A designed photoenzyme for enantioselective [2+2] cycloadditions.

The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains1-4. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present5-12. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref. 13) converts a de novo Diels-Alderase into a photoenzyme for [2+2] cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.

Recent Advances in Macrocyclic Drugs and Microwave-Assisted and/or Solid-Supported Synthesis of Macrocycles.

Macrocycles represent attractive candidates in organic synthesis and drug discovery. Since 2014, nineteen macrocyclic drugs, including three radiopharmaceuticals, have been approved by FDA for the treatment of bacterial and viral infections, cancer, obesity, immunosuppression, etc. As such, new synthetic methodologies and high throughput chemistry (e.g., microwave-assisted and/or solid-phase synthesis) to access various macrocycle entities have attracted great interest in this chemical space. This article serves as an update on our previous review related to macrocyclic drugs and new synthetic strategies toward macrocycles (Molecules, 2013, 18, 6230). In this work, I first reviewed recent FDA-approved macrocyclic drugs since 2014, followed by new advances in macrocycle synthesis using high throughput chemistry, including microwave-assisted and/or solid-supported macrocyclization strategies. Examples and highlights of macrocyclization include macrolactonization and macrolactamization, transition-metal catalyzed olefin ring-closure metathesis, intramolecular C-C and C-heteroatom cross-coupling, copper- or ruthenium-catalyzed azide-alkyne cycloaddition, intramolecular SNAr or SN2 nucleophilic substitution, condensation reaction, and multi-component reaction-mediated macrocyclization, and covering the literature since 2010.

Recent Advances in Copper-Based Solid Heterogeneous Catalysts for Azide-Alkyne Cycloaddition Reactions.

The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is considered to be the most representative ligation process within the context of the "click chemistry" concept. This CuAAC reaction, which yields compounds containing a 1,2,3-triazole core, has become relevant in the construction of biologically complex systems, bioconjugation strategies, and supramolecular and material sciences. Although many CuAAC reactions are performed under homogenous conditions, heterogenous copper-based catalytic systems are gaining exponential interest, relying on the easy removal, recovery, and reusability of catalytically copper species. The present review covers the most recently developed copper-containing heterogenous solid catalytic systems that use solid inorganic/organic hybrid supports, and which have been used in promoting CuAAC reactions. Due to the demand for 1,2,3-triazoles as non-classical bioisosteres and as framework-based drugs, the CuAAC reaction promoted by solid heterogenous catalysts has greatly improved the recovery and removal of copper species, usually by simple filtration. In so doing, the solving of the toxicity issue regarding copper particles in compounds of biological interest has been achieved. This protocol is also expected to produce a practical chemical process for accessing such compounds on an industrial scale.

Preparation and characterization of starch-cellulose interpenetrating network hydrogels based on sequential Diels-Alder click reaction and photopolymerization.

Herein, starch-cellulose interpenetrating network (IPN) hydrogels were fabricated by sequential Diels-Alder click reaction and photopolymerization in water. Moreover, ß-cyclodextrin, a commonly used host molecule in supramolecular chemistry, was also introduced to improve the performance of the IPN hydrogel. Firstly, the starch-based dienes were synthesized by modifying starch with N-maleoyl-ß-alanine, and the cellulose-based dienophiles were obtained by the reaction of cellulose and furfurylamide succinate; Secondly, the as-synthesized starch-based dienes, cellulose-based dienophiles, polymerizable ß-cyclodextrin, crosslinker, and acrylamide were dissolved in water and obtained a transparent solution. The solution was maintained in a water bath of 50 °C for 3 h, forming the first network via catalyst-free click Diels-Alder reaction, subsequently, the second network was formed by photopolymerization. Their preparation conditions were optimized via one-factor experiments and their properties and structures were characterized. Finally, 5- fluorouracil (5-Fu) was used as a model drug to study the sustained release behavior of the drug-loaded hydrogels. Release profile was found to fit in Ritger-Peppas kinetic model and polymer relaxation and drug diffusion made a valuable contribution to drug release. Taking into account the virtues of easily controllable photopolymerization and catalyst-free Diels-Alder reaction, the strategy described here has a potential application in the preparation of IPN hydrogels.

Chiral Brønsted acid-controlled intermolecular asymmetric [2 + 2] photocycloadditions.

Control over the stereochemistry of excited-state photoreactions remains a significant challenge in organic synthesis. Recently, it has become recognized that the photophysical properties of simple organic substrates can be altered upon coordination to Lewis acid catalysts, and that these changes can be exploited in the design of highly enantioselective catalytic photoreactions. Chromophore activation strategies, wherein simple organic substrates are activated towards photoexcitation upon binding to a Lewis acid catalyst, rank among the most successful asymmetric photoreactions. Herein, we show that chiral Brønsted acids can also catalyze asymmetric excited-state photoreactions by chromophore activation. This principle is demonstrated in the context of a highly enantio- and diastereoselective [2+2] photocycloaddition catalyzed by a chiral phosphoramide organocatalyst. Notably, the cyclobutane products arising from this method feature a trans-cis stereochemistry that is complementary to other enantioselective catalytic [2+2] photocycloadditions reported to date.

Direct access to spirocycles by Pd/WingPhos-catalyzed enantioselective cycloaddition of 1,3-enynes.

Spirocycles play an important role in drug discovery and development. The direct, catalytic, and enantioselective synthesis of spirocycles from readily available starting materials and in an atom economic manner remains a highly sought-after task in organic synthesis. Herein, an enantioselective Pd-hydride-catalyzed cycloaddition method for the synthesis of spirocyclic compounds directly from two classes of commonly available starting materials, 1,3-enynes and cyclic carbon-hydrogen (C-H) bonds, is reported. The reactions employ a chiral Pd/WingPhos catalyst to both suppress the formation of bis-allenyl by-products and control the stereoselectivity. 1,3-Enynes are used as dielectrophilic four-carbon units in the cycloaddition reactions, which also enables an enyne substrate-directed enantioselectivity switch with good levels of stereocontrol. The present spirocycle synthesis tolerates a broad range of functional groups of 1,3-enyne substrates, including alcohols, esters, nitriles, halides, and olefins. A variety of diverse cyclic nucleophiles, including pharmaceutically important heterocycles and carbocycles, can be flexibly incorporated with spiro scaffolds.

1- and 2-Azetines via Visible Light-Mediated [2 + 2]-Cycloadditions of Alkynes and Oximes.

Azetines, four-membered unsaturated nitrogen-containing heterocycles, hold great potential for drug design and development but remain underexplored due to challenges associated with their synthesis. We report an efficient, visible light-mediated approach toward 1- and 2-azetines relying on alkynes and the unique triplet state reactivity of oximes, specifically 2-isoxazolines. While 2-azetine products are accessible upon intermolecular [2 + 2]-cycloaddition via triplet energy transfer from a commercially available iridium photocatalyst, the selective formation of 1-azetines proceeds upon a second, consecutive, energy transfer process. Mechanistic studies are consistent with a stepwise reaction mechanism via N-O bond homolysis following the second energy transfer event to result in the formation of 1-azetine products. Characteristic for this method is its operational simplicity, mild conditions, and modular approach that allow for the synthesis of functionalized azetines and tetrahydrofurans (via in situ hydrolysis) from readily available precursors.

IEDDA: An Attractive Bioorthogonal Reaction for Biomedical Applications.

The pretargeting strategy has recently emerged in order to overcome the limitations of direct targeting, mainly in the field of radioimmunotherapy (RIT). This strategy is directly dependent on chemical reactions, namely bioorthogonal reactions, which have been developed for their ability to occur under physiological conditions. The Staudinger ligation, the copper catalyzed azide-alkyne cycloaddition (CuAAC) and the strain-promoted [3 + 2] azide-alkyne cycloaddition (SPAAC) were the first bioorthogonal reactions introduced in the literature. However, due to their incomplete biocompatibility and slow kinetics, the inverse-electron demand Diels-Alder (IEDDA) reaction was advanced in 2008 by Blackman et al. as an optimal bioorthogonal reaction. The IEDDA is the fastest bioorthogonal reaction known so far. Its biocompatibility and ideal kinetics are very appealing for pretargeting applications. The use of a trans-cyclooctene (TCO) and a tetrazine (Tz) in the reaction encouraged researchers to study them deeply. It was found that both reagents are sensitive to acidic or basic conditions. Furthermore, TCO is photosensitive and can be isomerized to its cis-conformation via a radical catalyzed reaction. Unfortunately, the cis-conformer is significantly less reactive toward tetrazine than the trans-conformation. Therefore, extensive research has been carried out to optimize both click reagents and to employ the IEDDA bioorthogonal reaction in biomedical applications.

Expanding the chemical functionality of DNA nanomaterials generated by rolling circle amplification.

Rolling circle amplification (RCA) is a powerful tool for the construction of DNA nanomaterials such as hydrogels, high-performance scaffolds and DNA nanoflowers (DNFs), hybrid materials formed of DNA and magnesium pyrophosphate. Such DNA nanomaterials have great potential in therapeutics, imaging, protein immobilisation, and drug delivery, yet limited chemistry is available to expand their functionality. Here, we present orthogonal strategies to produce densely modified RCA products and DNFs. We provide methods to selectively modify the DNA component and/or the protein cargo of these materials, thereby greatly expanding the range of chemical functionalities available to these systems. We have used our methodology to construct DNFs bearing multiple surface aptamers and peptides capable of binding to cancer cells that overexpress the HER2 oncobiomarker, demonstrating their potential for diagnostic and therapeutic applications.

[3 + n] Cycloaddition Reactions: A Milestone Approach for Elaborating Pyridazine of Potential Interest in Medicinal Chemistry and Optoelectronics.

During the last few decades, pyridazine derivatives have emerged as privileged structures in heterocyclic chemistry, both because of their excellent chemistry and because of their potential applications in medicinal chemistry and optoelectronics. This review is focused on the recent advances in [3 + n] cycloaddition reactions in the pyridazine series as well as their medicinal chemistry and optoelectronic applications over the last ten years. The stereochemistry and regiochemistry of the cycloaddition reactions are discussed. Applications in optoelectronics (in particular, as fluorescent materials and sensors) and medicinal chemistry (in particular, antimicrobials and anticancer) are also reviewed.

Toward Intracellular Bioconjugation Using Transition-Metal-Free Techniques.

Bioconjugation is the chemical strategy of covalent modification of biomolecules, using either an external reagent or other biomolecules. Since its inception in the twentieth century, the technique has grown by leaps and bounds, and has a variety of applications in chemical biology. However, it is yet to reach its full potential in the study of biochemical processes in live cells, mainly because the bioconjugation strategies conflict with cellular processes. This has mostly been overcome by using transition metal catalysts, but the presence of metal centers limit them to in vitro use, or to the cell surface. These hurdles can potentially be circumvented by using metal-free strategies. However, the very modifications that are necessary to make such metal-free reactions proceed effectively may impact their biocompatibility. This is because biological processes are easily perturbed and greatly depend on the prevailing inter- and intracellular environment. With this taken into consideration, this review analyzes the applicability of the transition-metal-free strategies reported in this decade to the study of biochemical processes in vivo.

Pyrrole-Aminopyrimidine Ensembles: Cycloaddition of Guanidine to Acylethynylpyrroles.

An efficient method for the synthesis of pharmaceutically prospective pyrrole-aminopyrimidine ensembles (in up to 91% yield) by the cyclocondensation of easily available acylethynylpyrroles with guanidine nitrate has been developed. The reaction proceeds under heating (110-115 °C, 4 h) in the KOH/DMSO system. In the case of 2-benzoylethynylpyrrole, the unexpected addition of the formed pyrrole-aminopyrimidine as N- (NH moiety of the pyrrole ring) and C- (CH of aminopyrimidine) nucleophiles to the triple bond is observed.

"Kobayashi Benzynes" as Hexadehydro-Diels-Alder Diynophiles.

We report here elaboration of a cascade strategy for naphthyne formation using Kobayashi benzynes as diynophiles in a subsequent in situ hexadehydro-Diels-Alder reaction. Density functional theory computations suggest that the strained benzynes act as "super-diynophiles" in this transformation. The reaction requires only mildly basic and ambient temperature conditions and allows for rapid construction of various naphthalenic products. The trapping efficiencies of several arynophiles were determined using the benzyne to naphthyne cyclization as an internal clock reaction.

A [6+4]-cycloaddition adduct is the biosynthetic intermediate in streptoseomycin biosynthesis.

Streptoseomycin (STM, 1) is a bacterial macrolactone that has a unique 5/14/10/6/6-pentacyclic ring with an ether bridge. We have previously identified the biosynthetic gene cluster for 1 and characterized StmD as [6 + 4]- and [4 + 2]-bispericyclase that catalyze a reaction leading to both 6/10/6- and 10/6/6-tricyclic adducts (6 and 7). The remaining steps, especially how to install and stabilize the required 10/6/6-tricyclic core for downstream modifications, remain unknown. In this work, we have identified three oxidoreductases that fix the required 10/6/6-tryciclic core. A pair of flavin-dependent oxidoreductases, StmO1 and StmO2, catalyze the direct hydroxylation at [6 + 4]-adduct (6). Subsequently, a spontaneous [3,3]-Cope rearrangement and an enol-ketone tautomerization result in the formation of 10/6/6-tricyclic intermediate 12b, which can be further converted to a stable 10/6/6-tricyclic alcohol 11 through a ketoreduction by StmK. Crystal structure of the heterodimeric complex NtfO1-NtfO2, homologues of StmO1-StmO2 with equivalent function, reveals protein-protein interactions. Our results demonstrate that the [6 + 4]-adduct instead of [4 + 2]-adduct is the bona fide biosynthetic intermediate.

Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD+ capping.

Recent findings regarding nicotinamide adenine dinucleotide (NAD+)-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ noncanonical RNA capping to regulate gene expression. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to label NAD-RNAs. However, copper ions can fragment/degrade RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) for labeling NAD-RNAs, followed by identification of tagged RNA by single-molecule direct RNA sequencing. We used this method to compare NAD-RNA and total transcript profiles of Escherichia coli cells in the exponential and stationary phases. We identified hundreds of NAD-RNA species in E. coli and revealed genome-wide alterations of NAD-RNA profiles in the different growth phases. Although no or few NAD-RNAs were detected from some of the most highly expressed genes, the transcripts of some genes were found to be primarily NAD-RNAs. Our study suggests that NAD-RNAs play roles in linking nutrient cues with gene regulation in E. coli.

The Azide-Allene Dipolar Cycloaddition: Is DFT Able to Predict Site- and Regio-Selectivity?

The site- and regio-selectivity of thermal, uncatalysed 1,3-dipolar cycloadditions between arylazides and mono- or tetra-substituted allenes with different electronic features have been investigated by both conceptual (reactivity indices) and computational (M08-HX, ωB97X-D, and B3LYP) DFT approaches. Both approaches show that these cycloadditions follow a nonpolar one-step mechanism. The experimental site- and regio-selectivity of arylazides towards methoxycarbonyl- and sulfonyl-allenes as well as tetramethyl- and tetrafluoro-allenes was calculated by DFT transition state calculations, achieving semiquantitative agreement to both previous and novel experimental findings. From the mechanistic standpoint, 1H-NMR evidence of a methylene-1,2,3-triazoline intermediate reinforces the reliability of the computational scheme.

Multiplexed CuAAC Suzuki-Miyaura Labeling for Tandem Activity-Based Chemoproteomic Profiling.

Mass-spectrometry-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional and potentially 'druggable' hotspots in proteins. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or 'click' chemistry. The absence of bio-orthogonal chemistries that are functionally equivalent and complementary to CuAAC for chemoproteomic applications has hindered the development of multiplexed chemoproteomic platforms capable of assaying multiple amino acid side chains in parallel. Here, we identify and optimize Suzuki-Miyaura cross-coupling conditions for activity-based protein profiling and mass-spectrometry-based chemoproteomics, including for target deconvolution and labeling site identification. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual labeling. Multiplexed targeted deconvolution identified the protein targets of bifunctional cysteine- and lysine-reactive probes.

Unexpected regio- and stereoselective [4 + 3] cycloaddition reaction of azomethine ylides with benzylidene thiazolidinediones: synthesis of pharmacologically active spiroindoline oxazepine derivatives and theoretical study.

An unexpected regio- and stereoselective [4 + 3] cycloaddition reaction of azomethine ylides with 5-benzylidenethiazolidine-2,4-diones has been successfully developed for the synthesis of the novel pharmacologically active 4',5'-dihydro-3'H-spiro[indoline-3,2'-[1, 3] oxazepin]-2-one derivatives in basic condition. Easy purification, high yield, short experimental time and operational simplicity are specific advantages of this protocol. Furthermore, all the synthesized compounds have been evaluated for antioxidant and antibacterial activities. According to the results, most of the synthesized compounds exhibited DPPH radical scavenging activity and nine of them showed antibacterial properties. The reaction mechanism and 1H NMR spectrum have been evaluated by B3LYP/6311G method.

Reactivity of 3-nitroindoles with electron-rich species.

The indol(in)e building block is one of the "privileged-structures" for the pharmaceutical industry since this fragment plays a central role in drug discovery. While the electron-rich character of the indole motif has been investigated for decades, exploiting the electrophilic reactivity of 3-nitroindole derivatives has recently been put at the heart of a wide range of new, albeit challenging, chemical reactions. In particular, dearomatization processes have considerably enriched the scope of C2[double bond, length as m-dash]C3 functionalizations of these scaffolds. This feature article showcases this remarkable electrophilic reactivity of 3-nitroindoles with electron-rich species and highlights their value in generating diversely substituted indol(in)es. This compilation underlines how these heteroaromatic templates have gradually become model substrates for electron-poor aromatic compounds in dearomatization strategies.

Vinyl sulfone-modified carbohydrates: Michael acceptors and 2π partners for the synthesis of functionalized sugars and enantiomerically pure carbocycles and heterocycles.

Increasing demands for molecules with skeletal complexity, including those of stereochemical diversity, require new synthetic strategies. Carbohydrates have been used extensively as chiral building blocks for the synthesis of various complex molecules. On the other hand, the vinyl sulfone group has been identified as a unique functional group, which acts either as a Michael acceptor or a 2π partner in cycloaddition reactions. A combination of the high reactivity of the vinyl sulfone group and the in-built chiralities of carbohydrates has the potential to function as a powerful tool to generate a wide variety of enantiomerically pure reactive intermediates. Since CS bond formation in carbohydrates is easily achieved with regioselectivity, further synthetic manipulations of these thiosugars has led to the generation of a wide range of vinyl sulfone-modified furanosyl, pyranosyl, acyclic, and bicyclic carbohydrates. Several approaches have been studied to standardize the preparative methods for accessing vinyl sulfone-modified carbohydrates at least on a gram scale. Reactions of these modified carbohydrates with appropriate reagents afford a large number of new chemical entities primarily via (i) Michael addition reactions, (ii) desulfostannylation, (iii) Michael-initiated ring-closure reactions, and (iv) cycloaddition reactions. A wide range of desulfonylating reagents in the context of sensitive molecules such as carbohydrates have also been extensively studied. Applications of these strategies have led to the synthesis of (a) amino sugars and branched-chain sugars, (b) C-glycosides, (c) enantiomerically pure cyclopropanes, five- and six-membered carbocycles, (d) saturated oxa-, aza-, and thio-monocyclic heterocycles, (e) bi-and tricyclic saturated oxa and aza heterocycles, (f) enantiomerically pure and trisubstituted pyrroles, (g) 1,5-disubstituted 1,2,3-triazolylated carbohydrates and the corresponding triazole-linked di- and trisaccharides, (h) divinyl sulfone-modified carbohydrates and densely functionalized S,S-dioxothiomorpholines, and (i) modified nucleosides. Details of reaction conditions were incorporated as much as possible and mechanistic discussions were included wherever necessary.