Fluorescence imaging techniques have been widely used to visualize biological molecules and phenomena. In particular, several studies on the development of small-molecule fluorescent probes have been carried out, because their fluorescence properties can be easily tuned by synthetic chemical modification. For this reason, various fluorescent probes have been developed for targeting biological components, such as proteins, peptides, amino acids, and ions, to the interior and exterior of cells. In this review, we cover advances in the development of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-based fluorescent probes for biological studies over the past decade.

BODIPY-based probes for the fluorescence imaging of biomolecules in living cells

The activation strain or distortion/interaction model is a tool to analyze activation barriers that determine reaction rates. For bimolecular reactions, the activation energies are the sum of the energies to distort the reactants into geometries they have in transition states plus the interaction energies between the two distorted molecules. The energy required to distort the molecules is called the activation strain or distortion energy. This energy is the principal contributor to the activation barrier. The transition state occurs when this activation strain is overcome by the stabilizing interaction energy. Following the changes in these energies along the reaction coordinate gives insights into the factors controlling reactivity. This model has been applied to reactions of all types in both organic and inorganic chemistry, including substitutions and eliminations, cycloadditions, and several types of organometallic reactions.

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Analyzing Reaction Rates with the Distortion/Interaction‐Activation Strain Model

O.B. thanks the European Commission (Marie Curie CIG) and Ministerio de Ciencia e Innovacion, Spain (Juan de la Cierva Fellowship). G.J.L.B. thanks his generous sources of funding: Royal Society, FCT Portugal (FCT Investigator), European Commission (Marie Curie CIG), and the EPSRC. G.J.L.B. is a Royal Society University Research Fellow. The authors thank Paula Boutureira Regla and Francisco Pinteus da Cruz Lopes Bernardes for inspiration.

Advances in Chemical Protein Modification

Macrocycles occupy a unique segment of chemical space. In the past decade, their chemical diversity expanded significantly, supported by advances in bioinformatics and synthetic methodology. As a consequence, this structural type has now been successfully tested on most biological target classes. The goal of this article is to put into perspective the current applications, opportunities, and challenges associated with synthetic macrocycles in drug discovery. Historically, macrocyclic drug candidates have originated primarily from two sources. The first, natural products, provided unique drugs such as erythromycin, rapamycin, vancomycin, cyclosporin, and epothilone. Excellent reviews are dedicated to this class and how it inspired further synthetic and medicinal chemistry efforts; thus, it will not be covered here. From a molecular evolution standpoint, the medicinal chemistry of macrocyclic natural products usually involved direct use as a therapeutic agent or functionalization of the natural product scaffold by hemisynthesis. It parallels significant advances in the total synthesis of macrocyclic natural products during the past 2 decades. The second traditional source of macrocycles stems from peptides, some of which are natural products and, hence, also belong to the first category. Macrocyclization was recognized early in peptide chemistry as an efficient way to restrict peptide conformation, reduce polarity, increase proteolytic stability, and consequently improve druggability. Chemists accessed macrocyclic peptides with different geometries (head to tail, side chain to side chain, head to side chain), including the incorporation of nonpeptidic groups. Compelling examples of macrocyclic scaffolding of peptides include the works on somatostatins, melanocortins, and integrins, among others. Macrocyclic peptides generated several drugs from synthetic or natural sources, including octreotide, cyclosporine, eptifibatide, and caspofungin. Purely peptidic, depsipeptidic, and peptoid macrocycles will also not be covered in this article; the reader is instead referred to previous reviews. It is well understood that the boundary between synthetic macrocycles and the above categories is not always clear-cut; as a result, examples presented in the following sections could occasionally belong to one of these categories. In these cases, they were selected owing to their relevance to the perspective. Macrocycles are defined herein as molecules containing at least one large ring composed of 12 or more atoms. On the basis of standard molecular descriptors, macrocycles as a class are at the outskirts of the window generally considered optimal for good PK-ADME properties using these criteria. Indeed, their molecular weights tend to be on the higher end (often in the 500-900 g 3mol -1 range), their numbers of H-bond donors and acceptors, as well as their polar surface area (PSA), tend to be on the far side of the accepted druglike spectrum. For an equal number of heavy atoms, macrocycles inherently possess a lower number of rotatable bonds than their acyclic analogues, a beneficial feature for oral bioavailability (in the following, “acyclic” will be used in the sense of “nonmacrocyclic”). As a result, macrocycles are more conformationally restricted than their acyclic analogues, which potentially can impart higher target binding and selectivity and improved oral bioavailability (in this assessment, endocyclic bonds are considered to be nonrotatable, which is only an approximation; see ref 18). For a systematic chemoinformatic analysis of biologically active macrocycles, the reader is referred to the recent review of Brandt et al. Topologically, macrocycles have the unique ability to span large surface areas while remaining conformationally restricted compared to acyclic molecules of equivalent molecular weight. This characteristic makes them especially suited for targets displaying shallow surfaces, which can prove to be quite challenging for acyclic small molecules. Medicinal chemistry relies strategically on robust synthetic methods capable of producing an acceptable chemical diversity to adequately interrogate the chemical space of a biological target. Macrocycles are often (and rightly so) perceived as difficult to synthesize and hence deterred many medicinal chemists because of the lack of versatile synthetic platforms. The macrocyclization step is regularly plagued by low yields and often requires high dilution conditions to counterbalance entropic loss. In other words, the reduction in entropy responsible for beneficial conformational restrictions to the final molecule comes at a price during synthesis: what goes around comes around. Accordingly, the first part of this article is dedicated to the drug discovery aspects ofmacrocycles and highlights salient features of their medicinal chemistry. This section is organized by target class, a choice aimed at providing the reader an appreciation of the structural diversity generated for each class. To give the reader an appreciation of the tools available to construct macrocyclic scaffolds, the site and method of the pivotal macrocyclization step are indicated in the figures. Readers are referred to the source articles for further details. In the second part, the technologies and synthetic approaches that already have demonstrated utility or possess a high potential for macrocycle-based

Macrocycles Are Great Cycles: Applications, Opportunities, and Challenges of Synthetic Macrocycles in Drug Discovery

The emerging inverse electron demand Diels–Alder (IEDDA) reaction stands out from other bioorthogonal reactions by virtue of its unmatchable kinetics, excellent orthogonality and biocompatibility. With the recent discovery of novel dienophiles and optimal tetrazine coupling partners, attention has now been turned to the use of IEDDA approaches in basic biology, imaging and therapeutics. Here we review this bioorthogonal reaction and its promising applications for live cell and animal studies. We first discuss the key factors that contribute to the fast IEDDA kinetics and describe the most recent advances in the synthesis of tetrazine and dienophile coupling partners. Both coupling partners have been incorporated into proteins for tracking and imaging by use of fluorogenic tetrazines that become strongly fluorescent upon reaction. Selected notable examples of such applications are presented. The exceptional fast kinetics of this catalyst-free reaction, even using low concentrations of coupling partners, make it amenable for in vivo radiolabelling using pretargeting methodologies, which are also discussed. Finally, IEDDA reactions have recently found use in bioorthogonal decaging to activate proteins or drugs in gain-of-function strategies. We conclude by showing applications of the IEDDA reaction in the construction of biomaterials that are used for drug delivery and multimodal imaging, among others. The use and utility of the IEDDA reaction is interdisciplinary and promises to revolutionize chemical biology, radiochemistry and materials science.

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Inverse electron demand Diels–Alder reactions in chemical biology

Bioorthogonal chemistries can be used to tag diverse classes of biomolecules in cells and other complex environments. With over 20 unique transformations now available, though, selecting an appropriate reaction for a given experiment is challenging. In this article, we compare and contrast the most common classes of bioorthogonal chemistries and provide a framework for matching the reactions with downstream applications. We also discuss ongoing efforts to identify novel biocompatible reactions and methods to control their reactivity. The continued expansion of the bioorthogonal toolkit will provide new insights into biomolecule networks and functions and thus refine our understanding of living systems.

Finding the Right (Bioorthogonal) Chemistry

Chemical reporters are unique functional groups that can be used to label biomolecules in living systems. Only a handful of broadly applicable reporters have been identified to date, owing to the rigorous demands placed on these functional groups in biological settings. We describe here a new chemical reporter-cyclopropene-that can be used to target biomolecules in vitro and in live cells. A variety of substituted cyclopropene scaffolds were synthesized and found to be stable in aqueous solution and in the presence of biological nucleophiles. Furthermore, some of the cyclopropene units were metabolically introduced into cell surface glycans and subsequently detected with covalent probes. The small size and selective reactivity of cyclopropenes will facilitate efforts to tag diverse collections of biomolecules in vivo.

Functionalized Cyclopropenes As Bioorthogonal Chemical Reporters

Bioorthogonal chemistries have provided tremendous insight into biomolecule structure and function. However, many popular bioorthogonal transformations are incompatible with one another, limiting their utility for studies of multiple biomolecules in tandem. We identified two reactions that can be used concurrently to tag biomolecules in complex environments: the inverse electron-demand Diels-Alder reaction of tetrazines with 1,3-disubstituted cyclopropenes, and the 1,3-dipolar cycloaddition of nitrile imines with 3,3-disubstituted cyclopropenes. Remarkably, the cyclopropenes used in these transformations differ by the placement of a single methyl group. Such orthogonally reactive scaffolds will bolster efforts to monitor multicomponent processes in cells and organisms.

Isomeric Cyclopropenes Exhibit Unique Bioorthogonal Reactivities

Synthesis of sansalvamide A peptidomimetics: triazole, oxazole, thiazole, and pseudoproline containing compounds.

Heat shock proteins (HSP) are a family of highly conserved proteins, whose expression increases in response to stresses that may threaten cell survival. Over the past decade, heat shock protein 90 (Hsp90) has emerged as a potential therapeutic target for cancer as it plays a vital role in normal cell maturation and acts as a molecular chaperone for proper folding, assembly, and stabilization of many oncogenic proteins. To date, a majority of Hsp90 inhibitors that have been discovered are macrocycles. The relatively rigid conformation provided by the macrocyclic scaffold allows for a selective interaction with a biological target such as Hsp90. This review highlights the discovery and development of nine macrocycles that inhibit the function of Hsp90, detailing their potency and the client proteins affected by Hsp90 inhibition.

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Lidia A. Nazarova

Papers

Finding the Right (Bioorthogonal) Chemistry

Functionalized Cyclopropenes As Bioorthogonal Chemical Reporters

Isomeric Cyclopropenes Exhibit Unique Bioorthogonal Reactivities

Synthesis of sansalvamide A peptidomimetics: triazole, oxazole, thiazole, and pseudoproline containing compounds.

Macrocyclic Inhibitors of Hsp90