3-羟基异恶唑的O-和N-酰化及其区域选择性研究 Ⅱ.3-羟基-5-甲基异恶唑O-和N-酰化产物的选择性合成

本文研究了不同反应条件下3-羟基-5-甲基异恶唑(1)与3-甲基-2- 对氯苯基丁酰氯(2a)的反应,提出了区域选择性合成O-和N-酰化产物的方法.在三乙胺存在下,乙腈为溶剂,1和2a～2e反应得到含量为88～96%的O-酰化产物3a～3e,而若将1 转化为相应的异恶唑硅醚4,再与2a～2i反应,则得到含量为86～97%的N-酰化产物5a～5i.试验表明,在DMAP催化下,3a和5a可发生O-/N-酰基转移反应     

Hydroxamic Acid‐Piperidine Conjugate is an Activated Catalyst for Lysine Acetylation under Physiological Conditions

Lysine acylation of proteins is an essential chemical reaction for posttranslational modification and as a means of protein modification in various applications. N,N‐Dimethyl‐4‐aminopyridine (DMAP) derivatives are widely‐used catalysts for lysine acylation of proteins; however, the DMAP moiety mostly exists in a protonated, and thus deactivated, form under physiological conditions due to its basicity. An alternative catalytic motif furnishing higher acylation activity would further broaden the possible applications of chemical lysine acylation. We herein report that the hydroxamic acid‐piperidine conjugate Ph‐HXA is a more active catalytic motif for lysine acetylation than DMAP under physiological conditions. In contrast to DMAP, the hydroxamic acid moiety is mostly deprotonated under aqueous neutral pH, resulting in a higher concentration of the activated form. The Ph‐HXA catalyst is also more tolerant of deactivation by a high concentration of glutathione than DMAP. Therefore, Ph‐HXA might be a suitable catalytic motif for target protein‐selective and site‐selective acetylation in cells.     

Regioselective lipase-catalyzed acylation of 41-desmethoxy-rapamycin without vinyl esters

Selective lipase-catalyzed acylation of 41-desmethoxyrapamycin has been achieved with a quaternary carboxylic acid avoiding the use of vinyl ester activation. Among the acyl donors investigated, the novel butanedione-monooxime and the N-acetylhydroxamate ester proved to be the most efficient donors, comparable in reactivity to the undesired vinyl ester and allowing selective, preparative acylation on gram scale in excellent yields. These new donors are proposed as sustainable and process-friendly alternatives to the widely used vinyl ester substrate activation in lipase-catalyzed acylations of secondary alcohols.     

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysyl‐tRNA synthetase‐tRNA^Pyl pair, azidonorleucine is genetically encoded in E. coli . Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site‐specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su‐H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post‐translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.     

Selective esterifications of alcohols and phenols through carbodiimide couplings

Esterification of carboxylic acids capable of forming ketene intermediates upon treatment with carbodiimides permits the selective acylation of alcohols in the presence of phenols lacking strong electron-withdrawing groups. The selectivity of acylations involving highly acidic phenols could be reversed through the addition of catalytic amount of acid. Esterification of other carboxylic acids was found to proceed through the formation of symmetric anhydrides and provide the opposite chemoselectivity. In both cases the relative acylation rates of substituted phenols are consistent with a reaction mechanism involving an attack of phenolate anions on electrophilic intermediates such as ketenes and symmetric anhydrides, with the carbodiimides serving both as an activating reagent and as a basic catalyst.     

Integrative Chemical Biology Approaches for Identification and Characterization of “Erasers” for Fatty‐Acid‐Acylated Lysine Residues within Proteins

Acylation of proteins with fatty acids is important for the regulation of membrane association, trafficking, subcellular localization, and activity of many cellular proteins. While significant progress has been made in our understanding of the two major forms of protein acylation with fatty acids, N‐myristoylation and S‐palmitoylation, studies of the acylation of lysine residues, within proteins, with fatty acids have lagged behind. Demonstrated here is the use of integrative chemical biology approaches to examine human sirtuins as de‐fatty‐acid acylases in vitro and in cells. Photo‐crosslinking chemistry is used to investigate enzymes which recognize fatty‐acid acylated lysine. Human Sirt2 was identified as a robust lysine de‐fatty‐acid acylase in vitro. The results also show that Sirt2 can regulate the acylation of lysine residues, of proteins, with fatty acids within cells.     

2‐Oxazoline Formation for Selective Chemical Labeling of 5‐Hydroxylysine

Hydroxylation of lysine, one of posttranslational modifications of proteins, generates 5‐hydroxylysine (Koh) and plays a crucial role in regulating protein functions in cellular activity. We have developed a chemical labeling method of Koh. The 1,2‐aminoalcohol moiety of Koh in synthetic peptide sequences was trapped by an alkyne‐containing benzimidate to form a 2‐oxazoline ring. An additional ammonia treatment process removed the undesirable amidine residue formed between benzimidate and lysine. During the ammonia treatment, the oxazoline residue formed at Koh mainly remained intact, and the ring opening to the amide form was observed for only part of oxazoline, indicating that the chemical labeling is amino acid selective. Azide‐substituted biotin or fluorescent dye was attached to the peptide through Huisgen cycloaddition at Koh and converted into an alkyne‐labeled oxazoline form. The Koh‐labeling assay could provide a platform to enhance proteomic research of lysine hydroxylation.     

The first intermolecular Friedel-Crafts acylation with beta-lactams

The first intermolecular Friedel-Crafts acylation of a variety of aromatic substrates with azetidinones is described. The Friedel-Crafts acylations are performed under very mild conditions, using trifluoromethanesulfonic acid to produce beta-amino aromatic ketones in excellent yields.     

Susceptibility of Methyl 3-Amino-1H-pyrazole-5-carboxylate to Acylation

In the search for a new method of synthesis of hybrid peptides with aminopyrazole carboxylic acid, we tried to force selective acylation at the aromatic amino group instead of at the ring nitrogen atom with fairly gentle acylating agents. The acylating agents used were acid anhydrides: acetic anhydride, tert-butyl pyrocarbonate, and 2-(2-methoxyethoxy)ethoxyacetic acid/dicyclohexylcarbodiimide. We succceded in acylation at this amino group with almost none at the ring nitrogen atom. Sometimes, however, acylation in small quantities at the ring nitrogen atom was observed as a by-product. To remove this by-product, imidazole was used. Thus, we were able to obtain the hybrid peptides in question with no protection and subsequent removal required. We synthesized a few these free peptides with no protection of the pyrazole ring. This is a simpler method than that being used currently.     

Highly efficient and enantioselective enzymatic acylation of amines in aqueous medium

A new strategy based on the unique catalytic properties, stability and enantioselectivity of the relatively unknown penicillin acylase from Alcaligenes faecalis has been developed for the effective and enantioselective acylation of amines in aqueous medium. In contrast to lipase-catalyzed acylations in organic solvents, the penicillin acylase-catalyzed acylation of amines in aqueous solution is a rapid and chemoselective process leading to a product which can subsequently be deacylated by the same enzyme, imposing secondary enantiocontrol and leading to effective resolution.     

Mild, efficient Friedel-Crafts acylations from carboxylic acids using cyanuric chloride and AlCl3

A mild method for Friedel-Crafts acylation with aromatic and aliphatic carboxylic acids using cyanuric chloride, pyridine, and AlCl(3) was developed. Both inter- and intramolecular acylations were achieved at room temperature in high yield and in very short reaction times.     

Highly Selective Lysine Acylation in Proteins Using a Lys-His Tag Sequence

Chemical modification of proteins has numerous applications, but it has been challenging to achieve the required high degree of selectivity on lysine amino groups. Recently, we described the highly selective acylation of proteins with an N-terminal Gly-His₆ segment. This tag promoted acylation of the N-terminal N^α-amine resulting in stable conjugates. Herein, we report the peptide sequences His_n-Lys-His_m, which we term Lys-His tags. In combination with simple acylating agents, they facilitate the acylation of the designated Lys N^ϵ-amine under mild conditions and with high selectivity over native Lys residues. We show that the Lys-His tags, which are 7 to 10 amino acids in length and still act as conventional His tags, can be inserted in proteins at the C-terminus or in loops, thus providing high flexibility regarding the site of modification. Finally, the selective and efficient acylation of the therapeutic antibody Rituximab, pure or mixed with other proteins, demonstrates the scope of the Lys-His tag acylation method.     

Liquid poly(ethylene glycol) and supercritical carbon dioxide as a biphasic solvent system for lipase-catalyzed esterification

The biphasic solvent system composed of poly(ethylene glycol)(PEG) and supercritical carbon dioxide (scCO2) is ideally suited for the lipase-catalyzed acylation of alcohols; batch or continuous flow acylations are possible, scCO2 being used to extract the products.     

Benzoylation of 2(3H)-benzothiazolones in the Presence of 8 Equivalents of Zinc Chloride in N,N-dimethylformamide

The use of the mixture of aluminum chloride-N,N-dimethylformamide 1,2 (AlCl 3 -DMF, 11:1) reagent in the Friedl-Crafts C acylation reaction of 2(3H)-benzothiazolones was previously reported. 3 These acylations reactions were found to proceed with high regioselectivity. The precise position of acylation was uniquivocally assigned by x-ray single-crystal defraction in the case of 6-benzoyl-2(3H)-benzothiazolone. 4 The assignment of the position of acylation was extended to other compounds by the use of high-field 1 H-NMR . 3     

Full Functionalization of the Imidazole Scaffold by Selective Metalation and Sulfoxide/Magnesium Exchange

A simple, flexible, and straightforward method for the functionalization of all the positions of the imidazole heterocycle through regioselective arylations, allylations, acylations, and additions to aldehydes is disclosed. Starting from the readily available key imidazole 1 , highly functionalized imidazole derivatives have been synthesized in a regioselective manner from directed metalations and a sulfoxide/magnesium exchange. Moreover, the selective N3‐alkylation followed by deprotection of N1 (trans‐N‐alkylation) allows the regioselective N‐alkylation of complex imidazoles.     

RNA Cloaking by Reversible Acylation

We describe a selective and mild chemical approach for controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide‐substituted acylating agent (100–200 mm ) yields several 2′‐OH acylations per RNA strand in as little as 10 min. This poly‐acylated (“cloaked”) RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA‐processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water‐soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups (“uncloaking”). This fully restores RNA folding and biochemical activity.     

Cyclizations of some furyl acid chlorides

Intramolecular Friedel-Crafts acylation of the furan ring has been accomplished in good yields with stannic chloride for both furyl isomers of butyryl acid chloride. The propionyl chloride isomers, however, failed to produce reasonable quantities of the cyclic ketone.     

Selective cleavage enhanced by acetylating the side chain of lysine

Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision‐induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N‐terminal of the peptide. After acetylation of the lysine side‐chain, ions were the most abundant primary fragment products and the Lys_(Ac)–Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side‐chain of lysine promoted the selective cleavage of Lys–Xxx amide bond and generated much more information of the peptide backbone sequence. The results re‐evaluate the selective cleavage due to the lysine basic side‐chain and provide information for studying the post‐translational modification of proteins and other bio‐molecules containing Lys residues. Copyright © 2013 John Wiley & Sons, Ltd.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.