feature of the 1,3-dipolar cycloaddition of azomethine ylides is the possibility to generate pyrrolidines with multiple stereocenters in a single step. The development of enantioselective cycloadditions became a subject of intensive and impressive studies in recent years. Among many modes of stereoinduction, the application of chiral metal-ligand complexes has emerged as the most viable option for control of enantioselectivity. In chemical biology research based on the principle of biology-oriented synthesis (BIOS), compound collections are prepared inspired by natural product scaffolds. In BIOS, biological relevance is employed as the key criterion to generate hypotheses for the design and synthesis of focused compound libraries. In particular, the underlying scaffolds of natural product classes provide inspiration for BIOS because they define the areas of chemical space explored by nature, and therefore, they can be regarded as "privileged". The scaffolds of natural products are frequently complex and rich in stereocenters, which necessitates the development of efficient enantioselective methodologies. This Account highlights examples, mostly from our work, of the application of 1,3-dipolar cycloaddition reactions of azomethine ylides for the catalytic enantioselective synthesis of complex products. We successfully applied the 1,3-dipolar cycloaddition in the synthesis of spiro-compounds such as spirooxindoles, for kinetic resolution of racemic compounds in the synthesis of an iridoid inspired compound collection and in the synthesis of a nitrogen-bridged bicyclic tropane scaffold by application of 1,3-fused azomethine ylides. Furthermore, we performed the synthesis of complex molecules with eight stereocenters using tandem cycloadditions. In a programmable sequential double cycloaddition, we demonstrated the synthesis of both enantiomers of complex products by simple changes in the order of addition of chemicals. Complex products were obtained using enantioselective higher order [6 + 3] cycloaddition of azomethine ylides with fulvenes followed by Diels-Alder reaction. The bioactivity of these compound collections is also discussed.

Catalytic aerobic oxidation and tandem enantioselective cycloaddition in cascade multicomponent synthesis.

An efficient multicomponent cascade transformation for the highly diastereo- and enantioselective synthesis of complex natural product inspired polycyclic products from simple starting materials is described. The cascade is initiated by copper-catalyzed aerobic CH oxidation of cyclopentadiene to cyclopentadienone followed by double catalytic asymmetric 1,3-dipolar cycloaddition of azomethine ylides. The cascade synthesis efficiently yields structurally complex 5,5,5-tricyclic products with eight stereocenters with good yields and excellent diastereo- and enantiocontrol using one catalyst.

Biology-oriented synthesis of benzopyrano[3,4-c]pyrrolidines.

A natural product inspired synthesis of 6,6,5-tricyclic compounds via a silver(I)-catalyzed formal 1,3-dipolar cycloaddition of coumarins with α-iminoesters was developed. The reaction proceeds in a stepwise reaction course under formation of the trans-substituted diastereomer with respect to the 1,3-dipole and shows a broad substrate scope.

Programmable enantioselective one-pot synthesis of molecules with eight stereocenters.

We developed an enantioselectively catalyzed tandem synthesis of structurally and stereochemically complex molecules that forms four carbon-carbon bonds and sets eight stereocenters with high regio-, diastereo- and enantioselectivity. It can be programmed to yield different stereoisomers by varying only the order of combination of a common set of reagents and catalysts. We report what is to our knowledge the first synthesis of both enantiomers of a chiral compound using the same chiral catalyst.

Navigating chemical reaction space - application to DNA-encoded chemistry.

Databases contain millions of reactions for compound synthesis, rendering selection of reactions for forward synthetic design of small molecule screening libraries, such as DNA-encoded libraries (DELs), a big data challenge. To support reaction space navigation, we developed the computational workflow Reaction Navigator. Reaction files from a large chemistry database were processed using the open-source KNIME Analytics Platform. Initial processing steps included a customizable filtering cascade that removed reactions with a high probability to be incompatible with DEL, as they would e.g. damage the genetic barcode, to arrive at a comprehensive list of transformations for DEL design with applicability potential. These reactions were displayed and clustered by user-defined molecular reaction descriptors which are independent of reaction core substitution patterns. Thanks to clustering, these can be searched manually to identify reactions for DEL synthesis according to desired reaction criteria, such as ring formation or sp3 content. The workflow was initially applied for mapping chemical reaction space for aromatic aldehydes as an exemplary functional group often used in DEL synthesis. Exemplary reactions have been successfully translated to DNA-tagged substrates and can be applied to library synthesis. The versatility of the Reaction Navigator was then shown by mapping reaction space for different reaction conditions, for amines as a second set of starting materials, and for data from a second database.

Investigations Into Chemically Stabilized Four-Letter DNA for DNA-Encoded Chemistry.

DNA-encoded libraries are a prime technology for target-based small molecule screening. Native DNA used as genetic compound barcode is chemically vulnerable under many reaction conditions. DNA barcodes that are composed of pyrimidine nucleobases, 7-deazaadenine, and 7-deaza-8-azaguanine have been investigated for their suitability for encoded chemistry both experimentally and computationally. These four-letter barcodes were readily ligated by T4 ligation, amplifiable by Taq polymerase, and the resultant amplicons were correctly sequenced. Chemical stability profiling showed a superior chemical stability compared to native DNA, though higher susceptibility to depurination than a three-letter code based on pyrimidine DNA and 7-deazaadenine.

Copper(I/II)-Promoted Diverse Imidazo[1,2-α]pyridine Synthesis on Solid-Phase Bound DNA Oligonucleotides for Encoded Library Design.

DNA-encoded libraries designed around heterocyclic scaffolds have proven highly productive in target-based screening. Here, we show the synthesis of imidazopyridines on a controlled pore glass-coupled DNA oligonucleotide for solid phase-initiated encoded library synthesis. The target compounds were synthesized by a variant of the A3 coupling reaction from aminopyridines, alkynes, and aldehydes promoted by copper(I/II) and furnished diverse substituted scaffolds with functionalities for library design. Although proceeding under forcing conditions, it produced minimal DNA damage.

Scanning Protein Surfaces with DNA-Encoded Libraries.

Understanding the ligandability of a target protein, defined as the capability of a protein to bind drug-like compounds on any site, can give important stimuli to drug-development projects. For instance, inhibition of protein-protein interactions usually depends on the identification of protein surface binders. DNA-encoded chemical libraries (DELs) allow scanning of protein surfaces with large chemical space. Encoded library selection screens uncovered several protein-protein interaction inhibitors and compounds binding to the surface of G protein-coupled receptors (GPCRs) and kinases. The protein surface-binding chemotypes from DELs are predominantly chemically modified and cyclized peptides, and functional small-molecule peptidomimetics. Peptoid libraries and structural peptidomimetics have been less studied in the DEL field, hinting at hitherto less populated chemical space and suggesting alternative library designs. Roughly a third of bioactive molecules evolved from smaller, target-focused libraries. They showcase the potential of encoded libraries to identify more potent molecules from weak, for example, fragment-like, starting points.

Synthesis of DNA-coupled isoquinolones and pyrrolidines by solid phase ytterbium- and silver-mediated imine chemistry.

DNA-encoded libraries of chemically synthesized compounds are an important small molecule screening technology. The synthesis of encoded compounds in solution is currently restricted to a few DNA-compatible and water-tolerant reactions. Encoded compound synthesis of short DNA-barcodes covalently connected to solid supports benefits from a broad range of choices of organic solvents. Here, we show that this encoded chemistry approach allows for the synthesis of DNA-coupled isoquinolones by an Yb(iii)-mediated Castagnoli-Cushman reaction under anhydrous reaction conditions and for the synthesis of highly substituted pyrrolidines by Ag(i)-mediated 1,3-dipolar azomethine ylide cycloaddition. An encoding scheme for these DNA-barcoded compounds based on a DNA hairpin is demonstrated.

Screening of metal ions and organocatalysts on solid support-coupled DNA oligonucleotides guides design of DNA-encoded reactions.

DNA-encoded compound libraries are a widely used technology for target-based small molecule screening. Generally, these libraries are synthesized by solution phase combinatorial chemistry requiring aqueous solvent mixtures and reactions that are orthogonal to DNA reactivity. Initiating library synthesis with readily available controlled pore glass-coupled DNA barcodes benefits from enhanced DNA stability due to nucleobase protection and choice of dry organic solvents for encoded compound synthesis. We screened the compatibility of solid-phase coupled DNA sequences with 53 metal salts and organic reagents. This screening experiment suggests design of encoded library synthesis. Here, we show the reaction optimization and scope of three sp3-bond containing heterocyclic scaffolds synthesized on controlled pore glass-connected DNA sequences. A ZnCl2-promoted aza-Diels-Alder reaction with Danishefsky's diene furnished diverse substituted DNA-tagged pyridones, and a phosphoric acid organocatalyst allowed for synthesis of tetrahydroquinolines by the Povarov reaction and pyrimidinones by the Biginelli reaction, respectively. These three reactions caused low levels of DNA depurination and cover broad and only partially overlapping chemical space though using one set of DNA-coupled starting materials.

Catalytic asymmetric exo-selective [6+3] cycloaddition of iminoesters with fulvenes.

A novel exo-selective [6+3] cycloaddition approach for the highly enantioselective synthesis of polysubstituted piperidines was developed. The developed methodology was applied in a one-pot [6+3]-[4+2] dicycloaddition, allowing the construction of structurally and stereochemically rich polycyclic compounds from simple building blocks.