RESUMO
The ability to predict chemical structure from DNA sequence has to date been a necessary cornerstone of DNA-encoded library technology. DNA-encoded libraries (DELs) are typically screened by immobilized affinity selection and enriched library members are identified by counting the number of times an individual compound's sequence is observed in the resultant dataset. Those with high signal reads (DEL hits) are subsequently followed up through off-DNA synthesis of the predicted small molecule structures. However, hits followed-up in this manner often fail to translate to confirmed ligands. To address this low conversion rate of DEL hits to off-DNA ligands, we have developed an approach that eliminates the reliance on chemical structure prediction from DNA sequence. Here we describe our method of combining non-combinatorial resynthesis on-DNA following library procedures as a rapid means to assess the probable molecules attached to the DNA barcode. Furthermore, we apply our Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS) technique to identify the true binders found within the mixtures of on-DNA synthesis products. Finally, we describe a Normalized Enrichment (NE) metric that allows for the quantitative assessment of affinity selection in these studies. We exemplify how this combined approach enables the identification of putative hit matter against a clinically relevant therapeutic target bisphosphoglycerate mutase, BPGM.
Assuntos
DNA/química , Descoberta de Drogas , Biblioteca Gênica , Espectrometria de Massas/métodos , Técnicas de Química Combinatória , Ligantes , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
This Personal Account describes the authors' foray into DNA-encoded libraries. The article addresses several key aspects of this hit generation technology, from the development of new synthetic methodology to the subsequent conception, design, and delivery of a DNA-encoded library. In particular, we have been engaged in adapting photocatalytic reactions to the idiosyncratic requirements of DNA-encoded chemistry. We have chosen one such methodology, namely a photocatalytic [2+2] cycloaddition reaction, to showcase how we employed property-based computational analyses to guide the selection and validation of building blocks for the production of a library. Ultimately, these novel bond disconnections and design principles led to the assembly of a DNA-encoded library that is composed of structurally diverse compounds within largely desirable property space and, therefore, well positioned to deliver novel chemical matter for drug discovery programs.
Assuntos
DNA/química , Desenho de Fármacos , Biblioteca Gênica , Bibliotecas de Moléculas Pequenas/síntese química , Catálise , Técnicas de Química Combinatória , Processos Fotoquímicos , Bibliotecas de Moléculas Pequenas/químicaRESUMO
A catalytic manifold that enables photoredox cross-electrophile coupling of alkyl bromides with DNA-tagged aryl iodides in aqueous solution is presented. This metallaphotoredox transformation was aided by the identification of a new pyridyl bis(carboxamidine) ligand, which proved critical to the nickel catalytic cycle. The described C(sp2)-C(sp3) coupling tolerates a wide range of both DNA-tagged aryl iodides as well as alkyl bromides. Importantly, this reaction was optimized for parallel synthesis, which is a paramount prerequisite for the preparation of combinatorial libraries, by using a 96-well plate-compatible blue LED array as the light source. Therefore, this mild and DNA-compatible transformation is well positioned for the construction of DNA-encoded libraries.
Assuntos
Alcanos/química , Brometos/química , DNA/química , Hidrocarbonetos Aromáticos/química , Iodetos/química , Alcanos/síntese química , Alquilação , Brometos/síntese química , Catálise , Técnicas de Química Combinatória , DNA/síntese química , Hidrocarbonetos Aromáticos/síntese química , Iodetos/síntese química , Ligantes , Níquel/química , Oxirredução , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The on-DNA synthesis of highly substituted cyclobutanes was achieved through a photocatalytic [2 + 2] cycloaddition reaction in aqueous solution. Readily available DNA-tagged styrene derivatives were reacted with structurally diverse cinnamates in the presence of an iridium-based photocatalyst, Ir(ppy)2(dtbbpy)PF6, to forge two new C(sp3)-C(sp3) bonds. This transformation was demonstrated to have excellent functional group tolerance and allowed for the facile installation of a variety of heteroaromatic substituents on a densely functionalized cyclobutane scaffold.
Assuntos
Ciclobutanos/química , DNA/síntese química , Catálise , Reação de Cicloadição , DNA/química , Estrutura Molecular , Processos FotoquímicosRESUMO
DNA encoded libraries (DEL) have shown promise as a valuable technology for democratizing the hit discovery process. Although DEL provides relatively inexpensive access to libraries of unprecedented size, their production has been hampered by the idiosyncratic needs of the encoding DNA tag relegating DEL compatible chemistry to dilute aqueous environments. Recently reversible adsorption to solid support (RASS) has been demonstrated as a promising method to expand DEL reactivity using standard organic synthesis protocols. Here we demonstrate a suite of on-DNA chemistries to incorporate medicinally relevant and C-S, C-P and N-S linkages into DELs, which are underrepresented in the canonical methods.
Assuntos
DNA/síntese química , Adsorção , Técnicas de Química Sintética , Técnicas de Química Combinatória , Descoberta de Drogas , Indicadores e Reagentes , Bibliotecas de Moléculas Pequenas , Solubilidade , Sulfonas/química , Sulfóxidos/químicaRESUMO
DNA-encoded library (DEL) technology has the potential to dramatically expedite hit identification in drug discovery owing to its ability to perform protein affinity selection with millions or billions of molecules in a few experiments. To expand the molecular diversity of DEL, it is critical to develop different types of DNA-encoded transformations that produce billions of molecules with distinct molecular scaffolds. Sequential functionalization of multiple C-H bonds provides a unique avenue for creating diversity and complexity from simple starting materials. However, the use of water as solvent, the presence of DNA, and the extremely low concentration of DNA-encoded coupling partners (0.001 M) have hampered the development of DNA-encoded C(sp3)-H activation reactions. Herein, we report the realization of palladium-catalyzed C(sp3)-H arylation of aliphatic carboxylic acids, amides and ketones with DNA-encoded aryl iodides in water. Notably, the present method enables the use of alternative sets of monofunctional building blocks, providing a linchpin to facilitate further setup for DELs. Furthermore, the C-H arylation chemistry enabled the on-DNA synthesis of structurally-diverse scaffolds containing enriched C(sp3) character, chiral centers, cyclopropane, cyclobutane, and heterocycles.
RESUMO
A new catalytic manifold that merges photoredox with nickel catalysis in aqueous solution is presented. Specifically, the combination of a highly active, yet air-stable, nickel precatalyst with a new electron-deficient pyridyl carboxamidine ligand was key to the development of a water-compatible nickel catalysis platform, which is a crucial requirement for the preparation of DNA-encoded libraries (DELs). Together with an iridium-based photocatalyst and a powerful light source, this dual catalysis approach enabled the efficient decarboxylative arylation of α-amino acids with DNA-tagged aryl halides. This C(sp2)-C(sp3) coupling tolerates a wide variety of functional groups on both the amino acid and the aryl halide substrates. Due to the mild and DNA-compatible reaction conditions, the presented transformation holds great potential for the construction of DELs. This was further evidenced by showing that well plate-compatible LED arrays can serve as competent light sources to facilitate parallel synthesis. Lastly, we demonstrate that this procedure can serve as a blueprint toward the adaptation of other established nickel metallaphotoredox transformations to the idiosyncratic requirements of a DEL.
Assuntos
DNA/química , Níquel/química , Água/química , Catálise , Técnicas de Química Combinatória , Descarboxilação , Ligantes , Estrutura Molecular , Oxirredução , Processos Fotoquímicos , SoluçõesRESUMO
In this communication, the synthesis of three unknown polyfluorinated cyanine dyes and their application as selective markers for mitochondria are presented. By incorporating fluorous side chains into cyanine dyes, their remarkable photophysical properties were enhanced. To investigate their biological application, several different cell lines were incubated with the synthesized cyanine dyes. It was discovered that the presented dyes can be utilized for selective near-infrared-light (NIR) staining of mitochondria, with very low cytotoxicity determined by MTT assay. This is the first time that polyfluorinated cyanine fluorophores are presented as selective markers for mitochondria. Due to the versatile applications of polyfluorinated fluorophores in bioimaging and materials science, it is expected that the presented fluorophores will be stimulating for the scientific community.
Assuntos
Carbocianinas/química , Corantes Fluorescentes/química , Hidrocarbonetos Fluorados/química , Microscopia de Fluorescência/métodos , Mitocôndrias/ultraestrutura , Imagem Óptica/métodos , Células A549 , Carbocianinas/síntese química , Linhagem Celular , Corantes Fluorescentes/síntese química , Halogenação , Células HeLa , Células Hep G2 , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrocarbonetos Fluorados/síntese química , Raios Infravermelhos , Microscopia Confocal/métodosRESUMO
A new procedure for the photoredox-mediated conjugate addition of radicals that can be conveniently generated from α-amino acids to DNA-tagged Michael acceptors and styrenes is presented. This C(sp3 )-C(sp3 ) coupling tolerates a broad array of structurally diverse radical precursors, including all of the 20 proteinogenic amino acids. Importantly, this reaction proceeds under mild conditions and in DNA-compatible aqueous media. Furthermore, the presented reaction conditions are compatible with DNA, making this reaction platform well suited for the construction of DNA-encoded libraries. The scope and limitations of the chemistry are discussed herein along with proposals for how this methodology might be used to construct DNA-encoded libraries.
Assuntos
Aminoácidos/química , DNA/química , Acrilamidas/química , Alquilação , Aminas/síntese química , Aminoácidos/efeitos da radiação , Catálise , Complexos de Coordenação/química , Complexos de Coordenação/efeitos da radiação , Descarboxilação , Radicais Livres/química , Irídio/química , Luz , Oxirredução , Estudo de Prova de ConceitoRESUMO
The advent of antibody-drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.
Assuntos
Insulina/síntese química , Alquilação , Catálise , Descarboxilação , Transporte de Elétrons , Insulina/química , Luz , Modelos Moleculares , Estrutura Molecular , OxirreduçãoRESUMO
We report on the two-photon absorption spectra of a series of 2,6-disubstituted BODIPY dyes. Depending on the substituents, we observe increasing two-photon absorption cross sections with values up to 350 GM compared to 70 GM in the unsubstituted dye. Quantum chemical calculations are performed to assign the absorption bands and to understand the factors controlling the size of the two-photon absorption cross section. Both the maximum of the two-photon absorption band as well as the red-shift of the whole spectrum correlate with the ability of the substituents to extend the π-electron system of the dye. The above-mentioned intense two-photon absorption band corresponds to the absorption of photons with 1.3 eV, which is at the first near-infrared transparency window for biological tissues. The dyes could thus be suitable for bio-imaging applications.
RESUMO
The formation of oximes and hydrazones is employed in numerous scientific fields as a simple and versatile conjugation strategy. This imine-forming reaction is applied in fields as diverse as polymer chemistry, biomaterials and hydrogels, dynamic combinatorial chemistry, organic synthesis, and chemical biology. Here we outline chemical developments in this field, with special focus on the past â¼10 years of developments. Recent strategies for installing reactive carbonyl groups and α-nucleophiles into biomolecules are described. The basic chemical properties of reactants and products in this reaction are then reviewed, with an eye to understanding the reaction's mechanism and how reactant structure controls rates and equilibria in the process. Recent work that has uncovered structural features and new mechanisms for speeding the reaction, sometimes by orders of magnitude, is discussed. We describe recent studies that have identified especially fast reacting aldehyde/ketone substrates and structural effects that lead to rapid-reacting α-nucleophiles as well. Among the most effective new strategies has been the development of substituents near the reactive aldehyde group that either transfer protons at the transition state or trap the initially formed tetrahedral intermediates. In addition, the recent development of efficient nucleophilic catalysts for the reaction is outlined, improving greatly upon aniline, the classical catalyst for imine formation. A number of uses of such second- and third-generation catalysts in bioconjugation and in cellular applications are highlighted. While formation of hydrazone and oxime has been traditionally regarded as being limited by slow rates, developments in the past 5 years have resulted in completely overturning this limitation; indeed, the reaction is now one of the fastest and most versatile reactions available for conjugations of biomolecules and biomaterials.
Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/síntese química , Hidrazonas/química , Oximas/química , Catálise , Iminas/síntese química , Iminas/química , Estrutura MolecularRESUMO
We describe a photoswitchable DNA-based dimeric dye that visibly changes fluorescence from green to blue upon UV irradiation. A novel bis-alkyne-dependent [2+2+2] cycloaddition is proposed as a mechanism for the color change in air. The photoinduced structural switching results in spatial separation of stacked pyrene units, thereby causing selective loss of the excimer emission. We demonstrate and suggest several applications for this novel photoswitch.
Assuntos
Alcinos/química , Cor , Corantes Fluorescentes/química , Oxigênio/química , Pirenos/química , Reação de Cicloadição , Dimerização , Células HEK293 , Humanos , Lasers de Excimer , Microscopia de Fluorescência , Saccharomyces cerevisiae/metabolismo , Raios UltravioletaRESUMO
Here, we report a new modification of doxorubicin based on an amphiphilic stearoylspermine anchor, enabling loading into liposomal membranes. Doxorubicin is coupled with stearoylspermine through an acid-labile hydrazone linker to ensure the release of the drug in the acidic interstitium of tumors. Using ATR-FTIR spectroscopy (Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy), the mechanism of interaction of doxorubicin with the anionic liposomal membrane was studied: incorporation of stearoyl chains leads to an increase in local microfluidity, and the amino groups of spermine interact with the phosphate groups of lipids. To stabilize liposomes against aggregation, we applied the copolymer PEG-chitosan as a coating: complex formation leads to charge neutralization, and the liposomes grow in size. According to MTT tests and confocal microscopy for cell lines A459 and Caco-2, PEG-chitosan-coated liposomes are as effective as neutral liposomes but are much more stable.
RESUMO
A facile and general procedure for the preparation of alkynyl C-nucleosides with varied fluorophores is presented. Sonogashira coupling was used as a key reaction to conjugate the dyes to an easily accessible ethynyl functionalized deoxyribose derivative. The new C-nucleosides were used for the preparation of DNA-based polyfluorophores.
Assuntos
Alcinos/química , DNA/química , Corantes Fluorescentes/química , Nucleosídeos/química , Nucleosídeos/síntese química , Técnicas de Química SintéticaRESUMO
A new family of pentadecanuclear coordination cluster compounds (from now on simply referred to as clusters) [{Ln15 (OH)20 (PepCO2 )10 (DBM)10 Cl}Cl4 ] (PepCO2 =2-[{3-(((tert-butoxycarbonyl)amino)methyl)benzyl}amino]acetate, DBM=dibenzoylmethanide) with Ln=Y and Dy was obtained by using the cell-penetrating peptoid (CPPo) monomer PepCO2 H and dibenzoylmethane (DBMH) as supporting ligands. The combination of an inorganic cluster core with an organic cell-penetrating peptoid in the coordination sphere resulted in a core component {Ln15 (µ3 -OH)20 Cl}(24+) (Ln=Y, Dy), which consists of five vertex-sharing heterocubane {Ln4 (µ3 -OH)4 }(8+) units that assemble to give a pentagonal cyclic structure with one Cl atom located in the middle of the pentagon. The solid-state structures of both clusters were established by single-crystal X-ray crystallography. MS (ESI) experiments suggest that the cluster core is robust and maintained in solution. Pulsed gradient spin echo (PGSE) NMR diffusion measurements were carried out on the diamagnetic yttrium compound and confirmed the stability of the cluster in its dicationic form [{Y15 (µ3 -OH)20 (PepCO2 )10 (DBM)10 Cl}Cl2 ](2+) . The investigation of both static (dc) and dynamic (ac) magnetic properties in the dysprosium cluster revealed a slow relaxation of magnetization, indicative of single-molecule magnet (SMM) behavior below 8â K. Furthermore, the χT product as a function of temperature for the dysprosium cluster gave evidence that this is a ferromagnetically coupled compound below 11â K.
RESUMO
During the last decade peptoid-based molecular transporters have been broadly applied. They are highly valued for their easy synthesis and their superior stability against enzymatic degradation. The special structure of peptoids generally allows introducing a variety of different side chains. Yet, the cationic side chains of cell-penetrating peptoids displayed solely lysine- or arginine-like structures. Thus this report is intended to extend the spectrum of cationic peptoid side chains. Herein, we present novel functional groups, like polyamines, aza-crown ethers, or triphenylphosphonium ions that are introduced into peptoids for the first time. In addition, the obtained peptoids were tested for their cell-penetrating properties.
Assuntos
Compostos Aza/química , Peptídeos Penetradores de Células/química , Éteres de Coroa/química , Compostos Organofosforados/química , Poliaminas/química , Cátions/química , Peptídeos Penetradores de Células/síntese química , Cristalografia por Raios X , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Células Tumorais CultivadasRESUMO
A novel pentadecanuclear lanthanide hydroxy cluster [{Ln15(µ3-OH)20(PepCO2)10(DBM)10Cl}Cl4] (Ln = Eu (1), Tb (2)) featuring the first example with peptoids as supporting ligands was prepared and fully characterized. The solid-state structures of 1 and 2 were established via single-crystal X-ray crystallography. ESI-MS experiments revealed the retention of the cluster core in solution. Although OH groups are present, 1 showed intense red fluorescence with 11(1)% absolute quantum yield, whereas the emission intensity and the quantum yield of 2 were significantly weaker. In vitro investigations on 1 and 2 with HeLa tumor cells revealed an accumulation of the clusters in the endosomal-lyosomal system, as confirmed by confocal microscopy in the TRLLM mode. The cytotoxicity of 1 and 2 toward the HeLa cells is moderate.
Assuntos
Elementos da Série dos Lantanídeos/química , Luminescência , Compostos Organometálicos/química , Cristalografia por Raios X , Células HeLa , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Incorporation of fluorous ponytails such as polyfluorinated alkyl residues (CH2)m(CF2)nCF3 leads to a novel class of bright rhodamine-based fluorescence dyes. These dyes combine the excellent photophysical properties of the frequently used rhodamine dyes with the unique features of "light" fluorous molecules. One of those features is the possibility to separate substances utilizing fluorous solid-phase extraction (F-SPE), which is based on the specific intermolecular interaction between fluorous compounds. Thus, molecules, which are labeled with these new dyes, are not only accessible to fluorescence experiments, but can also be easily purified (via so-called FluoroFlash columns) prior to use. The dyes were bound to a cell penetrating peptoid (polycationic oligo(N-substituted) glycine) on solid supports. These conjugates were purified with F-SPE before their photophysical and biological properties were investigated.
Assuntos
Corantes Fluorescentes/química , Rodaminas/química , Corantes Fluorescentes/síntese química , Estrutura Molecular , Rodaminas/síntese química , TermodinâmicaRESUMO
Cell penetrating peptoids (CPPos) are potent mimics of the corresponding cell penetrating peptides (CPPs). The synthesis of diverse oligomeric libraries that display a variety of backbone scaffolds and side-chain appendages are a very promising source of novel CPPos, which can be used to either target different cellular organelles or even different tissues and organs. In this study we established the submonomer-based solid phase synthesis of a "proof of principle" peptoid library in IRORI MiniKans to expand the amount for phenotypic high throughput screens of CPPos. The library consisting of tetrameric peptoids [oligo(N-alkylglycines)] was established on Rink amide resin in a split and mix approach with hydrophilic and hydrophobic peptoid side chains. All CPPos of the presented library were labeled with rhodamine B to allow for the monitoring of cellular uptake by fluorescent confocal microscopy. Eventually, all the purified peptoids were subjected to live cell imaging to screen for CPPos with organelle specificity. While highly charged CPPos enter the cells by endocytosis with subsequent endosomal release, critical levels of lipophilicity allow other CPPos to specifically localize to mitochondria once a certain lipophilicity threshold is reached.