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DNA-encoded library technologies empower the screening of synthetic molecules but have thus far not tapped into the power of Darwinian selection with iterative cycles of selection, amplification and diversification. Herein we report a simple strategy to rapidly assemble libraries of conformationally constrained peptides that are paired in a combinatorial fashion (suprabodies). We demonstrate that the pairing can be shuffled after each amplification cycle in a process akin to DNA shuffling or mating to regenerate diversity. Using simulations, we show the benefit of this recombination in yielding a more accurate correlation of selection fitness with affinity after multiple rounds of selection, particularly if the starting library is heterogeneous in the concentration of its members. The method was validated with selections against streptavidin and applied to the discovery of PD-L1 binders. We further demonstrate that the binding of self-assembled suprabodies can be recapitulated by smaller (ca. 7 KDa) synthetic products that maintain the conformational constrain of the peptides. A selective suprabody binding PD-L1 with an affinity of 70nM was identified.

Sesquiterpenes are a rich source of covalent inhibitors with a long history in traditional medicine and include several important therapeutics and tool compounds. Herein, we report the total synthesis of 16 sesquiterpene lactones via a build/couple/pair strategy, including goyasensolide. Using an alkyne-tagged cellular probe and proteomics analysis, we discovered that goyazensolide selectively targets the oncoprotein importin-5 (IPO5) for covalent engagement. We further demonstrate that goyazensolide inhibits the translocation of RASAL-2, a cargo of IPO5, into the nucleus and perturbs the binding between IPO5 and two specific viral nuclear localization sequences., Goyazensolide covalently binds to importin-5 and inhibits interaction with cargo proteins. This was found with alkyne-tagged cell probes obtained via synthesis of 16 sesquiterpene natural products.

The early phase of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic was exacerbated by a diagnostic challenge of unprecedented magnitude. In the absence of effective therapeutics or vaccines, breaking the chain of transmission through early disease detection and patient isolation was the only means to control the growing pandemic. While polymerase chain reaction (PCR)-based methods and rapid-antigen tests rose to the occasion, the analytical challenge of rapid and sequence-specific nucleic acid-sensing at a point-of-care or home setting stimulated intense developments. Herein we report a method that combines recombinase polymerase amplification and a DNA-templated reaction to achieve a dual readout with either fluorescence (microtiter plate) or naked eye (lateral flow assay: LFA) detection. The nucleic acid templated reaction is based on an SNAr that simultaneously transfers biotin from one Peptide Nucleic Acid (PNA) strand to another PNA strand, enabling LFA detection while uncaging a coumarin for fluorescence readout. This methodology has been applied to the detection of a DNA or RNA sequence uniquely attributed to the SARS-CoV-2.

Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPI). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide’s conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53 / MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads and the best binders were identified by mass spectrometry.

Withaferin A, a natural steroidal lactone found in the extracts of Withania somnifera, is used extensively in traditional medicine and part of an ancient remedy in ayurvedic medicine. Prior investigations into its mode of action have shown withaferin to be a polyfunctional pharmacophore with the covalent engagement of a multitude of therapeutic targets. Herein, we report that withaferin A is also a covalent inhibitor of IPO5, an importin that translocates cargos from the cytosol to the nucleus. We show that withaferin inhibits influenza A replication in epithelial cells (A549). Using a panel of inhibitors that selectively recapitulate part of withaferin A’s pharmacological profile (goyazensolide, withaferin A derivatives, FiVe1, and bardoxolone methyl), we show that IPO5 inhibition contributes to the influenza replication inhibition but is not essential for the observed activity of withaferin A. We show that bardoxolone methyl, a semisynthetic triterpenoid in clinical development to treat chronic kidney disease and that shares some of the pharmacological profile of withaferin, also inhibits influenza A replication effectively. The inhibitory activity against influenza A replication should stimulate further studies to repurpose this therapeutic.

Background Unravelling autoimmune targets triggered by SARS‐CoV‐2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti‐SARS‐CoV‐2 and anti‐apoA‐1 humoral response and (b) the degree of linear homology between SARS‐CoV‐2, apoA‐1 and Toll‐like receptor 2 (TLR2) epitopes. Design Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti‐SARS‐CoV‐2 and anti‐apoA‐1 IgG as well as cytokines were assessed by immunoassays on a case‐control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post‐pandemic period. Results Using bioinformatics modelling, linear sequence homologies between apoA‐1, TLR2 and Spike epitopes were identified but without experimental evidence of cross‐reactivity. Overall, anti‐apoA‐1 IgG levels were higher in COVID‐19 patients or anti‐SARS‐CoV‐2 seropositive individuals than in healthy donors or anti‐SARS‐CoV‐2 seronegative individuals (P

A dual Bcl-X

AimsUnravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights in the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining i) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response, ii) their relationship to prognosis, and iii) the degree of linear homology between SARS-CoV-2, apoA-1, and Toll-like receptor-2 (TLR2) epitopes.Methods and ResultsImmunoreactivity against different engineered peptides as well as cytokines were assessed by immunoassays, on a case-control (n=101), an intensive care unit (ICU; n=126) with a 28-days follow-up, and a general population cohort (n=663) with available samples in the pre and post-pandemic period. Using bioinformatics modelling a linear sequence homologies between apoA-1, TLR2, and Spike epitopes were identified. Overall, anti-apoA-1IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (pConclusionCOVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.

A dual Bcl-XL / Bcl-2 inhibitor was discovered from DNA-encoded libraries using a two steps process. In the first step, DNA was used to pair PNA-encoded fragments exploring > 250 000 combinations. In the second step, a focused library combining the selected fragments with linkers of different lengths and geometries led to the identification of tight binding adducts that were further investigated for their selective target engagement in pull-down assays, for their affinity by SPR, and their selectivity in a cytotoxicity assay. The best compound showed comparable cellular activity to venetoclax, the first-in-class therapeutic targeting Bcl-2. &nbsp

Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPIs). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide's conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53/MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads, and the best binders were identified by mass spectrometry.

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients, we identified immunodominant linear epitopes corresponding to key proteolytic sites on the spike protein.

Kilian Muniz passed away unexpectedly on March 16th, 2020, at the age of only 49. Kilian was a leading figure in the field of catalytic (di-)amination reactions. He will be remembered as one of the finest, most passionate chemists, a dear colleague, and, most of all, as a close friend.

Selection from dynamic combinatorial libraries (DCL) benefit from the dynamic nature of the library that can change constitution upon addition of a selection pressure, such as ligands binding to a protein. This technology has been predominantly used with small molecules interacting with each other through reversible covalent interaction. However, application of this technology in biomedical research and drug discovery has been limited by the reversibility of covalent exchange and the analytical deconvolution of small molecule fragments. Here we report a supramolecular approach based on the use of a constant short PNA tag to direct the combinatorial pairing of fragment. This PNA tag yields fast exchange kinetics, while still delivering the benefits of cooperativity, and provides favourable properties for analytical deconvolution by MALDI. A selection of > 6 000 assemblies of glycans (mono-, di-, tri-saccharides) targeting AFL, a lectin from pathogenic fungus, yielded a 95 nM assembly, nearly three orders of magnitude better affinity than the corresponding glycan alone (41 µM).

DNA-encoded library technologies have emerged as a powerful platform to rapidly screen for binders to a protein of interest. These technologies are underpinned by the ability to encode a rich diversity of small molecules. While large libraries are accessible by cycles of mix and split synthesis, libraries based on single chemistries tend to be redundant. Furthermore, the quality of libraries generally decreases with the number of synthetic transformations performed in its synthesis. An alternative approach is to use hybridization to program the combinatorial assembly of fragment pairs onto a library of DNA templates. A broad molecular diversity is more easily sampled since it arises from the pairing of diverse fragments. Upon identification of productive fragment pairs, a focused library covalently linking the fragments is prepared. This focused library includes linker of different length and geometry and offers the opportunity to enrich the selected fragment set with close neighbors. Herein we describe detailed protocols to covalently link diverse fragments and screen fragment-based libraries using commercially available microarray platform.

VCP/p97 belongs to the AAA+ ATPase family and has an essential role in several cellular processes ranging from cell division to protein homeostasis. Compounds targeting p97 inhibit the main ATPase domain and cause cell death. Here, using PNA-encoded chemical libraries, we have identified two small molecules that target the regulatory domain of p97, comprising the N-terminal and the D1 ATPase domains, and do not cause cell death. One molecule, NW1028, inhibits the degradation of a p97-dependent reporter, whereas the other, NW1030, increases it. ATPase assays show that NW1028 and NW1030 do not affect the main catalytic domain of p97. Mapping of the binding site using a photo-affinity conjugate points to a cleft at the interface of the N-terminal and the D1 ATPase domains. We have therefore discovered two new compounds that bind to the regulatory domain of p97 and modulate specific p97 cellular functions. Using these compounds, we have revealed a role for p97 in the regulation of mitotic spindle orientation in HeLa cells.

Cellular uptake mediated by cyclic oligochalcogenides (COCs) is emerging as a conceptually innovative method to penetrate mammalian cells. Their mode of action is based on dynamic covalent oligochalcogenide exchange with cellular thiols. To test thiol-mediated uptake in bacteria, five antibiotics have been equipped with up to three different COCs: One diselenolane and two dithiolanes. We found that the COCs do not activate antibiotics in Gram-negative bacteria. In Gram-positive bacteria, the COCs inactivate antibiotics that act in the cytoplasm and reduce the activity of antibiotics that act on the cell surface. These results indicate that thiol-mediated uptake operates in neither of the membranes of bacteria. COCs are likely to exchange with thiols on the inner, maybe also on the outer membrane, but do not move on. Concerning mammalian cells, the absence of a COC-mediated uptake into bacteria observed in this study disfavors trivial mechanisms, such as passive diffusion, and supports the existence of more sophisticated, so far poorly understood uptake pathways.

DNA-encoded library technologies have emerged as a powerful platform to rapidly screen for binders to a protein of interest. These technologies are underpinned by the ability to encode a rich diversity of small molecules. While large libraries are accessible by cycles of mix and split synthesis, libraries based on single chemistries tend to be redundant. Furthermore, the quality of libraries generally decreases with the number of synthetic transformations performed in its synthesis. An alternative approach is to use hybridization to program the combinatorial assembly of fragment pairs onto a library of DNA templates. A broad molecular diversity is more easily sampled since it arises from the pairing of diverse fragments. Upon identification of productive fragment pairs, a focused library covalently linking the fragments is prepared. This focused library includes linker of different length and geometry and offers the opportunity to enrich the selected fragment set with close neighbors. Herein we describe detailed protocols to covalently link diverse fragments and screen fragment-based libraries using commercially available microarray platform.

DNA display of PNA-encoded libraries was used to pair fragments containing different phosphotyrosine surrogates with diverse triazoles. Microarray-based screening of the combinatorially paired fragment sets (62,500 combinations) against a prototypical phosphatase, PTP1B, was used to identify the fittest fragments. A focused library (10,000 members) covalently pairing identified fragments with linkers of different length and geometry was synthesized. Screening of the focused library against PTP1B and closely related TCPTP revealed orthogonal inhibitors. The selectivity of the identified inhibitors for PTP1B versus TCPT was confirmed by enzymatic inhibition assay.

DNA-encoded chemical libraries are increasingly used to identify leads for drug discovery or chemical biology. Despite the resurging interest in covalent inhibitors, libraries are typically designed with synthon filtered out for reactive functionalities that can engage a target through covalent interactions. Herein, we report the synthesis of two libraries containing Michael acceptors to identify cysteine reactive ligands. We developed a simple procedure to discriminate between covalent and high affinity non-covalent inhibitors using DNA display of the library in a microarray format. The methodology was validated with known covalent and high affinity non-covalent kinase inhibitors. Screening of the library revealed novel covalent inhibitors for MEK2 and ERBB2.

Die Regulierung von transkriptionellen Programmen durch epigenetische Leser (Bromodomanen) wurde mit der Entwicklung von mehreren Krankheiten in Zusammenhang gebracht. Sie ist vor allem bei der Regulierung des Zellwachstums, Umgehung der Apoptose, bei Krebs sowie bei Entzundungskrankheiten beteiligt. Die Entdeckung niedermolekularer Sonden, um die Rolle der Bromodomanen zu studieren, hat daher eine grose Bedeutung. Wir zeigen, dass spezifische konservierte Cysteine in den Bromodomanen kovalent abgefangen werden konnen. Wir berichten uber die Entdeckung von zwei niedermolekularen Substanzen, die eine kovalente Bindung mit konservierten Cysteinen in der Familie von Bromodomanen eingehen, analysieren die Teilmenge an Bromodomanen die durch die kovalente Bindung angesprochen werden kann und demonstrieren Proteomanalysen durch Anreicherung von Bromodomanen aus nativen Zell-Lysaten.

Identification of selective and synthetically tractable ligands to glycan-binding proteins is important in glycoscience. Carbohydrate arrays have had a tremendous impact on profiling glycan-binding proteins and as analytical tools. We report a highly miniaturized synthetic format to access nucleic-acid-encoded hetero-glycoconjugate libraries with an unprecedented diversity in the combinations of glycans, linkers, and capping groups. Novel information about plant and bacterial lectin specificity was obtained by microarray profiling, and we show that a ligand identified on the array can be converted to a high-affinity soluble ligand by straightforward chemistry.

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody–oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

A concise and modular synthesis of pochonin E and F, and their epimers at C-6 established the correct stereochemistry of these two natural products. Several members of the pochonin family have been shown to bind the heat shock protein 90 (Hsp90), which has been the focus of intense drug discovery efforts. Pochonin E and F as well as their epimers were derivatized into the corresponding pochoximes and further modified at the C-6 position. Molecular dynamics simulations, docking studies, and Hsp90 affinity measurements were performed to evaluate the impact of these modifications.

Natural selection: An acyclic chain containing an ene-yne-ene motif, in analogy to farnesyl diphosphate, was cyclized to obtain six distinct scaffolds using different transition-metal catalysts (see scheme). Notably, the guaianolide framework was accessed through enynene metathesis enabling the synthesis of three natural products.

The transcription factor nuclear factor κB (NF-κB) plays a central role in regulating inflammation in response to several external signals. The TGFβ-associated kinase 1 (TAK1) is an upstream regulator of NF-κB signaling. In TGFβ-stimulated cells, TAK1 undergoes Lys-63-linked polyubiquitination at Lys-34 by TNF receptor-associated factor 6 and is thereby activated. The aim of this study was to investigate whether TAK1 polyubiquitination at Lys-34 is also essential for NF-κB activation via TNF receptor, IL-1 receptor and toll-like receptor 4. We observed that TAK1 polyubiquitination occurred at Lys-34 and required the E3 ubiquitin ligase TNF receptor-associated factor 6 after stimulation of cells with IL-1β. Polyubiquitination of TAK1 also occurred at Lys-34 in cells stimulated by TNF-α and LPS, which activates TLR4, as well as in HepG2 and prostate cancer cells stimulated with TGFβ, which in all cases resulted in NF-κB activation. Expression of a K34R-mutant TAK1 led to a reduced NF-κB activation, IL-6 promoter activity, and proinflammatory cytokine secretion by TNF-α-stimulated PC-3U cells. Similar results were obtained in the mouse macrophage cell line RAW264.7 after LPS treatment. In conclusion, polyubiquitination of TAK1 is correlated with activation of TAK1 and is essential for activation of NF-κB signaling downstream of several receptors.

The concise nature and modularity of the synthesis described for deguelin and tephrosin (retrosynthetic analysis depicted) should facilitate access to labeled analogues to dissect the mechanism of action of this important pharmacophore.

Hypothemycin and related resorcylic acid lactones (RAL) bearing a cis-enone moiety have emerged as an alternative pharmacophore to heterocyclic motifs for kinase inhibition, and are endowed with a unique selectivity filter based on the irreversible reaction with a subset of the kinome bearing a suitably positioned cysteine residue. Two prototypical examples of "edited" RAL were evaluated for antitumoral, antimetastatic and antiangiogenic efficacy in an orthotopic murine renal cell carcinoma (RENCA) model. Both compounds (3 and 5) are good inhibitors of VEGFRs in vitro, and inhibited tumor growth in vivo with comparable efficacy to sunitinib, an FDA-approved VEGFRs inhibitor. Compound 3 promoted lung metastasis to a similar extent as sunitinib, while compound 5 strongly inhibited lung metastasis. This study attests to the potential of irreversible kinase inhibitors and molecular editing of natural pharmacophores and provides encouraging results to a clinically significant problem.

The pochoximes, based on the radicicol pharmacophore, are potent inhibitors of heat shock protein 90 (HSP90) that retain their activity in vivo. Herein we report an extended library that broadly explores the structure–activity relationship (SAR) of the pochoximes with four points of diversity. Several modifications were identified that afford improved cellular efficacy, new opportunities for conjugation, and further diversifications. Cocrystal structures of pochoximes A and B with HSP90 show that pochoximes bind to a different conformation of HSP90 than radicicol and provide a rationale for the enhanced affinity of the pochoximes relative to radicicol and the pochonins.

The resorcylic acid lactones (RAL) are endowed with diverse biological activity ranging from transcription factor modulators (zearalenone and zearalenol) to HSP90 inhibitors (radicicol and pochonin D) and reversible (aigialomycin D) as well as irreversible kinase inhibitors (hypothemycin and other RAL containing a cis-enone). Our interest in broadening the diversity of this family beyond naturally occurring diversity has led us to seek a general approach that could be used to address the entire spectrum of functionalities present within this family. Herein, we present our efforts on accessing macrocycles bearing an alkane, alkene, or epoxide at the benzylic position from a common benzylic sulfide intermediate to access L-783277, LL-Z1640-2, and hypothemycin.

Pochoximes are potent inhibitors of heat shock protein 90 (HSP90) based on the radicicol pharmacophores. Herein we present a pharmacokinetics and pharmacodynamics evaluation of this compound series as well as a phosphate prodrug strategy to facilitate formulation and improve oral bioavailability.

Short and sweet: A concise and modular synthesis of radicicol A and related resorcylic acid lactones using fluorous isolation technology and immobilized reagents is reported (see scheme, RF=C3H6C6F13, TMSE=2-(trimethylsilyl)ethyl). The compounds are found to be potent (low-nanomolar) inhibitors of selected kinases. Despite their irreversible inactivation of kinases, they show good selectivity amongst a panel of 127 kinases.

While resorcylic acid lactones (RALs) have been known for a long time, the more recent discoveries that radicicol is a potent and selective HSP90 inhibitor while other members such as hypothemycin, LL-Z1640-2 and LL-783,277 are potent kinase inhibitors have stimulated a renewed interest in this family of natural products. The recent developments regarding the chemistry and biology of RALs are reviewed.

Peptide Nucleic Acids (PNA) are non-natural oligonucleotides mimics. {\gamma}-PNA backbone are formed by standard nucleic acids nucleobases connected by a neutral N-(2-aminoethyl)glycine backbone linked by a peptide bond. In this study, we use Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) to explore the properties of the supramolecular duplexes formed by these species. We show that standard Watson-Crick base pair as well as non-standard ones are formed in solution. The duplexes thus formed present marked melting transition temperatures substantially higher than their nucleic acid homologs. Moreover, the presence of a chiral group on the {\gamma}-peptidic backbone increases further more this transition temperature, leading to very stable duplexes., Comment: 13 pages, 6 figures, plus 30 pages of Supp. Mat

Pochonins A-F were recently characterized as six new members of the naturally occurring family of 14-membered resorcylic acid lactones. As there are a high number of ATPase and kinase inhibitors among natural resorcylic lactones, a library based on the pochonin scaffold, with five points of diversity, was prepared which includes diversity beyond that of the natural analogues. The library was synthesized by using solid-supported reagents amenable to automation. Testing the library for its inhibition against a panel of 24 kinases at 10 microM afforded a14 % hit rate. These results demonstrate the potential of the resorcylides towards the inhibition of therapeutically relevant kinases.

A modular synthesis for pochonin C and radicicol is reported. The two natural products were prepared in seven and eight steps, respectively, from three readily available fragments. Alternative syntheses of these compounds were achieved using a combination of polymer-bound reagents and solid phase reactions. The conformation of the two natural products was studied and compared by using 2D NMR spectroscopy.

Back to its origins: Pochonin C was synthesized in 11 steps from the four fragments shown in the scheme. This asymmetric synthesis and the conversion of pochonin C into radicicol defines the configuration at the center bearing the chlorine atom as S. Ipc=isopenocampheyl, PG=protecting group.

RNA recognition by natural aminoglycoside antibiotics depends on the 2-deoxystreptamine (2-DOS) scaffold which participates in specific hydrogen bonds with the ribosomal decoding-site target. Three-dimensional structure information has been used for the design of azepane-monoglycosides, building blocks for novel antibiotics in which 2-DOS is replaced by a heterocyclic scaffold. Azepane-glycosides showed target binding and translation inhibition in the low micromolar range and inhibited growth of Staphylococcus aureus, including aminoglycoside-resistant strains.

An efficient and highly versatile synthesis of two libraries 1(x,y) and 2-Ar(x,y,z) or 2-R 2 (x,y,w) based on the privileged benzimidazole scaffold is described. Our design is aimed at obtaining molecules, biased for binding to RNA targets, by incorporating functionalities, which are frequently found in natural RNA-ligands. The library construction was realized with the use of SPOS in high average yields and purity. Monitoring and quantitation of intermediates and final products were performed by the use of NMR spectroscopy using DMFu as an internal standard.

The programmability of oligonucleotide hybridization offers an attractive platform for the design of assemblies with emergent properties or functions. Developments in DNA nanotechnologies have transformed our thinking about the applications of nucleic acids. Progress from designed assemblies to functional outputs will continue to benefit from functionalities added to the nucleic acids that can participate in reactions or interactions beyond hybridization. In that respect, peptide nucleic acids (PNAs) are interesting because they combine the hybridization properties of DNA with the modularity of peptides. In fact, PNAs form more stable duplexes with DNA or RNA than the corresponding natural homoduplexes. The high stability achieved with shorter oligomers (an 8-mer is sufficient for a stable duplex at room temperature) typically results in very high sequence fidelity in the hybridization with negligible impact of the ionic strength of the buffer due to the lack of electrostatic repulsion between the duplex strands. The simple peptidic backbone of PNA has been shown to be tolerant of modifications with substitutions that further enhance the duplex stability while providing opportunities for functionalization. Moreover, the metabolic stability of PNAs facilitates their integration into systems that interface with biology. Over the past decade, there has been a growing interest in using PNAs as biosupramolecular tags to program assemblies and reactions. A series of robust templated reactions have been developed with functionalized PNA. These reactions can be used to translate DNA templates into functional polymers of unprecedented complexity, fluorescent outputs, or bioactive small molecules. Furthermore, cellular nucleic acids (mRNA or miRNA) have been harnessed to promote assemblies and reactions in live cells. The tolerance of PNA synthesis also lends itself to the encoding of small molecules that can be further assembled on the basis of their nucleic acid sequences. It is now well-established that hybridization-based assemblies displaying two or more ligands can interact synergistically with a target biomolecule. These assemblies have now been shown to be functional in vivo. Similarly, PNA-tagged macromolecules have been used to prepare bioactive assemblies and three-dimensional nanostructures. Several technologies based on DNA-templated synthesis of sequence-defined polymers or DNA-templated display of ligands have been shown to be compatible with reiterative cycles of selection/amplification starting with large libraries of DNA templates, bringing the power of in vitro evolution to synthetic molecules and offering the possibility of exploring uncharted molecular diversity space with unprecedented scope and speed.

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

Natural aminoglycoside antibiotics recognize an internal loop of bacterial ribosomal-decoding-site RNA by binding to the deep groove of the RNA structure. We have designed, synthesized, and tested RNA-targeted paromamine derivatives that exploit additional interactions on the shallow groove face of the decoding-site RNA. An in vitro transcription-translation assay of a series of 6'-derivatives showed the 6'-position to be very sensitive to substitution. This result suggests that the group at the 6'-position plays a pivotal role in RNA target recognition.

The ribosomal decoding site is the target of aminoglycoside antibiotics that specifically recognize an internal loop RNA structure. We synthesized RNA-targeted 2,5-dideoxystreptamine-4-amides in which a sugar moiety in natural aminoglycosides is replaced by heterocycles.

A series of bromotyrosine-derived compounds, including marine natural products and members of a psammaplin A-inspired combinatorial synthetic library, were screened for their ability to inhibit the Mycobacterium tuberculosis detoxification enzyme mycothiol- S -conjugate amidase (MCA). Correlations between the structures and their respective IC 50 values (which range from 3 μM to 2.7 mM) should prove valuable when optimizing more potent inhibitors of MCA.