Applications In Chemical Biology Research Articles

Modular click chemistry libraries for functional screens using a diazotizing reagent.

Click chemistry is a concept in which modular synthesis is used to rapidly find new molecules with desirable properties1. Copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) triazole annulation and sulfur(VI) fluoride exchange (SuFEx) catalysis are widely regarded as click reactions2-4, providing rapid access to their products in yields approaching 100% while being largely orthogonal to other reactions. However, in the case of CuAAC reactions, the availability of azide reagents is limited owing to their potential toxicity and the risk of explosion involved in their preparation. Here we report another reaction to add to the click reaction family: the formation of azides from primary amines, one of the most abundant functional groups5. The reaction uses just one equivalent of a simple diazotizing species, fluorosulfuryl azide6-11 (FSO2N3), and enables the preparation of over 1,200 azides on 96-well plates in a safe and practical manner. This reliable transformation is a powerful tool for the CuAAC triazole annulation, the most widely used click reaction at present. This method greatly expands the number of accessible azides and 1,2,3-triazoles and, given the ubiquity of the CuAAC reaction, it should find application in organic synthesis, medicinal chemistry, chemical biology and materials science.

Applications of guanine quartets in nanotechnology and chemical biology

Guanine and related nucleobases such as guanosine, deoxyguanosine and isoguanosine are notable molecular tools for designing functional supramolecular assemblies. This popularity originates in their ability to self-assemble via a unique topological pluralism — as isolated nucleobases, discrete macrocyclic quartets and virtually infinite linear ribbons — that endows them with a considerable functional versatility. Many programmes have been launched to fine-tune the chemical properties of guanine derivatives, to make them usable under different experimental conditions, such as in organic or aqueous environments, and responsive to external stimuli, such as ionic strength, pH, light or temperature. These strategies aim to translate the chemical information encoded in a basic guanine unit into programmable, higher-order supramolecular architectures. Spectacular results have been recently obtained in various chemical fields, from supramolecular chemistry to chemical biology, from soft matter to catalysis. In this Review, we detail these advances and demonstrate how these multidisciplinary investigations cast a bright light on the diversity that guanines, synthetic guanines and related nucleobases uniquely offer in terms of both structure and function. The propensity of guanine and its derivatives to assemble into guanine quartets makes them a privileged tool in the design of functional supramolecular assemblies. This Review describes the production of such functional suprastructures and their applications in nanotechnology, soft matter and chemical biology.

"Close-to-Release": Spontaneous Bioorthogonal Uncaging Resulting from Ring-Closing Metathesis.

Bioorthogonal uncaging reactions offer versatile tools in chemical biology. In recent years, reactions have been developed to proceed efficiently under physiological conditions. We present herein an uncaging reaction that results from ring-closing metathesis (RCM). A caged molecule, tethered to a diolefinic substrate, is released via spontaneous 1,4-elimination following RCM. Using this strategy, which we term “close-to-release”, we show that drugs and fluorescent probes are uncaged with fast rates, including in the presence of mammalian cells or in the periplasm of Escherichia coli. We envision that this tool may find applications in chemical biology, bioengineering and medicine.

A Physical Organic Approach to Tuning Reagents for Selective and Stable Methionine Bioconjugation.

We report a data-driven, physical organic approach to the development of new methionine-selective bioconjugation reagents with tunable adduct stabilities. Statistical modeling of structural features described by intrinsic physical organic parameters was applied to the development of a predictive model and to gain insight into features driving the stability of adducts formed from the chemoselective coupling of oxaziridine and methionine thioether partners through Redox Activated Chemical Tagging (ReACT). From these analyses, a correlation between sulfimide stabilities and sulfimide ν (C═O) stretching frequencies was revealed. We exploited the rational gains in adduct stability exposed by this analysis to achieve the design and synthesis of a bis-oxaziridine reagent for peptide stapling. Indeed, we observed that a macrocyclic peptide formed by ReACT stapling at methionine exhibited improved uptake into live cells compared to an unstapled congener, highlighting the potential utility of this unique chemical tool for thioether modification. This work provides a template for the broader use of data-driven approaches to bioconjugation chemistry and other chemical biology applications.

Data structures for compound promiscuity analysis: promiscuity cliffs, pathways and promiscuity hubs formed by inhibitors of the human kinome.

Aim:A large collection of promiscuity cliffs (PCs), PC pathways (PCPs) and promiscuity hubs (PHs) formed by inhibitors of human kinases is made freely available.Methodology:Inhibitor PCs were systematically identified and organized in network representations, from which PCPs were extracted. PH compounds were classified and their neighborhoods analyzed.Data & exemplary results:Nearly 16,000 PCs covering the human kinome were identified, which yielded more than 600 PC clusters and 8900 PCPs. Moreover, 520 PHs were obtained.Limitations & next steps:PC and PCP data structures capture structure–promiscuity relationships. Promiscuity assessment is also affected by data sparseness. Given the rapid growth of kinase inhibitor data, the relevance of PC/PCP/PH information for medicinal chemistry and chemical biology applications will further increase.

Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ

NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.

Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology.

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Site-Resolved and Quantitative Characterization of Very Weak Protein-Ligand Interactions.

Very weak interactions between small organic molecules and proteins have long been predicted and are expected to have dissociation constants of hundreds of millimolar and above. Unfortunately, quantitative evaluation of binding in a high-resolution structural context for this affinity regime is particularly difficult and often impossible using existing experimental approaches. Here, we show that nanoscale encapsulation of single protein molecules within the water core of reverse micelles enables the detection and evaluation of weak binding interactions at atomic resolution using solution NMR spectroscopy. This strategy is used to survey the interactions of a set of small molecules with the cytokine interleukin-1β (IL-1β). The interaction of IL-1β with these molecules is found to vary from more diffuse and weak binding modes to more specific and with a relatively higher affinity. The interactions detected here cover a large portion of the protein surface and have dissociation constants mostly in the low molar range. These results illustrate the ability of a protein to interact productively with a variety of small molecule functional groups and point to a broader potential to target even relatively featureless protein surfaces for applications in chemical biology and drug discovery.

Cover Feature: New Size‐Expanded Fluorescent Thymine Analogue: Synthesis, Characterization, and Application (Chem. Eur. J. 42/2019)

A new size-expanded fluorescent T-mimic deoxyribonucleoside is reported, based on a dioxoloquinazoline core, named as dioxT. dioxT showed comparable biophysical properties and displayed notable photophysical characteristics including an excellent quantum yield (0.36) in water as a fluorescent thymine surrogate. Interestingly, when the dioxT-containing strand was hybridized with a complementary strand, the duplex exhibited higher fluorescence than a single strand. dioxT can be used for a broad range of applications in chemical biology. More information can be found in the Full Paper by S. Park, H. Sugiyama, et al. on page 9913.

New Size-Expanded Fluorescent Thymine Analogue: Synthesis, Characterization, and Application.

Here, the synthesis, photophysical characterization, and application of a new size-expanded thymine nucleoside, diox T, is described. diox T has desirable qualities as a T surrogate, including excellent quantum yield (0.36) and high environmental sensitivity. When incorporated into single- and double-stranded DNA, diox T showed excellent photophysical characteristics including a high quantum yield (average 0.20), and unlike BgQ, demonstrated dependence on neighboring bases without significant destabilization of the duplex. Interestingly, the matched base pair of adenine (A) and diox T has the unique property that it exhibits higher fluorescence than mismatched base pairs, and diox T has self-quenching effects. As one example of the possible applications of these promising features, single nucleoside polymorphism typing is demonstrated for discrimination of A by using diox T. The results suggest that diox T can be used for a broad range of applications in chemical biology.

Steroid diversification by multicomponent reactions.

Reports on structural diversification of steroids by means of multicomponent reactions (MCRs) have significantly increased over the last decade. This review covers the most relevant strategies dealing with the use of steroidal substrates in MCRs, including the synthesis of steroidal heterocycles and macrocycles as well as the conjugation of steroids to amino acids, peptides and carbohydrates. We demonstrate that steroids are available with almost all types of MCR reactive functionalities, e.g., carbonyl, carboxylic acid, alkyne, amine, isocyanide, boronic acid, etc., and that steroids are suitable starting materials for relevant MCRs such as those based on imine and isocyanide. The focus is mainly posed on proving the amenability of MCRs for the diversity-oriented derivatization of naturally occurring steroids and the construction of complex steroid-based platforms for drug discovery, chemical biology and supramolecular chemistry applications.

Synthesis, characterization and biological applications of selenoureas having ferrocene and substituted benzoyl functionalities

In this article we have comprehensively reported the synthesis, complete chemical characterization (FTIR, multinuclear NMR, single crystal XRD, CHNS, AAS) and biological applications of seventeen ferrocene incorporated selenoureas. Biological applications include comparative DNA binding studies (with, cyclic voltammetry), antioxidant activities (DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging), anti-inflammatory activities (nitrite inhibition), phase I enzyme inhibition activities (aromatase inhibition), phase II enzyme induction studies (quinone reductase induction) and cytotoxic studies against neuroblastoma (MYCN2 and SK-N-SH), liver cancer (Hepa 1c1c7) breast cancer (MCF-7) and human cervical carcinoma HeLa cells (ATCC® CCL-2). All the compounds showed decent structure dependent biological applications.

The unexplored potential of quinone methides in chemical biology.

Quinone methides (QMs) are transient reactive species that can be efficiently generated from stable precursors under a variety of biocompatible conditions. Due to their electrophilic nature, QMs have been widely explored as cross-linking agents of DNA and proteins under physiological conditions. However, QMs also have a diene character and can irreversibly react via Diels-Alder reaction with electron-rich dienophiles. This particular reactivity has been recently exploited to label biomolecules with fluorophores in living cells. QMs are characterised by two unique properties that make them ideal candidates for chemical biology applications: i) they can be efficiently generated in situ from very stable precursors by means of bio-orthogonal protocols ii) they are reversible cross-linking agents, making them suitable for "catch and release" target-enrichment experiments. Nevertheless, there are only few examples reported to date that truly take advantage of QMs unique chemistry in the context of chemical-biology assay development. In this review, we will examine the most relevant examples that illustrate the benefit of using QMs for chemical biology purposes and we will anticipate novel approaches to further their applications in biologically relevant contexts.

Peptides for Infectious Diseases: From Probe Design to Diagnostic Microarrays.

Peptides and peptidomimetics have attracted revived interest regarding their applications in chemical biology over the last few years. Their chemical versatility, synthetic accessibility and the ease of storage and management compared to full proteins have made peptides particularly interesting in diagnostic applications, where they proved to efficiently recapitulate the molecular recognition properties of larger protein antigens, and were proven to be able to capture antibodies circulating in the plasma and serum of patients previously exposed to bacterial or viral infections. Here, we describe the development, integration and application of strategies for computational prediction and design, advanced chemical synthesis, and diagnostic deployment in multiplexed assays of peptide-based materials which are able to bind antibodies of diagnostic as well as therapeutic interest. By presenting successful applications of such an integrated strategy, we argue that they will have an ever-increasing role in both basic and clinical realms of research, where important advances can be expected in the next few years.

Aqueous olefin metathesis: recent developments and applications.

Olefin metathesis is one of the most powerful C–C double-bond-forming reactions. Metathesis reactions have had a tremendous impact in organic synthesis, enabling a variety of applications in polymer chemistry, drug discovery and chemical biology. Although challenging, the possibility to perform aqueous metatheses has become an attractive alternative, not only because water is a more sustainable medium, but also to exploit biocompatible conditions. This review focuses on the progress made in aqueous olefin metatheses and their applications in chemical biology.

Structure-guided design of a high-affinity ligand for a riboswitch.

We have designed structure-based ligands for the guanidine-II riboswitch that bind with enhanced affinity, exploiting the twin binding sites created by loop–loop interaction. We synthesized diguanidine species, comprising two guanidino groups covalently connected by Cn linkers where n = 4 or 5. Calorimetric and fluorescent analysis shows that these ligands bind with a 10-fold higher affinity to the riboswitch compared to guanidine. We determined X-ray crystal structures of the riboswitch bound to the new ligands, showing that the guanidino groups are bound to both nucleobases and backbone within the binding pockets, analogously to guanidine binding. The connecting chain passes through side openings in the binding pocket and traverses the minor groove of the RNA. The combination of the riboswitch loop–loop interaction and our novel ligands has potential applications in chemical biology.

A Multicomponent Stapling Approach to Exocyclic Functionalized Helical Peptides: Adding Lipids, Sugars, PEGs, Labels, and Handles to the Lactam Bridge.

Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversity-generating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels, and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology.

Strategy and Effects of Polyproline Peptide Stapling by Copper(I)-Catalyzed Alkyne-Azide Cycloaddition Reaction.

Polyproline is a unique type of peptide that has a stable, robust, and well-defined helical structure in an aqueous environment. These features have allowed polyproline to be used as a nanosized scaffold for applications in chemical biology and related fields. To understand its structural properties and to expand the applications, this secondary structure was tested systematically by stapling the peptide at different locations with staples of various lengths. Using the efficient copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), we successfully prepared stapled polyproline and investigated the impact of this peptide macrocyclization through circular dichroism analysis. Whereas the stapling seems to have no significant effect on polyproline helix II (PPII) conformation in water, the location and the length of the staple affect the transformation of conformation in n-propanol. These results provide valuable information for future research using peptide stapling to manipulate polyproline conformation for various applications.

Emerging and Re-Emerging Warheads for Targeted Covalent Inhibitors: Applications in Medicinal Chemistry and Chemical Biology.

Targeted covalent inhibitors (TCIs) are designed to bind poorly conserved amino acids by means of reactive groups, the so-called warheads. Currently, targeting noncatalytic cysteine residues with acrylamides and other α,β-unsaturated carbonyl compounds is the predominant strategy in TCI development. The recent ascent of covalent drugs has stimulated considerable efforts to characterize alternative warheads for the covalent-reversible and irreversible engagement of noncatalytic cysteine residues as well as other amino acids. This Perspective article provides an overview of warheads-beyond α,β-unsaturated amides-recently used in the design of targeted covalent ligands. Promising reactive groups that have not yet demonstrated their utility in TCI development are also highlighted. Special emphasis is placed on the discussion of reactivity and of case studies illustrating applications in medicinal chemistry and chemical biology.

Fast Magic-Angle Spinning 19 F NMR Spectroscopy of HIV-1 Capsid Protein Assemblies.

19 F NMR spectroscopy is an attractive and growing area of research with broad applications in biochemistry, chemical biology, medicinal chemistry, and materials science. We have explored fast magic angle spinning (MAS) 19 F solid-state NMR spectroscopy in assemblies of HIV-1 capsid protein. Tryptophan residues with fluorine substitution at the 5-position of the indole ring were used as the reporters. The 19 F chemical shifts for the five tryptophan residues are distinct, reflecting differences in their local environment. Spin-diffusion and radio-frequency-driven-recoupling experiments were performed at MAS frequencies of 35 kHz and 40-60 kHz, respectively. Fast MAS frequencies of 40-60 kHz are essential for consistently establishing 19 F-19 F correlations, yielding interatomic distances of the order of 20 Å. Our results demonstrate the potential of fast MAS 19 F NMR spectroscopy for structural analysis in large biological assemblies.