Publications

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. Using 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.

The photoreduction of azide-based immolative linker by Ru(II) conjugates to uncage rhodamine was achieved using different oligomeric protein templates. The generality of the approach was validated with three sets of ligand having varying affinity to their target (biotin, desthiobiotin and raloxifene). The reaction rates of the templated reaction was found to be at least 30-fold faster than the untemplated reaction providing a clear fluorescent signal in response to the protein oligomer within 30 min. The templated reaction was found to also proceed in cellulo and could be used to identify acetyl coenzyme A carboxylase (ACC) in Pseudomonas aeruginosa and human cell lines as well the and estrogen receptor (ER).

Nucleic acid-encoded libraries are emerging as an attractive and highly miniaturized format for the rapid identification of protein ligands. An important criterion in the synthesis of nucleic acid encoded libraries is the scope of reactions that can be used to introduce molecular diversity and devise divergent pathways for diversity-oriented synthesis (DOS). To date, the protecting group strategies that have been used in peptide nucleic acid (PNA) encoded synthesis (PES) have limited the choice of reactions used in the library synthesis to just a few prototypes. Herein, we describe the preparation of PNA monomers with a protecting group combination (Mtt/Boc) that is orthogonal to Fmoc-based synthesis and compatible with a large palette of reactions that have been productively used in DOS (palladium cross-couplings, metathesis, reductive amination, amidation, heterocycle formation, nucleophilic addition, conjugate additions, Pictet–Spengler cyclization). We incorporate γ-modifications in the PNA backbone that are known to enhance hybridization and solubility. We demonstrate the robustness of this strategy with a library synthesis that is characterized by MALDI MS analysis at every step.

Over the past decade, several technologies have emerged to access nucleic acid-tagged libraries and select the fittest compound within such libraries. This perspective focuses on recent development with PNA-tagged small molecules displayed on DNA templates for screening purposes and to probe the optimal geometry in multivalent interactions.

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.

Dehydrocholic acid was identified as a selective streptavidin binder from a PNA-tagged library. Isothermal calorimetry titration measurements showed this interaction to be entropically driven. Peptides tagged with dehydrocholic acid can be captured on a streptavidin resin and released under thermal conditions.

Hybridization-based reactions have attracted significant attention. The nucleic acid templated photocatalyzed azide reduction using catalytic amounts of a [Ru(bpy)₂phen]²⁺ conjugate is reported. The reaction could be performed with as little as 2% of the Ru nucleic acid probe and was shown to productively unquench 7-azido-coumarin as well as uncage a small molecule.

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.

Deguelin, a rotenoid, has emerged as an attractive pharmacophore for chemoprevention showing in vivo activity in several xenografts. Recently, several lines of evidence have suggested its mode of action may involve inhibition of HSP90, however binding in a different mode than known pharmacophores. To further probe the target of deguelin and related rotenoids, several biotin conjugates were prepared. None of the conjugates showed significant affinity for HSP90, however two conjugates showed a strong cellular co-localization with mitochondria, consistent with binding to mitochondrial complex 1. Contrarily to rotenone, deguelin and tephrosin were not found to inhibit tubulin polymerization demonstrating a dramatic pharmacological difference between these closely related rotenoids.

The photocleavage of a nitrobenzyl-type linker (NPPOC) at 405 nm wavelength was enabled by nucleic acid-templated energy transfer from a sensitizer (thioxanthenone) to the linker. This strategy was used to release profluorescent rhodamine, which facilitated monitoring of the reaction via fluorescence measurement in a nonoverlapping window with the sensitizer/photocleavage reaction. The rate acceleration of the templated reaction was greater than 20-fold over the background reaction. The templated reaction was used in conjunction with strand displacement to design four-component systems that responded to an analyte (DNA). Programming a specific hierarchical relationship among the four components enabled the design of a system that responded first positively and then negatively to increasing levels of an analyte.

We report the synthesis of a nucleic acid-encoded carbohydrate library, its combinatorial self-assembly into 37485 pairs and a screen against DC-SIGN leading to the identification of consensus ligand motifs. A prototypical example from the selected pairs was shown to have enhanced binding. A dendrimer incorporating the selected motifs inhibited gp120's binding to dendritic cells with higher efficiency than mannan.

It is generally accepted that microRNAs (miRNAs) play a crucial role in gene expression regulation and that their aberrant expression is intimately linked with pathologies, most notably cancer. There is thus significant interest in detecting and quantifying these important regulators. Herein, we report the fluorescence imaging of miRNAs within a few hours using a nucleic-acid templated Staudinger reaction. A good correlation between the level of miRNAs and the fluorescence intensity was observed across different cell lines. This method was shown to also be applicable for suspended cells with fluorescence quantification by flow cytometry.

The predictable nature of nucleic acid hybridization offers a simple platform to program assemblies with emerging function. This review will highlight some examples from our laboratory towards the use of this concept in chemical biology. These developments capitalize on peptide nucleic acids (PNAs), a functional analogue of natural oligonucleotides to program assemblies. In the first example, hybridization is used to organize mixtures of molecules tagged with PNAs into a microarray format; in the second example, the PNA tags are used to program the dimerization of ligands onto a nucleic acid template which translates into enhanced affinity for a biomolecule; in the third one, hybridization is used to program a reaction by aligning reactive functional groups and translated into a fluorescent signal. While these examples are only a prelude in translating instructions into a function, we hope that they can be the basis of more complex network and inspire further developments in the broader context of system chemistry.

Kinases have emerged as one of the most prolific therapeutic targets. An important criterion in the therapeutic success of inhibitors targeting the nucleotide binding pocket of kinases is the inhibitor residence time. Recently, covalent kinase inhibitors have attracted attention since they confer terminal inhibition and should thus be more effective than reversible inhibitors with transient inhibition. The most robust approach to design irreversible inhibitors is to capitalize on the nucleophilicity of a cysteine thiol group present in the target protein. Herein, we report a systematic analysis of cysteine residues present in the nucleotide binding site of kinases, which could be harnessed for irreversible inhibition, taking into consideration the different kinase conformations. We demonstrate the predictive power of this analysis with the design and validation of an irreversible inhibitor of KIT/PDGFR kinases. This is the first example of a covalent kinase inhibitor that combines a pharmacophore addressing the DFG-out conformation with a covalent trap.

Nucleic acid templated reactions have attracted significant attention for nucleic acid sensing. Herein we report a general design which extends the potential of nucleic acid templated reactions to unleash the function of a broad diversity of small molecules such as a transcription factor agonist, a cytotoxic or a fluorophore.

The discovery of small molecule probes which selectively modulate biological pathways is a cornerstone in the development of new therapeutics. Progress in our ability to access libraries of biologically relevant small molecules in conjunction with streamlined screening technologies have also enabled a more systematic approach to chemical biology. Nevertheless, the current state of the art still requires a large infrastructure and only a small fraction of the proteome has been addressed thus far. The emergence of technologies based on nucleic acid encoding of small molecules presents a new screening paradigm. We describe a method based on DNA-templated combinatorial display of PNA-encoded drug fragments affording 62500 combinations which can be amplified following a selection. This concept was demonstrated with a screen against a representative target, carbonic anhydrase, by iterative cycles of affinity selection, amplification of DNA template and "translation" back into selected library members. The results show a clear convergence towards combinations which, upon resynthesis as covalent adducts, proved to bind cooperatively to carbonic anhydrase.

Complex glycan arrays: A general method for preparing thioglycosides from native oligosaccharides and coupling them to peptide nucleic acids (PNAs) is reported. An array of 625 glycans was assembled by hybridization of two PNA-encoded libraries of 25 glycan fragments. The cooperativity of the fragment for carbohydrate binding proteins was validated with lectins of known specificity.

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.

Oligomerization of receptors induced or stabilized by polyvalent ligands is a fundamental mechanism in cellular recognition and signal transduction. Herein we report a general approach to encode complex peptide macrocycles with peptide nucleic acid (PNA) tags and program their oligomerization through hybridization as exemplified with a ligand binding to oligomeric DR5, a receptor of TRAIL cytokine.

Glioblastoma multiforme (GBM) has an abysmal prognosis. We now know that the epidermal growth factor receptor (EGFR) signaling pathway and the loss of function of the tumor suppressor genes p16Ink4a/p19ARF and PTEN play a crucial role in GBM pathogenesis: initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. We have recently shown that this genetic combination is sufficient to promote the development of GBM in adult mice. Therapeutic agents raised against single targets of the EGFR signaling pathway have proven rather inefficient in GBM therapy, showing the need for combinatorial therapeutic approaches. An effective strategy for concurrent disruption of multiple signaling pathways is via the inhibition of the molecular chaperone heat shock protein 90 (Hsp90). Hsp90 inhibition leads to the degradation of so-called client proteins, many of which are key effectors of GBM pathogenesis. NXD30001 is a novel second generation Hsp90 inhibitor that shows improved pharmacokinetic parameters. Here we show that NXD30001 is a potent inhibitor of GBM cell growth in vitro consistent with its capacity to inhibit several key targets and regulators of GBM biology. We also show the efficacy of NXD30001 in vivo in an EGFR-driven genetically engineered mouse model of GBM. Our findings establish that the Hsp90 inhibitor NXD30001 is a therapeutically multivalent molecule, whose actions strike GBM at the core of its drivers of tumorigenesis and represent a compelling rationale for its use in GBM treatment.

Transforming growth factor-β-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain αG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic αG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of αG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.

Emphasizing the impact of metathesis in natural product synthesis through the different types of key reactions, this is a comprehensive view of a hot topic. Written by top international authors, this ready reference is clearly structured and packed with important information, including representative experimental procedures for practical applications.
A real must-have for anyone working in natural product synthesis.

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab-on-Bead^TM library, a one-bead, one-sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy-aided identification of single target-bound beads and the extraction - wet or dry - of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target-binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target-binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead-based design allows for easy screening over a variety of prepared surfaces that can extend this technique's application to the discovery of diagnostic reagents and disease markers.

The screening for treatment-induced enzyme activities offers the opportunity to discover important regulatory mechanisms and the identification of potential targets for anti-cancer therapies. A novel screening technique was applied to screen substrate peptide sequences for proteolytic activities up- or down-regulated by ionizing radiation in tumor cells. One specific substrate sequence was cleaved in control cell extracts but to a smaller extent in irradiated cell extracts and investigated in detail. Based on protease-class-specific inhibitory studies and cleavage site analysis a potent warhead-inhibitor was synthesized and used to identify the proteasome as the protease of interest. The investigated sequence shows high homology to a regulatory site of nucleoporin 50, an element of the nuclear pore complex, and site specific cleavage of nucleoporin 50 was determined in vitro suggesting a novel link between the ionizing radiation-regulated proteasome and nuclear protein shuttling.

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.

Inspirational kinase inhibitors: The terminal inhibition achieved with irreversible inhibitors has frequently been harnessed by Nature. Resorcyclic acid lactones (RAL) bearing a cis-enone moiety have emerged as promising pharmacophores for kinase inhibition. Two prototypical edited RALs were evaluated on an orthotopic murine carcinoma. Despite their similar biochemical activity profile, remarkable differences were observed with respect to metastasis inhibition.

Peptide nucleic acids (PNAs) are functional analogues of natural oligonucleotides. Herein, we report the synthesis of PNAs bearing a triazole in lieu of the amide bond assembled using a "click" cycloaddition, their hybridization properties as well as the DNA-templated coupling of the azide and alkyne PNA fragments.

Molecular editing: The resorcylic acid lactones (RAL) are known small-molecule irreversible inhibitors of select kinases, and represent a unique pharmacophore with potential for further development in kinase research. The basic pharmacophore was "edited" to improve the properties and to diversify the scaffold. Two fluoroenones were synthesized, and their preliminary biological evaluation revealed interesting activity.

Heat shock protein 90 (Hsp90) is an ATP-dependent chaperone which is involved in the post-translational maturation and stabilization of over one hundred proteins ("its clients"). In the absence of Hsp90's chaperoning, its clients are misfolded and degraded via ubiquitin-proteasome pathway. HSP90 has become the focus of intense drug discovery efforts as its activity has been implicated in diverse pathologies ranging from oncology to neurodegenerative and infectious diseases. The most promising inhibitors reported to date inhibit the ATPase activity by binding to the Nterminal ATP pocket. Radicicol, a member of the resorcylic acid lactones (RALs), represents an important pharmacophore to this end. Efforts towards the development of this pharmacophore and its SAR are reviewed herein.

Potent pochoximes: We generated an extended library of pochoxime inhibitors of heat shock protein 90 and identified a pochoxime that has a cellular efficacy of less than 10 nm. By examining cocrystal structures of pochoximes with human HSP90α, we found that these compounds bind to a different conformation of HSP90 than the closely related radicicol.

Browsing through the library: A library of cis-enone resorcyclic acids offering important structural activity relationships is reported. Two modifications were found to independently and synergistically improve the activity of this series of compounds, whereas a modification which dramatically simplifies the synthetic accessibility of these compounds was established.

Extension of the scope of the sulfide linker to access all the functionalities present in the natural members of the resorcylic acid lactones is reported (see scheme). The use of the benzylic sulfide was efficient in providing access to both LL-Z1640-2 and L-783277 by alkylation followed by oxidative elimination or reductive cleavage, respectively.

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.

All under control: The programmability of hybridization has been utilized to generate a combinatorial library of structures that emulate the topologies of complex carbohydrates interacting with an antibody that shows broad-spectrum activity against HIV. This simple method involves attaching oligosaccharides tagged with peptide nucleic acids onto DNA templates in a controlled manner

Nucleic acid-templated reactions leading to a fluorescent product represent an attractive strategy for the detection and imaging of cellular nucleic acids. Herein we report the use of a Staudinger reaction to promote the reduction of profluorescent azidorhodamine. The use of two cell-permeable GPNA probes, one labeled with the profluorescent azidorhodamine and the other with trialkylphosphine, enabled the detection of the mRNA encoding O-6-methylguanine-DNA methyltransferase in intact cells.

We determine the binding mode of a macrocyclic radicicol-like oxime to yeast HSP90 by combining computer simulations and experimental measurements. We sample the macrocyclic scaffold of the unbound ligand by parallel tempering simulations and dock the most populated conformations to yeast HSP90. Docking poses are then evaluated by the use of binding free energy estimations with the linear interaction energy method. Comparison of QM/MM-calculated NMR chemical shifts with experimental shift data for a selective subset of backbone ¹⁵N provides an additional evaluation criteria. As a final test we check the binding modes against available structure-activity-relationships. We find that the most likely binding mode of the oxime to yeast HSP90 is very similar to the known structure of the radicicol-HSP90 complex.

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.

Several technologies have been described to immobilize libraries of small molecules or peptides in a microarray format. Herein, we describe protocols for an alternative strategy whereby each small molecule or peptide within a library is labeled with a peptide nucleic acid (PNA) tag such that they self-assemble in a microarray format upon hybridization with readily available DNA arrays. An important asset of the method is that it allows the library to be used in solution prior to hybridizing and as such offers the opportunity to separate the inhibitors bound to the protein from the rest of the library. Two methods based on size exclusion filtration or gel electrophoresis to separate protein-bound inhibitors from the remaining library are described.

The recent discoveries of potent HSP90 and MAP kinase inhibitors amongst the resorcylic acid lactones (RALs) have revived interest in this family of natural products. Both HSP90 and MAP kinase inhibition hold tremendous therapeutic potential, particularly in the treatment of cancer. Our synthetic efforts towards the RALs and, in particular, selective inhibitors of HSP90 and kinases are reviewed.

We present a thorough investigation of six types of protecting groups for the terminal nitrogen atom and five protecting groups on the nucleobases of peptide nucleic acids for fully orthogonal synthesis with Fmoc.

Peptide nucleic acids (PNAs) hybridize to natural oligonucleotides according to Watson and Crick base-pairing rules. The robustness of PNA oligomers and ease of synthesis have made them an attractive platform to encode small or macromolecules for microarraying purposes and other applications based on programmable self assembly. A cornerstone of these endeavors is the orthogonality of PNA synthesis with other chemistries. Herein, we present a thorough investigation of six types of protecting groups for the terminal nitrogen atom (Alloc, Teoc, 4-N₃Cbz, Fmoc, 4-OTBSCbz, and Azoc) and five protecting groups on the nucleobases (Cl-Bhoc, F-Bhoc, Teoc, 4-OMeCbz, and Boc).

A concise sequence utilizing a Petasis three component reaction followed by a tandem aza-Cope-Mannich cyclization afforded novel polycyclic heterocycles in good yield; alternative iminium cyclization based on a Pictet-Spengler reaction or aminal formation led to divergent pathways affording skeletal diversity.

Make a clean breast of it: The generation of a library of pochonin D derivatives by a solid-phase approach has led to the discovery of pochoxime (see scheme), a potent inhibitor of the heat-shock protein 90, with over 100-fold improved incellular activity. Pochoxime was found to be effective in breast tumor xenografts, leading to a reduction in the tumor size.

This tutorial review serves as an introduction to the use of oligonucleotides and in particular peptide nucleic acids (PNAs) to encode function beyond heredity. Applications in chemical biology are reviewed starting with the use of nucleic acid tags to program self-assembled microarrays of small and macromolecules, followed by the use of nucleic acid templated reactions for the purpose of DNA or RNA sensing and finally, the use of nucleic acid templates to display ligands.

Templated reduction of low fluorescence azidocoumarin-PNA conjugate to high fluorescence aminocoumarin was achieved using a catalytic amount of DNA with single nucleotide resolution.

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, R^F=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.

Seven PNA-encoded combinatorial libraries targeting proteases and phosphatases with covalent reversible and irreversible mechanism-based inhibitors were prepared. The libraries were synthesized using modified PNA monomers, which dramatically increase the water solubility of the libraries in biologically relevant buffers. The libraries were shown to selectively inhibit targeted enzymes.

The azide-based carbamate or carbonate protecting group (Azoc) shown above can be removed in less than 2 min under neutral conditions using trimethyl or tributyl phosphine as well as polymer-bound triphenyl phosphine. It was shown to be orthogonal to Fmoc and Mtt for peptide synthesis and to afford β-glycoside with a 2-aminoglucosyl donor by virtue of the neighboring group participation.

A facile method to convert thioglycosides to glycosyl fluorides with Ipy₂BF₄ (py = pyridine) is presented. Alternatively, activation of thioglycosides with Ipy₂BF₄ in the presence of acids and glycosyl acceptors led to glycosylation reactions. Perbenzylated (armed) glycosyl donors yielded predominantly the β-anomeric product. This methodology is compatible with one-pot sequential glycosylation.

In the continuous drive to increase screening throughput and reduce sample requirement, microarray-based technologies have risen to the occasion. In the past 7 years, a number of new methodologies have been developed for preparing small molecule microarrays from combinatorial and natural product libraries with the goal of identifying new interactions or enzymatic activities. Recent advances and applications of small molecule microarrays are reviewed.

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.

From libraries to arrays. Peptide nucleic acid encoded libraries can be converted into organized microarrays by hybridization to readily available DNA arrays (see images). Here this method has been applied to the discovery of specific inhibitors for cathepsin K and cathepsin F.

Small-molecule microarrays are attractive for chemical biology as they permit the analysis of hundreds to thousands of interactions in a highly miniaturized format. Methods to prepare small-molecule microarrays from combinatorial libraries by a self-assembly process based on the sequence-specific hybridization of peptide nucleic acid (PNA) encoded libraries to oligonucleotide arrays are presented. A systematic study of the dynamic range for multiple detection agents, including direct fluorescence of attached fluorescein and cyanine-3 dyes, antibody-mediated fluorescence amplification, and biotin-gold nanoparticle detection, demonstrated that individual PNA-encoded probes can be detected to concentrations of 10 pm on the oligonucleotide microarrays. Furthermore, a new method for parallel processing of biological samples by using gel-based separation of probes is presented. The methods presented in this report are exemplified through profiling two closely related cysteine proteases, cathepsin K and cathepsin F, across a 625-member PNA-encoded tetrapeptide acrylate library. A series of the specific cathepsin K and F inhibitors identified from the library were kinetically characterized and shown to correlate with the observed microarray profile, thus validating the described methods. Importantly, it was shown that this method could be used to obtain orthogonal inhibitors that displayed greater than tenfold selectivity for these closely related cathepsins.

Kinase inhibitors: Despite the lack of homology to purine analogues, pochonins have been shown to be ATPase inhibitors. A library of pochonins extending beyond the natural analogues was prepared by using solid-supported reagents. Screening the library against a panel of 24 kinases revealed a high number of inhibitors against therapeutically relevant kinases, such as Src, Aurora, and EGF-R, in contrast to pochonin D, which has no measurable kinase activity.

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 μm afforded a >14 % hit rate. These results demonstrate the potential of the resorcylides towards the inhibition of therapeutically relevant kinases.

On solid ground: Despite no obvious resemblance to adenosine analogues, the family of resorcyclic macrolides contains a high proportion of kinase and ATPase inhibitors. A solid-phase total synthesis of aigialomycin D extends the diversity of this class of natural product. Aigialomycin was found to inhibit CDK1/5 and GSK. EOM=ethoxymethyl.

An expedient synthesis of (-)-pochonin A is reported (seven steps). This natural product is closely related to radicicol and was shown to be a 90 nM inhibitor of HSP90.

The availability of complete genome sequences from numerous organisms has provided investigators with the challenge of assigning physiologic functions to the encoded gene products. To facilitate this process, multiple technologies have been developed to profile the transcriptome and the proteome, including methods to monitor the function of enzymes in complex biologic systems. These methods typically target specific classes of enzymes and attempt to correlate the enzymatic activity with the specific phenotype of interest. Here, technologies to measure enzymatic activity on a subproteomic scale are reviewed, including the authors' own efforts, which are based on self-assembled microarrays utilizing peptide nucleic acid-encoded small-molecule libraries.

Microarrays have become an indispensable technology in post-genomic research and a number of strategies have been developed to immobilize analytes in the microarray format. This concept article describes a supramolecular preparation based on sequence specific hybridization of PNA. This approach allows the use of the libraries as mixtures in solution which can be converted to an organized microarray in one step by a self-sorting process.

Microarray-based technologies have attracted attention in chemical biology by virtue of their miniaturized format, which is well suited to probe ligand-protein interactions or investigate enzymatic activity in complex biological mixtures. A number of research groups have reported the preparation of surfaces on microarrays with specific functional groups to chemoselectively attach small molecules from libraries. We have developed an alternative method whereby libraries are encoded with peptide nucleic acid (PNA), such that libraries which exist as mixtures in solution self-assemble into an organized microarray through hybridization to produce readily available DNA arrays. This allows libraries synthesized by split and mix methods to be decoded in a single step. An asset of this method compared to direct spotting is that libraries can be used in solution for bioassays prior to self-assembly into the microarray format.

Macrolides with a broad biological activity based on recorcinol-based compounds are described. An expedient and modular synthesis of radicicol (Monorden) and pochonin C is reported. A combination of solid-phase chemistry and polymer-bound reagents was used to assemble both natural products in seven and eight steps, respectively, from the intermediates depicted.

The molecular chaperone HSP90 is an attractive target for chemotherapy because its activity is required for the functional maturation of a number of oncogenes. Among the know inhibitors, radicicol, a 14-member macrolide, stands out as the most potent. A molecular dynamics/minimization of radicicol showed that there were three low energy conformers of the macrocycle. The lowest of these is the bioactive conformation observed in the cocrystal structure of radicicol with HSP90. Corresponding conformational analyses of several known analogues gave a good correlation between the bioactivity and the energy of the bioactive conformer, relative to other conformers. Based on this observation, a number of proposed analogues were analyzed for their propensity to adopt the bioactive conformation prior to synthesis. This led to the identification of pochonin D, a recently isolated secondary metabolite of Pochonia chlamydosporia, as a potential inhibitor of HSP90. Pochonin D was synthesized using polymer-bound reagents and shown to be nearly as potent an HSP90 inhibitor as radicicol.

Enzymatic activity in the fecal droppings from the house dust mite has been postulated to contribute to the elicited allergic response. Screening dust mite extracts through 137,180 tetrapeptide fluorogenic substrates allowed for the characterization of proteolytic substrate specificity from the potential cysteine and serine proteases in the extract. The extract was further screened against a 4000 member peptide nucleic acid (PNA) encoded inhibitor library designed to target cysteine proteases using microarray detection. Affinity chromatography coupled with mass spectrometry identified Der p 1 as one of the proteases targeted by the PNA inhibitors in the dust mite lysate. A phenotypic readout of Der p 1 function in allergy progression was demonstrated by the inhibition of CD25 cleavage from T cells by dust mite extract that had been treated with the Der p 1 inhibitor identified from the PNA-encoded inhibitor library.

Our current understanding of the role and regulation of protease activity in normal and pathogenic processes is limited by our ability to measure and deconvolute their enzymatic activity. To address this limitation, an approach was developed that utilizes rhodamine-based fluorogenic substrates encoded with PNA tags. The PNA tags address each of the substrates to a predefined location on an oligonucleotide microarray through hybridization, thus allowing the deconvolution of multiple signals from a solution. A library of 192 protease substrates was prepared by split and mix combinatorial synthesis. The methodology and validation of this approach for profiling proteolytic activity from single proteases and from those in crude cell lysates as well as clinical blood samples is described.

NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

Peptide nucleic acids (PNAs) have been used to encode a combinatorial library whereby each compound is labeled with a PNA tag which reflects its synthetic history and localizes the compound upon hybridization to an oligonucleotide array. We report herein the full synthetic details for a 4000 member PNA-encoded library targeted towards cysteine protease.

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.

The chemical shift sensitivity and significant signal enhancement afforded by laser-polarized ¹²⁹Xe have motivated the application of ¹²⁹Xe NMR to biological imaging and spectroscopy. Recent research done by our group has used laser-polarized ¹²⁹Xe in biomolecular assays that detect ligand-binding events and distinguish protein conformations. The successful application of unfunctionalized and functionalized ¹²⁹Xe NMR to in vitro biomolecular assays suggests the potential future use of a functionalized xenon biosensor for in vivo imaging.

A practical and efficient method for PNA synthesis using an azide group to mask the N-terminus is reported. The deprotection was carried out in 5 min, while couplings were complete within 60 min. The near neutral conditions of the phosphine deprotection combined with the base-free coupling using hydroxybenzotriazole-activated monomers make this approach very mild.

Using a polymer-bound selenenyl bromide resin, o-allyl and o-prenyl anilines were cycloaded to afford a series of solid-supported indoline and indole scaffolds. These scaffolds were then functionalized and cleaved via four distinct methods, namely traceless reduction, radical cyclization, radical rearrangement, and oxidative elimination, to afford 2-methyl indolines, polycyclic indolines, 2-methyl indoles, and 2-propenyl indolines, respectively. A number of small combinatorial libraries of compounds reminiscent of certain designed ligands of biological interest were constructed demonstrating the potential utility of the developed methodology to chemical biology studies and the drug discovery process.

The regulation of protein function through posttranslational modification, local environment, and protein-protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile.

Small molecules encoded with peptidonucleic acid (PNA) were used to probe protein function in a microarray format. The PNA tag served to encode the synthetic history of the small molecule and to positionally encode the identity of the small molecule by its location upon hybridization to an oligonucleotide microarray (see picture).

A number of highly potent antibiotics have been identified in the screening of compound libraries based on the notion that dimerization (an example is shown here) and/or variation of amino acid one of vancomycin could potentially enhance biological activity.

Based on the notion that dimerization and/or variation of amino acid 1 of vancomycin could potentially enhance biological activity, a series of synthetic and chemical biology studies were undertaken in order to discover potent antibacterial agents. Herein we describe two ligation methods (disulfide formation and olefin metathesis) for dimerizing vancomycin derivatives and applications of target-accelerated combinatorial synthesis (e.g. combinatorial synthesis in the presence of vancomycin's target Ac₂-l-Lys-d-Ala-d-Ala) to generate libraries of vancomycin dimers. Screening of these compound libraries led to the identification of a number of highly potent antibiotics effective against vancomycin-suspectible, vancomycin-intermediate resistant and, most significantly, vancomycin-resistant bacteria.

The development of a new synthetic technology based on the design of a novel selenium safety catch linker is described for the solid-phase semisynthesis of monomeric vancomycin (see for example 1, R¹ = tert-butyldimethylsilyl, R² = protected carbohydrates), along with the solid- and solution-phase synthesis of vancomycin libraries, which were further tested for their biological activity.

Vancomycin, the prototypical member of the glycopeptide family of antibiotics, is a clinically used antibiotic employed against a variety of drug-resistant bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA). The recent emergence of vancomycin resistance, viewed as a growing threat to public health, prompted us to initiate a program aimed at restoring the potency of this important antibiotic through chemical manipulation of the vancomycin structure. Herein, we describe the development of synthetic technology based on the design of a novel selenium safety catch linker, application of this technology to a solid-phase semisynthesis of vancomycin, and the solid- and solution-phase synthesis of vancomycin libraries. Biological evaluation of these compound libraries led to the identification of a number of in vitro highly potent antibacterial agents effective against vancomycin-resistant bacteria. In addition to aiding these investigations, the solid-phase chemistry described herein is expected to enhance the power of combinatorial chemistry and facilitate chemical biology and medicinal chemistry studies.

A new photolabile linker enabling nucleophilic cleavage of a carboxyl functionality upon irradiation with UV light (> 290 nm) was developed. When the photocleavage is carried out in the presence of primary or secondary amines, amides are obtained in high yields and purities, while the intramolecular version of this reaction leads to heterocycles via a cyclorelease mechanism.

The epothilones and sarcodictyins have shown to be potent cytotoxic compounds with a Taxol^TM-like mode of action. The clinical importance of Taxol^TM has stimulated intense research into finding compounds with prospective superior chemotherapeutic properties to Taxol^TM. We have developed enabling synthetic technologies for the construction of combinatorial libraries of these two potentially important chemotherapeutics, which led, in both cases to compounds with superior biological activity.

Biological activity can be predicted at a prescreening stage by using a target-accelerated combinatorial synthesis. The rate of dimerization of vancomycin analogues (see picture, X=CH=CH₂, SAc) in the presence of vancomycin's targets Ac-d-Ala-d-Ala and Ac₂-l-Lys-d-Ala-d-Ala correlated well with the observed biological activity. From this study three highly potent antibacterial agents effective against both vancomycin-susceptible and vancomycin-resistant bacteria strains were identified.

The macrolactonization-based strategy for the total synthesis of epothilones has been streamlined and improved to a high level of efficiency and stereoselectivity. This strategy has been applied to the construction of vinyl iodide 19 which served as a common intermediate for the synthesis of a series of natural and designed epothilones including an epothilone B₁₀ (3), epothilone F (5), 16-desmethylepothilone B (14), pyridine epothilones 57 a-57 g, dimeric epothilones 59 and 61, and benzenoid epothilones 63 a-63 g.

Pro-allyl and pro-alloc linkers can be formed by alkylation or esterification of a selenium-bound resin (the example shown is for the formation of a polymer-bound pro-allyl derivative) and can be readily cleaved in excellent yields under mild conditions. The scope of the pro-allyl linker has been demonstrated with the solid-phase semisynthesis of vancomycin. Alloc=allyloxycarbonyl.

A new millenium has begun-grounds enough to question the present state of the total synthesis of natural products. In this review we answer this question by tracing the evolution of this fine art and science from its birth to the present time. This retrospective on total synthesis should serve to demonstrate how far we have come, yet show that the science of total synthesis is still in its infancy.

The sarcodictyins A-F and eleutherobin comprise a family of marine-derived diterpenoids with potent cytotoxicities against various tumor cell lines. Investigations have revealed that several of thess compounds exert their cytotoxic effects through tubulin binding in a mechanism analogous to that of the clinical anticancer drug taxolTM. The biological importance, challenging molecular architecture, and relative scarcity of these natural products have prompted several groups to undertake their total chemical synthesis. In this review, we summarize the current synthetic efforts and examine the preliminary structure-activity relationships which have emerged from early combinatorial libraries.

A number of valuable new synthetic strategies, such as the triazene-driven biaryl ether synthesis, have been developed during the total synthesis of vancomycin (1). Modern catalytic asymmetric reactions were employed for the construction of the required amino acid building blocks, which were then assembled to the appropriate peptide fragments, whose cyclization in the order C-O-D→AB/C-O-D→AB/C-O-D-E led to framework of the vancomycin aglycon (2). Sequential attachment of the required sugar moieties onto a suitably protected aglycon derivative, followed by deprotection, allowed the stereoselective total synthesis of the glycopeptide antibiotic vancomycin (1).

The war against infectious bacteria is not over! Although vancomycin and glycopeptide antibiotics have provided a strong last line of defence against many drug-resistant bacteria, their overuse has given rise to more dangerous strains of bacteria. An understanding of the chemistry and biology of these highly complex glycopeptides are destined to play a crucial role in the discovery of new antibiotics.

The synthesis of the macrocyclic core of sanglifehrin A, a newly discovered natural product, is described.

The fine-tuning of the protecting groups and the glycosidation conditions were the key to the successful coupling of the carbohydrate units and vancomycin aglycon in the last steps of the total synthesis of vancomycin 1. The aglycon was converted into a suitably protected acceptor and the sugar donors were sequentially attached. Removal of all the protecting groups gave synthetic vancomycin that was indentical to the natural product (¹H and ¹³C NMR, HPLC).

Organoselenium resins 1-4 were prepared from polystyrene via lithiation and quenching with MeSeSeMe, and shown to react with a variety of substrates, aiding in useful functionalizations.

Isolated from certain species of soft corals, the sarcodictyins, eleutherobin, and eleuthosides have become important synthetic targets due the their novel molecular architectures, important biological activities, and potential in medicine. Of particular interest is their Taxol-like mechanism of action involving disturbance of the tubulin-microtubule interplay resulting in tumor cell death. Their scarcity and biological profile prompted us to initiate a program directed at exploring their chemical synthesis and chemical biology. Herein we report (a) the first total synthesis of sarcodictyins A (7) and B (8) by a combination of solution and solid-phase methods through the attachment of the common precursors 18 or 20 on solid support, thus generating conjugates 23 and 24, followed by standard chemical manipulations; (b) the construction of a combinatorial library of sarcodictyins by solution and solid-phase chemistry modifying the C-8 ester, C-15 ester, and C-4 ketal functionalities, and, therefore, producing analogues of the general structures 33, 37, and 40; (c) the tubulin polymerization properties of all members of the library; and (d) the cytotoxic actions of a selected number of these compounds against a number of tumor cells including Taxol-resistant lines. Several of the synthesized analogues were identified to be of equal or superior biological activities (e.g. 60, 61, 63, 66-70, 73, 76, 85, 92) as compared to the natural products, setting the stage for further developments in the field of cancer chemotherapy.

In this article, the history of the art and science of organic and natural products synthesis is briefly reviewed and the state of the art is discussed. The impact of this discipline on biology and medicine is amply demonstrated with examples, and projections for future developments in the field are made.

No heteroatom required! In many solid-phase syntheses, after the release from the polymer support a heteroatom (e.g. O, S, N) remains in the substrate as a residue of a linking protecting group. With polymer-bound tin reagents cleavage and cyclization of the substrate with C-C bond formation (see picture) can now be achieved by intramolecular Stille coupling. S=substrate.

Complicated oligosaccharides such as dodecasaccharide 1 can be constructed by a new solid-phase strategy. The attachment to the polymeric support (gray sphere) is through a photolabile linker (structure I), and thioglycosides serve as carbohydrate donors. Bn=benzyl, Bz=benzoyl.

The authors describe the construction of a muscone library using the powerful intramol. ketophosphonate-aldehyde condensation reaction. This adds another chem. to the solid-phase reaction library and demonstrates its power in the synthesis of natural products.

A library of epothilone A and B analogues, which was constructed by solid-phase combinatorial synthesis using SMART Microreactors and solution chemistry, was screened in two different tubulin binding assays. Selected compounds were subjected to cytotoxicity studies against a number of cell lines, including Taxol-resistant cells. Important structure-activity relationship emerged from these studies, which sets the stage for further discoveries and developments in the anticancer field.

Epothilones A and B, two compounds that have been recently isolated from myxobacterium Sorangium cellulosum strain 90, have generated intense interest among chemists, biologists and clinicians owing to the structural complexity, unusual mechanism of interaction with microtubules and anticancer potential of these molecules.

A new solid phase method for the synthesis of complex oligosaccharides is described. The method involves attachment to phenolic polystyrene of the first carbohydrate unit, through its anomeric position and via a photolabile linker, using dimethylthiomethylsulfonium triflate as an activator. Reiteration of the glycosidation process allows stereocontrolled growth of linear or branched oligosaccharides in high yield. Cleavage from the resin is efficiently achieved photolytically. The application of the method to the total prepn. of the heptasaccharide phytoalexin elicitor (HPE) is described.

A series of analogous arrays of small, non-peptidyl, non-oligomeric compounds were synthesized on polystyrene resin. With the aid of a functionally differentiated phenolic scaffold, the batch preparation of unique benzamide and urea resins was accomplished, which were further derivatized in modified 96-well plates. An efficient cleavage reaction of the phenyl benzoate link enabled the isolation of more than 600 phenolic compounds in milligram quantities that were suitable for direct biological screening. The technology described herein represents a facile, economical approach to non-peptidyl chemical diversity.