Aminotrifluoromethylation of Olefins via Cyclic Amine Formation: Mechanistic Study and Application to Synthesis of Trifluoromethylated Pyrrolidines

We examined the mechanism of our previously reported aminotrifluoromethylation reaction, which proceeds via intramolecular cyclization of alkenylamines in the presence of the combination of copper catalyst and Togni reagent (<b>1</b>). Kinetic studies revealed that the initial rate of the reaction was first order with respect to Togni reagent and CuI, as well as the substrate. Changes of the ¹⁹F NMR chemical shift of Togni reagent during the reaction suggested the existence of a dynamic equilibrium involving coordination of not only Togni reagent, but also the substrate amine and the product aziridine to copper. ESI-MS analysis provided evidence of involvement of reactive Cu­(II) intermediates in the catalytic cycle. Overall, our results indicate that the reaction proceeds at the hypervalent iodine moiety of Togni reagent, which is activated by Cu­(II) species acting as a Lewis acid catalyst. On the basis of these mechanistic considerations, we developed an efficient synthesis of trifluoromethylated pyrrolidine derivatives. This transformation exhibited a remarkable rate enhancement upon addition of Et₃N

Aminotrifluoromethylation of Olefins via Cyclic Amine Formation: Mechanistic Study and Application to Synthesis of Trifluoromethylated Pyrrolidines

We examined the mechanism of our previously reported aminotrifluoromethylation reaction, which proceeds via intramolecular cyclization of alkenylamines in the presence of the combination of copper catalyst and Togni reagent (<b>1</b>). Kinetic studies revealed that the initial rate of the reaction was first order with respect to Togni reagent and CuI, as well as the substrate. Changes of the ¹⁹F NMR chemical shift of Togni reagent during the reaction suggested the existence of a dynamic equilibrium involving coordination of not only Togni reagent, but also the substrate amine and the product aziridine to copper. ESI-MS analysis provided evidence of involvement of reactive Cu­(II) intermediates in the catalytic cycle. Overall, our results indicate that the reaction proceeds at the hypervalent iodine moiety of Togni reagent, which is activated by Cu­(II) species acting as a Lewis acid catalyst. On the basis of these mechanistic considerations, we developed an efficient synthesis of trifluoromethylated pyrrolidine derivatives. This transformation exhibited a remarkable rate enhancement upon addition of Et₃N

Convergent Synthesis of the <i>ent</i>-ZA′B′C′D′-Ring System of Maitotoxin

Stereoselective synthesis of the <i>ent</i>-ZA′B′C′D′-ring system of maitotoxin has been accomplished through a convergent strategy utilizing Suzuki–Miyaura cross coupling reaction of ZA′-ring alkylborane and C′D′-ring (<i>Z</i>)-vinyl iodide, and subsequent construction of the B′-ring by reduction of the <i>O</i>,<i>S</i>-acetal

Proteomic identification of substrates for seven-beta-strand MTases.

<p>A, Schematic protocol for proteomic identification. HEK293T cell lysates were added to either propargylic Se-adenosyl-l-selenomethionine (ProSeAM) alone (1) or ProSeAM plus recombinant KMT (lysate:enzyme ratio was 10∶1) (2). After the <i>in vitro</i> reaction, labeled proteins were tagged with biotin and then precipitated with streptavidin beads. The precipitants were then digested with trypsin, and the trypsinized protein fragments were analyzed by LC-MS/MS. B, ProSeAM competes with SAM in the labeling reaction. HEK293T cell lysates were incubated with ProSeAM (250 µM) in the presence or absence of the indicated amount of SAM (0 to 2.5 mM). Modified proteins were biotinylated and detected with streptavidin-HRP (top). Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). C, western blot of labeled proteins. A 5% input of precipitated proteins without ProSeAM (1), with ProSeAM alone (2), with ProSeAM plus GST-G9a (3), with ProSeAM plus His-METTL21A (4) or with ProSeAM plus His-METTL10 was separately analyzed with western blotting with streptavidin-HRP (top) prior to the MS analysis, to compare the labeled proteins. Equal protein loading was confirmed by western blotting with anti-α-tubulin antibody (bottom). D, Doughnut chart of the subcellular distribution of proteins labeled with ProSeAM. HEK293T lysates alone (lane 1 in Fig. 2C) and HEK293T lysates with ProSeAM (lane 2 in Fig. 2C) were analyzed as described in A and Experimental procedures (n = 3). In total, 318 proteins were identified as ProSeAM-labeled proteins. E, List of METTL21A substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL21A (lane 2 and lane 4 in Fig. 2C), and analyzed as above. Molecular weight, peptide area (reflecting the quantity of detected protein), and fold enrichment of the peptide area are listed: ND, not determined because the substrate was detected only in the condition for lane 4 of B. The total numbers of identified proteins, 2-fold increase (compared to control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone.0105394.s003" target="_blank">Table S2</a>.</p

ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates.

<p>A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).</p

METTL10 knockdown reduces the level of lysine 318 methylation in EF1A1 <i>in vivo</i>.

<p>A, HEK293T cells were transfected with either scramble siRNA (Scr) or siRNA against METTL10 (siMETTL10). Twenty-four hours after the transfection, the plasmid for FLAG-EF1A1 was transfected and cultured for an additional 48 hours. Cells were harvested and the METTL10 expression level was analyzed with RT-PCR. n = 3, mean ± SD, **p<0.01. B, FLAG-EF1A1 was purified with anti-FLAG agarose beads from siRNA treated cells. EF1A1 lysine 318 mono-methylation (K318-Me1), di-methylation (K318-Me2), tri-methylation (K318-Me3), and unmethylation (K318-Me0) were analyzed by using a triple stage quadrupole mass spectrometer. The intensity of each peptide was normalized to the quantity of each sample's EF1A1 as determined by the presence of five EF1A1 peptide fragments, and the relative intensity was normalized to Scr of K318-Me0; n = 3, mean ± SD, *p<0.05, **p<0.01. C, <i>In vivo</i> METTL10 knockdown is required for <i>in vitro</i> robust methylation by METTL10. FLAG-EF1A1 purified from Scr or siMETTL10 treated cells was incubated with or without His-METTL10 in the presence of ¹⁴C-labeled SAM. Proteins were separated with SDS-PAGE and stained with Coomassie blue (bottom), the autoradiography was performed using a BAS-5000 image analyzer (top).</p

<i>N</i>‑Heterocycle-Forming Amino/Carboperfluoroalkylations of Aminoalkenes by Using Perfluoro Acid Anhydrides: Mechanistic Studies and Applications Directed Toward Perfluoroalkylated Compound Libraries

This work describes a practical and efficient method for synthesizing a diverse array of perfluoroalkylated amines, including <i>N</i>-heterocycles, to afford perfluoroalkylated chemical libraries as potential sources of drug candidates, agrochemicals, and probe molecules for chemical-biology research. Perfluoro acid anhydrides, which are commonly used in organic synthesis, were employed as a perfluoroalkyl source for intramolecular amino- and carbo-perfluoroalkylations of aminoalkenes, affording perfluoroalkylated <i>N</i>-heterocycles, including: aziridines, pyrrolidines, benzothiazinane dioxides, indolines, and hydroisoquinolinones. Diacyl peroxides were generated <i>in situ</i> from the perfluoro acid anhydrides with urea·H₂O₂, and allowed to react with aminoalkenes in the presence of copper catalyst to control the product selectivity between amino- and carbo-perfluoroalkylations. To illustrate the synthetic utility of bench-stable trifluoromethylated aziridine, which was prepared on a gram scale, we used it to synthesize a wide variety of trifluoromethylated amines including complex molecules, such as trifluoromethylated tetrahydroharmine and spiroindolone. A mechanistic study of the role of the copper catalyst in the aminotrifluoromethylation of allylamine suggested that Cu­(I) accelerates CF₃ radical formation via decomposition of diacyl peroxide, which appears to be the turnover-limiting step, while Cu­(II) controls the product selectivity

Mammalian METTL10 is an EF1A1 KMT.

<p>A, Phylogenic tree of human KMTs. Proteins were clustered based on DNA sequence by the maximum likelihood method using MEGA version 6 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone.0105394-Tamura1" target="_blank">[45]</a>. The nucleotide sequences used here were as follows: G9a (NCBI ID; NM_006709), GLP (NCBI ID; NM_024757), SUV39H1 (NCBI ID; NM_001282166), ESET (NCBI ID; NM_001145415), DOT1L (NCBI ID; NM_032482), VCPKMT (NCBI ID; NM_024558), CAMKMT (NCBI ID; NM_024766), SMYD2 (NCBI ID; NM_020197), METTL21A (NCBI ID; NM_145280), METTL10 (NCBI ID; NM_212554), and METTL20 (NCBI ID; NM_001135863). B, Schematic structure of human METTL10. A conserved MTase domain, Methyltransf_31 (Pfam ID; PF13847) is located in the middle region. C, Subcellular localization of METTL10. The plasmid for FLAG-tagged METTL10 was transfected into HeLa cells, and the FLAG-tagged proteins were visualized under immunofluorescence microscopy. D, List of METTL10 substrates. HEK293T cell lysates and ProSeAM were incubated with or without METTL10 (lane 2 and lane 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone-0105394-g002" target="_blank">Fig. 2C</a>) and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone-0105394-g002" target="_blank">Figure 2</a>. Molecular weight, peptide area which reflects the protein amount, and fold enrichment of the peptide area are listed. ND, not determined because substrate was detected only in the condition for lane 5 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone-0105394-g002" target="_blank">Figure 2C</a>. The total numbers of identified proteins, 2-fold increase (compared to the control in each experiment), and overlapped identified numbers of 3 independent experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105394#pone.0105394.s004" target="_blank">Table S3</a>.</p

METTL10 tri-methylates K318 in EF1A1.

<p>A, METTL10 methylates EF1A1 <i>in vitro</i>. FLAG-tagged EF1A1 and His-METTL10 were incubated with or without 14C-labeled SAM. Proteins were separated with SDS-PAGE and stained with Coomassie blue (bottom), the autoradiography was detected with the image analyzer BAS-5000 (top). B, A conserved SAM-binding domain is important for MTase activity of METTL10. The MTase domain of METTL10 (WT, 85-DIGTGNG-91) was replaced with alanines (4A, 85-AIATANA-91). The representative gel and its autoradiography was detected as in A. C, The relative EF1A1 methylation was quantified by the intensity of autoradiography, and normalized to the intensity by WT 0 µg as 1 (n = 3, mean ± SD, *p<0.05, **p<0.01). D, MS/MS spectrum of peptide fragments containing trimethylated lysine 318. Box; Asterisks represent b- and y- ions detected. E. Table of the peptide fragment corresponding to amino acids 306-318. Bold numbers represents fragment ions detected in the experiment. F, METTL10 specifically methylates lysine 318. Five lysine methylation sites (lysine 36, lysine 55, lysine 79, lysine 165 and lysine 318) on EF1A1 were substituted with arginine, and their methylations were examined by autoradiography. G, FLAG-EF1A1 (WT and K318R) were incubated with or without His-METTL10 in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-FLAG antibody for loading control (bottom).</p

Kinetically Controlled One-Pot Formation of DEFGH-Rings of Type B Physalins through Domino-Type Transformations

The characteristic DEFGH-ring system of type B physalins has been synthesized by means of a one-pot procedure incorporating domino-type ring transformations. Unexpectedly, we found that introduction of an α-hydroxyester functionality at C17 in ring E allowed the key 7-<i>endo</i> oxy-Michael reaction to proceed. Originally this was thought to be an unfavored process. This afforded the desired caged ring system to be formed in a kinetically controlled manner. Consecutive treatment with AcOH at 100 °C furnished the DEFGH-ring system in one pot