Histidine N1-position-specific methyltransferase CARNMT1 targets C3H zinc finger proteins and modulates RNA metabolism.

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (ßAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation.

The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

Structure-activity relationship study of nitrogen signaling factors.

We have synthesized 10 analogs of oxylipins, which are nitrogen signaling factors (NSFs) that mediate cell-to-cell communication in the fission yeast Schizosaccharomyces pombe, and evaluated their structure-activity relationships with the aim of developing molecular probes for NSFs. We found that the OH or OAc group at C10 could be replaced with a compact amide (17) or carbamate (19). Introducing an alkyne as a detection tag at C10 led to decreased, though still sufficient, activity. Introducing an alkyne at the C18 position showed a similar trend, suggesting tolerance is relatively low even for compact functional groups such as alkynes. Although introduction of a diazirine moiety as a photoreactive group at the C5 position decreased the activity, we found that introducing diazirine at the C13 position was acceptable, and compound 38 exhibited potent NSF activity. These findings will be helpful in the development of molecular probes for NSFs.

Synthesis and biological activity of ganglioside GM3 analogues with a (S)-CHF-Sialoside linkage and an alkyne tag.

The alkyne tag, consisting of only two carbons, is widely used as a bioorthogonal functional group due to its compactness and nonpolar structure, and various probes consisting of lipids bearing an alkyne tag have been developed. Here, we designed and synthesized analogues of ganglioside GM3 bearing an alkyne tag in the fatty acid moiety and evaluated the effect of the alkyne tag on the biological activity. To eliminate the influence of other factors such as degradation of the glycan chain when evaluating biological activity in a cellular environment, we introduced the tag into sialidase-resistant (S)-CHF-linked GM3 analogues developed by our group. The designed analogues were efficiently synthesized by tuning the protecting group of the glucosylsphingosine acceptor. The growth-promoting effect of these analogues on Had-1 cells was dramatically altered depending upon the position of the alkyne tag.

Understanding and Controlling Fluorinated Diacyl Peroxides and Fluoroalkyl Radicals in Alkene Fluoroalkylations.

The demand for practical methods for the synthesis of novel fluoroalkyl molecules is increasing owing to their diverse applications. Our group has achieved efficient difunctionalizing fluoroalkylations of alkenes using fluorinated carboxylic anhydrides as user-friendly fluoroalkyl sources. Fluorinated diacyl peroxide, prepared in situ from carboxylic anhydrides, enables the development of novel reactions when used as a radical fluoroalkylating reagent. In this account, we aim to provide an in-depth understanding of the structure, bonding, and reactivity of fluorinated diacyl peroxides and radicals as well as their control in fluoroalkylation reactions. In the first part of this account, the physical properties and reactivity of diacyl peroxides and fluoroalkyl radicals are described. In the subsequent part, we categorize the reactions into copper-catalyzed and metal-free methods utilizing the oxidizing properties of fluorinated diacyl peroxides. We also outline examples and mechanisms.

Experimental and Computational Investigation of Facial Selectivity Switching in Nickel-Diamine-Acetate-Catalyzed Michael Reactions.

Chiral Ni complexes have revolutionized both asymmetric acid-base and redox catalysis. However, the coordination isomerism of Ni complexes and their open-shell property still often hinder the elucidation of the origin of their observed stereoselectivity. Here, we report our experimental and computational investigations to clarify the mechanism of ß-nitrostyrene facial selectivity switching in Ni(II)-diamine-(OAc)2-catalyzed asymmetric Michael reactions. In the reaction with a dimethyl malonate, the Evans transition state (TS), in which the enolate binds in the same plane with the diamine ligand, is identified as the lowest-energy TS to promote C-C bond formation from the Si face in ß-nitrostyrene. In contrast, a detailed survey of the multiple potential pathways in the reaction with α-keto esters points to a clear preference for our proposed C-C bond-forming TS, in which the enolate coordinates to the Ni(II) center in apical-equatorial positions relative to the diamine ligand, thereby promoting Re face addition in ß-nitrostyrene. The N-H group plays a key orientational role in minimizing steric repulsion.

Raman Spectroscopy for Chemical Biology Research.

In chemical biology research, various fluorescent probes have been developed and used to visualize target proteins or molecules in living cells and tissues, yet there are limitations to this technology, such as the limited number of colors that can be detected simultaneously. Recently, Raman spectroscopy has been applied in chemical biology to overcome such limitations. Raman spectroscopy detects the molecular vibrations reflecting the structures and chemical conditions of molecules in a sample and was originally used to directly visualize the chemical responses of endogenous molecules. However, our initial research to develop "Raman tags" opens a new avenue for the application of Raman spectroscopy in chemical biology. In this Perspective, we first introduce the label-free Raman imaging of biomolecules, illustrating the biological applications of Raman spectroscopy. Next, we highlight the application of Raman imaging of small molecules using Raman tags for chemical biology research. Finally, we discuss the development and potential of Raman probes, which represent the next-generation probes in chemical biology.

Chemoproteomic Identification of Blue-Light-Damaged Proteins.

Visible light, particularly in the blue region of the spectrum, can cause cell dysfunction through the generation of singlet oxygen, contributing to cellular aging and age-related pathologies. Although photooxidation of nucleic acids, lipids, and amino acids has been extensively studied, the magnitude and span of blue-light-induced protein damages within proteome remain largely unknown. Herein we present a chemoproteomic approach to mapping blue-light-damaged proteins in live mammalian cells by exploiting a nucleophilic alkyne chemical probe. A gene ontology enrichment analysis revealed that cell surface proteins are more readily oxidized than other susceptible sets of proteins, including mitochondrial proteins. In particular, the integrin family of cell surface receptors (ITGs) was highly ranked in the mammalian cells tested, including human corneal endothelial cells. The blue-light-oxidized ITGB1 protein was functionally inactive in promoting cell adhesion and proliferation, suggesting that the photodamage of integrins contributes to the blue-light-induced cell dysfunction. Further application of our method to various cells and tissues should lead to a comprehensive analysis of light-sensitive proteins.

Propargylic Se-adenosyl-l-selenomethionine: A Chemical Tool for Methylome Analysis.

Devising synthetic strategies to construct a covalent bond is a common research topic among synthetic chemists. A key driver of success is the high tunability of the conditions, including catalysts, reagents, solvents, and reaction temperature. Such flexibility of synthetic operations has allowed for the rapid exploration of a myriad of artificial synthetic transformations in recent decades. However, if we turn our attention to chemical reactions controlled in living cells, the situation is quite different; the number of hit substrates for the reaction-type is relatively small, while the crowded environment is chemically complex and inflexible to control.A specific objective of this Account is to introduce our chemical methylome analysis as an example of bridging the gap between chemistry and biology. Protein methylation, catalyzed by protein methyltransferases (MTases) using S-adenosyl-l-methionine (SAM or AdoMet) as a methyl donor, is a simple but important post-translational covalent modification. We aim to efficiently identify MTase substrates and methylation sites using activity-based protein profiling (ABPP) with propargylic Se-adenosyl-l-selenomethionine (ProSeAM, also called SeAdoYn). Specifically, we draw heavily from quantitative proteomics that yields information about the differences between two samples utilizing LC-MS/MS analysis. By exploiting the use of ProSeAM, we have prepared the requisite two samples for quantitative methylome analysis. The structural difference between ProSeAM and the parent SAM is so small that the quantity of modification of the protein substrate with this artificial cofactor reflects, to a large extent, levels of activity of the MTase of interest with SAM. First, we identified that the addition of exogenous recombinant MTase (methylation accel), a natural catalyst, enhances the generation of the corresponding propargylated product even in the cell lysate. Then, we applied the principle to isotope label-free quantification with HEK293T cell lysates. By comparing the intensity of LC-MS/MS signals in the absence and presence of the MTase, we have successfully correlated the MTase substrates. We have currently applied the concept to the stable isotope label-based quantification, SILAC (stable isotope labeling by amino acids in cell culture). The strategy merging ProSeAM/MTase/SILAC (PMS) is uniquely versatile and programmable. We can choose suitable cell lines, subcellular fractions (i.e.; whole lysate or mitochondria), and genotypes as required. In particular, we would like to emphasize that the use of cell lysates derived from disease-associated MTase knockouts (KOs) holds vast potential to discover functionally unknown but biologically important methylation events. By adding ProSeAM and a recombinant MTase to the lysates derived from KO cells, we successfully characterized unprecedented nonhistone substrates of several MTases. Furthermore, this chemoproteomic procedure can be applied to explore MTase inhibitors (methylation brake). The combined strategy with ProSeAM/inhibitor/SILAC (PIS) offers intriguing opportunities to explore nonhistone methylation inhibitors.Considering that SAM is the second most widely used enzyme-substrate following ATP, the interdisciplinary research between chemistry and biology using SAM analogs has a potentially huge impact on a wide range of research fields associated with biological methylation. We hope that this Account will help to further delineate the biological function of this important class of enzymatic reaction.

Effect of Alkynyl Group on Reactivity in Photoaffinity Labeling with 2-Thienyl-Substituted α-Ketoamide.

Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.

Theoretical Insights into the Substrate-Dependent Diastereodivergence in (3 + 2) Cycloaddition of α-Keto Ester Enolates with Nitrones.

Controlling catalytic asymmetric space has received increasing attention for the on-demand synthesis of chiral molecules of interest. However, the identification of the key parameters controlling the stereo-determining step in transition metal catalysis is challenging and involves the thorough characterization of the rate- and stereo-determining transition state(s). In this paper, we describe the computational analysis of the (3 + 2) cycloaddition of Ni(II)-enolate with cyclic (E)-nitrone to provide a comprehensive analysis of how the bond-forming processes are regulated in the two-electron manifold in the triplet state. Our molecular orbital analysis, in particular, reveals the occurrence of the singly occupied molecular orbital-highest occupied molecular orbital (SOMO-HOMO) level inversion in the Ni(II)-enolate. Further, distortion and interaction analysis are also used to explain the substrate-dependent diastereodivergence in this reaction by alternating the structure of the nitrone. Using a range of computational analyses, we show that the rate- and stereo-determining step in the (3 + 2) cycloaddition of (E)-nitrone is regulated integrally by (1) isomerism of the octahedral Ni(II) complex, (2) E/Z isomerism of the Ni(II)-enolate, and (3) steric repulsion between the reactants and ligand.

Dynamics in Catalytic Asymmetric Diastereoconvergent (3 + 2) Cycloadditions with Isomerizable Nitrones and α-Keto Ester Enolates.

Reaction design in asymmetric catalysis has traditionally been predicated on a structurally robust scaffold in both substrates and catalysts, to reduce the number of possible diastereomeric transition states. Herein, we present the stereochemical dynamics in the Ni(II)-catalyzed diastereoconvergent (3 + 2) cycloadditions of isomerizable nitrile-conjugated nitrones with α-keto ester enolates. Even in the presence of multiple equilibrating species, the catalytic protocol displays a wide substrate scope to access a range of CN-containing building blocks bearing adjacent stereocenters with high enantio- and diastereoselectivities. Our computational investigations suggest that the enantioselectivity is governed in the deprotonation process to form (Z)-Ni-enolates, while the unique syn addition is mainly controlled by weak noncovalent bonding interactions between the nitrone and ligand.

Design, Synthesis, and Antifungal Activity of 16,17-Dihydroheronamide C and ent-Heronamide C.

16,17-Dihydroheronamide C (8) and ent-heronamide C (ent-1) were designed as probes for the mode-of-action analysis of heronamide C (1). These molecules were synthesized by utilizing a highly modular strategy developed in the preceding paper. The evaluation of the antifungal activity of these compounds revealed the exceptional importance of the C16-C17 double bond for the antifungal activity of heronamide C and the existence of chiral recognition between heronamide C (1) and cell membrane components.

Detecting nitrile-containing small molecules by infrared photothermal microscopy.

The use of infrared (IR) photothermal microscopy (IR-PTM) is emerging for imaging chemical substances in various samples. In this research, we demonstrated the use of a nitrile group as a vibrational tag to image target molecules in the low water-background region. We performed IR photothermal imaging of trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) in cells and confirmed the high spatial resolution by photothermal detection using visible light as a probe beam. We imaged FCCP-treated HeLa cells and confirmed that the photothermal signal was indeed produced from the vibrational tag in lipid droplets. We also compared the results with nitrile imaging by stimulated Raman scattering (SRS) microscopy. From both the calculated and experimental results, IR-PTM demonstrated a signal-to-noise ratio (SNR) several tens of times better than that of SRS microscopy on the basis of the same power input.

Deuteration of terminal alkynes realizes simultaneous live cell Raman imaging of similar alkyne-tagged biomolecules.

Alkynes were employed as tags to observe small molecules in cells by Raman microscopy. Herein, simple deuteration was found to shift the vibrational frequency of the alkyne by 135 cm-1. Two-color Raman imaging of D-alkynes and H-alkynes made it possible to distinguish between and observe similar small molecules in live cells.

1,2-Bis-perfluoroalkylations of alkenes and alkynes with perfluorocarboxylic anhydrides via the formation of perfluoroalkylcopper intermediates.

A novel, Cu-mediated protocol toward the 1,2-bis-perfluoroalkyaltion of alkenes/alkynes was developed. The method proceeded with perfluorocarboxylic anhydrides as inexpensive and readily available perfluoroalkyl sources. Diacyl peroxide was generated in situ from the perfluorocarboxylic anhydrides and H2O2. The key step in this reaction is the formation of a stable perfluoroalkylcopper intermediate that is achieved with the aid of a bipyridyl ligand. Subsequent reaction of the intermediate with perfluoroalkyl-containing alkyl or vinyl radicals affords the desired products.

Recent advances in reactions using diacyl peroxides as sources of O- and C-functional groups.

Diacyl peroxides, (RCO2)2, are readily available and widely used reagents for organic synthesis, because they can serve as electrophiles, oxidants, and radical sources. Recently, they have been used extensively as sources of O- and C-functional groups, in contrast to their classical applications as radical initiators. These novel reaction modes have greatly expanded the synthetic utility of diacyl peroxides by making it possible to simultaneously utilize plural functionalities of diacyl peroxides in unprecedented ways, with or without the aid of transition-metal catalysts. Here, we review recent advances in reactions utilizing diacyl peroxides as O- and C-sources, with examples illustrating how the reactivity of diacyl peroxides in organic reactions can be controlled.

Recent Advances on the Halo- and Cyano-Trifluoromethylation of Alkenes and Alkynes.

Incorporation of fluorine into organic molecules is a well-established strategy in the design of advanced materials, agrochemicals, and pharmaceuticals. Among numerous modern synthetic approaches, functionalization of unsaturated bonds with simultaneous addition of trifluoromethyl group along with other substituents is currently one of the most attractive methods undergoing wide-ranging development. In this review article, we discuss the most significant contributions made in this area during the last decade (2012-2021). The reactions reviewed in this work include chloro-, bromo-, iodo-, fluoro- and cyano-trifluoromethylation of alkenes and alkynes.

Modern Approaches for Asymmetric Construction of Carbon-Fluorine Quaternary Stereogenic Centers: Synthetic Challenges and Pharmaceutical Needs.

New methods for preparation of tailor-made fluorine-containing compounds are in extremely high demand in nearly every sector of chemical industry. The asymmetric construction of quaternary C-F stereogenic centers is the most synthetically challenging and, consequently, the least developed area of research. As a reflection of this apparent methodological deficit, pharmaceutical drugs featuring C-F stereogenic centers constitute less than 1% of all fluorine-containing medicines currently on the market or in clinical development. Here we provide a comprehensive review of current research activity in this area, including such general directions as asymmetric electrophilic fluorination via organocatalytic and transition-metal catalyzed reactions, asymmetric elaboration of fluorine-containing substrates via alkylations, Mannich, Michael, and aldol additions, cross-coupling reactions, and biocatalytic approaches.

Formal Aerobic Oxidative Cross-Coupling of Benzofuranones with Azo Compounds Using Pd-µ-hydroxo Complex.

We have developed a catalytic aerobic oxidative dimerization reaction of benzofuranones using a Pd(II)-µ-hydroxo complex. Radical-radical cross-coupling of the resulting dimers with azo compounds enabled the one-pot synthesis of structurally congested benzofuranones having two distinct vicinal all-carbon quaternary centers.