An exception of eschweiler‐clarke methylation: Cyclocondensation of α‐amino amides with formaldehyde and formic acid

An exception of Eschweiler‐Clarke methylation was found for α‐amino amides. The α‐amino amides on treatment with formaldehyde and formic acid produce cyclocondensation products, imidazolidin‐4‐ones, but N‐methylation process becomes important when three substituents of the α‐amino amides are very bulky. On the other hand, N‐methylation is the only product for Eschweiler‐Clarke methylation of the α‐amino amides with N,N‐disubstituted amide.     

Synthesis of Cryptolepine and Cryptoteckieine from a Common Intermediate

Both cryptolepine 1 and cryptotackieine 3 have been synthesized from 1,3‐bis(2‐nitrophenyl)propan‐2‐one. The approach to 1 involved reduction of the NO₂ groups, oxidative cyclization with PhI(OAc)₂, and N‐methylation, whereas 3 was obtained via bromination, Favorskii rearrangement, reduction (in situ cyclization), and N‐methylation.     

Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation

A method for the determination of DNA global methylation, taken as the ratio (%) of 5‐methylcytosine (5mCyt) versus the sum of cytosine (Cyt) and 5mCyt, via gas chromatography/mass spectrometry (GC/MS), was developed and validated. DNA (2.5 µg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine‐2,4‐¹³C₂,¹⁵N₃ and 5‐methyl‐²H₃‐cytosine‐6‐²H₁ as internal standards, and derivatized with N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide and 1% tert‐butyldimethylchlorosilane, in the presence of acetonitrile and pyridine. GC/MS, operating in single ion monitoring mode, separated and specifically detected all nucleobases as tert‐butyldimethylsilyl derivatives, without interferences, with the exception of guanosine. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 3.2 pmol for Cyt and 0.056 pmol for 5mCyt, the latter corresponding to a methylation level of 0.41%. Intra‐ and inter‐day precision and accuracy were below 4.0% for both analytes and methylation. The matrix absolute effect, process efficiency and coefficient of variation ranged from 96.5 to 101.2%. The matrix relative effect was below 1%. The method was applied to the analysis of different human DNAs, including: nonmethylated DNA from PCR (methylation 0.00%), hypermethylated DNA prepared using M.SssI CpG methyltransferase (methylation 18.05%), DNA from peripheral blood leukocytes of healthy subjects (N = 6, median methylation 5.45%), DNA from bone marrow of leukemia patients (N = 5, 3.58%) and DNA from myeloma cell lines (N = 4, 2.74%). Copyright © 2009 John Wiley & Sons, Ltd.     

Improved Synthesis and Reaction of 11‐Chloroneocryptolepines,Strategic Scaffold for Antimalaria Agent,and Their 6‐Methyl Congener from Indole‐3‐carboxylate

Various 11‐chloro‐5‐methyl‐5H‐indolo[2,3‐b]quinolines (neocryptolepines) with different substituents on the quinoline ring, key intermediates for antimalaria agents, are prepared from the substituted N‐methylanilines, easily accessible by the N‐methylation of anilines, and indole‐3‐carboxylate as a counterpart. This protocol is benign in terms of the reduced number of steps to reach the target, compared with the known method using anilines, and easy product purification. Alternatively, their 6‐methyl congener is prepared by N‐methylation of the indole moiety of 2‐arylaminoindole‐3‐carboxylate followed by successive cyclization and chlorination. 11‐Chloroneocryptolepines are found more reactive than their 6‐methyl congener in the nucleophilic substitution at the C11 position.     

Determination of five nucleosides by LC‐MS/MS and the application of the method to quantify N6‐methyladenosine level in liver messenger ribonucleic acid of an acetaminophen‐induced hepatotoxicity mouse model

Ribonucleic acid N⁶‐methyladenosine methylation plays an important role in a variety of biological processes and diseases. Acetaminophen‐induced hepatotoxicity is one of the major challenges faced by clinicians. To date, the link between N⁶‐methyladenosine and acetaminophen‐induced hepatotoxicity has not been studied. In this study, a simple ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of five nucleosides (adenosine, uridine, cytidine, guanosine, and N⁶‐methyladenosine) in messenger ribonucleic acid. After enzymatic digestion of messenger ribonucleic acid, the nucleosides sample was separated on an Acquity UPLC column with gradient elution using methanol and 0.02% formic acid water, and detected by a Qtrap 4500 mass spectrometer with an electrospray ionization mode. The method was validated over the concentration ranges of 4–800 ng/mL for adenosine, uridine, cytidine, and guanosine and 0.1–20 ng/mL for N⁶‐methyladenosine. It was successfully applied to the determination of N⁶‐methyladenosine levels in liver messenger ribonucleic acid in an acetaminophen‐induced hepatotoxicity mouse model and a control group. This study offers a method for the determination of nucleoside contents in epigenetic studies and constitutes the first step toward the investigation of ribonucleic acid methylation in acetaminophen‐induced hepatotoxicity, which will facilitate the elucidation of its mechanism.     

Di‐N‐Methylation of Anti‐Gram‐Positive Aminoglycoside‐Derived Membrane Disruptors Improves Antimicrobial Potency and Broadens Spectrum to Gram‐Negative Bacteria

The effect of di‐N‐methylation of bacterial membrane disruptors derived from aminoglycosides (AGs) on antimicrobial activity is reported. Di‐N‐methylation of cationic amphiphiles derived from several diversely structured AGs resulted in a significant increase in hydrophobicity compared to the parent compounds that improved their interactions with membrane lipids. The modification led to an enhancement in antibacterial activity and a broader antimicrobial spectrum. While the parent compounds were either modestly active or inactive against Gram‐negative pathogens, the corresponding di‐N‐methylated compounds were potent against the tested Gram‐negative as well as Gram‐positive bacterial strains. The reported modification offers a robust strategy for the development of broad‐spectrum membrane‐disrupting antibiotics for topical use.     

Mechanism and Control in Biological Amine Methylation

Biological methylation is a subject that has fascinated mechanistically minded chemists for over 50 years. While early studies were usually directed at C‐methylation in natural products, more recent work on N‐methylation in DNA and proteins is being supported by the results of X‐ray crystallography. From this source, significant mechanistic detail can be gleaned and powerful insights gained into the nature of enzyme catalysis and selectivity in methyl‐transfer processes. The case of the human histone H3 transmethylase SET7/9 is considered in detail and compared to cognate histone lysine methylases. It provides an analysis of Nature's solution to the task of avoiding over‐methylation.     

Characterization of complexation of PVP copolymer with DNA

Interactions of amphiphilic copolymer poly(N‐vinylpyrrolidone‐co‐N‐t‐Boc‐ tryptophanamido‐N′‐methacrylthiourea) [P(NVP‐co‐TrpAMT)] and poly(N‐vinylpyrrlidone) (PVP) with DNA were studied with the aim to understand the difference in their complexation efficiencies when considered as a potential non‐viral delivery vector. Fluorescence spectroscopy, ethidium bromide (EB) quenching, ζ potential measurement, and agarose gel electrophoresis were all carried out in Tris‐HCl (pH 7.4) solution to assess the degree of interaction between the (co)polymers P(NVP‐co‐TrpAMT) and DNA. All of these measurements show that the high affinity of the copolymer for DNA can be demonstrated. We also found that P(NVP‐co‐TrpAMT)‐I (PVP/PTrpAMT molar ratio was 100:2.88) exhibited a high DNA cleavage activity and induced the supercoiled form of the plasmid cleavage in the nicked and linear forms. Moreover, P(NVP‐co‐TrpAMT)‐I exhibited stronger affinity for DNA than PVP and P(NVP‐co‐TrpAMT)‐II (PVP/PTrpAMT molar ratio of 100:6.35). This phenomenon is believed to be related to the structure of TrpAMT and the hydrophobic nature of the copolymer. P(NVP‐co‐TrpAMT)‐II with higher hydrophobic segments creates a strong hydrophobic interaction and induces a steric barrier which hinders their complexation with DNA. The study reveals important information about the inner mechanisms involved in the interaction of the PVP copolymer with DNA, which aids in the design of novel efficient non‐ionic gene carriers. Copyright © 2008 John Wiley & Sons, Ltd.     

The Catalytic Mechanism of the Class C Radical S‐Adenosylmethionine Methyltransferase NosN

S ‐Adenosylmethionine (SAM) is one of the most common co‐substrates in enzyme‐catalyzed methylation reactions. Most SAM‐dependent reactions proceed through an S_N2 mechanism, whereas a subset of them involves radical intermediates for methylating non‐nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM‐dependent methyltransferases, NosN does not produce S ‐adenosylhomocysteine (SAH) as a co‐product. Instead, NosN converts SAM into 5′‐methylthioadenosine as a direct methyl donor, employing a radical‐based mechanism for methylation and releasing 5′‐thioadenosine as a co‐product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme‐catalyzed methylation reactions.     

A Doubly Zwitterionic Antiaromatic [28]Hexaphyrin Formed upon Deprotonation of 5,20‐Di(N‐methyl‐4‐pyridinium)‐Substituted [28]Hexaphyrin

A rectangular 5,20‐di(4‐pyridyl) [26]hexaphyrin was reduced with NaBH₄ to give the corresponding twisted Möbius aromatic [28]hexaphyrin. Subsequent double N‐methylation gave a dicationic 5,20‐di(N‐methyl‐4‐pyridinium) [28]hexaphyrin, which was converted to a doubly zwitterionic and Hückel antiaromatic [28]hexaphyrin upon deprotonation with sodium methoxide.     

ESI‐MS/MS analysis of protonated N‐methyl amino acids and their immonium ions

Methylation is one of the important posttranslational modifications of biological systems. At the metabolite level, the methylation process is expected to convert bioactive compounds such as amino acids, fatty acids, lipids, sugars, and other organic acids into their methylated forms. A few of the methylated amino acids are identified and have been proved as potential biomarkers for several metabolic disorders by using mass spectrometry–based metabolomics workstation. As it is possible to encounter all the N‐methyl forms of the proteinogenic amino acids in plant/biological systems, it is essential to have analytical data of all N‐methyl amino acids for their detection and identification. In earlier studies, we have reported the ESI‐MS/MS data of all methylated proteinogenic amino acids, except that of mono‐N‐methyl amino acids. In this study, the N‐methyl amino acids of all the amino acids ( 1 ‐ 21 ; including one isomeric pair) were synthesized and characterized by ESI‐MS/MS, LC/MS/MS, and HRMS. These data could be useful for detection and identification of N‐methyl amino acids in biological systems for future metabolomics studies. The MS/MS spectra of [M + H]⁺ ions of most N‐methyl amino acids showed respective immonium ions by the loss of (H₂O, CO). The other most common product ions detected were [MH‐(NH₂CH₃]⁺, [MH‐(RH)]⁺ (where R = side chain group) ions, and the selective structure indicative product ions due to side chain and N‐methyl group. The isomeric/isobaric N‐methyl amino acids could easily be differentiated by their distinct MS/MS spectra. Further, the MS/MS of immonium ions inferred side chain structure and methyl group on α‐nitrogen of the N‐methyl amino acids.     

Selective Targeting of the KRAS Codon 12 Mutation Sequence by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Mutation of KRAS is a key step in many cancers. Mutations occur most frequently at codon 12, but the targeting of KRAS is notoriously difficult. We recently demonstrated selective reduction in the volume of tumors harboring the KRAS codon 12 mutation in a mouse model by using an alkylating hairpin N‐methylpyrrole–N‐methylimidazole polyamide seco‐1,2,9,9a‐tetrahydrocyclopropa[1,2‐c]benz[1,2‐e]indol‐4‐one conjugate (conjugate 4 ) designed to target the KRAS codon 12 mutation sequence. Herein, we have compared the alkylating activity of 4 against three other conjugates that were also designed to target the KRAS codon 12 mutation sequence. Conjugate 4 displayed greater affinity for the G12D mutation sequence than for the G12V sequence. A computer‐minimized model suggested that conjugate 4 could bind more efficiently to the G12D match sequence than to a one‐base‐pair mismatch sequence. Conjugate 4 was modified for next‐generation sequencing. Bind‐n‐Seq analysis supported the evidence showing that conjugate 4 could target the G12D mutation sequence with exceptionally high affinity and the G12V mutation sequence with much higher affinity than that for the wild‐type sequence.     

The Scaffold Design of Trivalent Chelator Heads Dictates Affinity and Stability for Labeling His‐tagged Proteins in vitro and in Cells

Small chemical/biological interaction pairs are at the forefront in tracing protein function and interaction at high signal‐to‐background ratios in cellular pathways. However, the optimal design of scaffold, linker, and chelator head still deserve systematic investigation to achieve the highest affinity and kinetic stability for in vitro and especially cellular applications. We report on a library of N‐nitrilotriacetic acid (NTA)‐based multivalent chelator heads (MCHs) built on linear, cyclic, and dendritic scaffolds and compare these with regard to their binding affinity and stability for the labeling of cellular His‐tagged proteins. Furthermore, we describe a new approach for tracing cellular target proteins at picomolar probe concentrations in cells. Finally, we outline fundamental differences between the MCH scaffolds and define a cyclic trisNTA chelator that displays the highest affinity and kinetic stability of all reported reversible, low‐molecular‐weight interaction pairs.     

Synthesis and NMR characteristics of N‐acetyl‐4‐nitro,N‐acetyl‐5‐nitro,N‐acetyl‐6‐nitro and N‐acetyl‐7‐nitrotryptophan methyl esters

N‐acetyl‐4‐nitrotryptophan methyl ester (2), N‐acetyl‐5‐nitrotryptophan methyl ester (3), N‐acetyl‐6‐nitrotryptophan methyl ester (4) and N‐acetyl‐7‐nitrotryptophan methyl ester (5) were synthesized through a modified malonic ester reaction of the appropriate nitrogramine analogs followed by methylation with BF₃‐methanol. Assignments of the ¹H and ¹³C NMR chemical shifts were made using a combination of ¹H–¹H COSY, ¹H–¹³C HETCOR and ¹H–¹³C selective INEPT experiments. Copyright © 2008 Crown in the right of Canada. Published by John Wiley & Sons, Ltd     

N‐Alkyl Ammonium Resorcinarene Salts as High‐Affinity Tetravalent Chloride Receptors

N‐Alkyl ammonium resorcinarene salts (NARYs, Y=triflate, picrate, nitrate, trifluoroacetates and NARBr) as tetravalent receptors, are shown to have a strong affinity for chlorides. The high affinity for chlorides was confirmed from a multitude of exchange experiments in solution (NMR and UV/Vis), gas phase (mass spectrometry), and solid‐state (X‐ray crystallography). A new tetra‐iodide resorcinarene salt (NARI) was isolated and fully characterized from exchange experiments in the solid‐state. Competition experiments with a known monovalent bis‐urea receptor ( 5 ) with strong affinity for chloride, reveals these receptors to have a much higher affinity for the first two chlorides, a similar affinity as 5 for the third chloride, and lower affinity for the fourth chloride. The receptors affinity toward chloride follows the trend K₁?K₂?K₃≈ 5 >K₄, with K_a=5011 m ^?1 for 5 in 9:1 CDCl₃/[D₆]DMSO.     

Recent advances in methods for the analysis of protein o‐glycosylation at proteome level

O‐Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post‐translational modifications. Compared with N‐linked glycosylation, O‐glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O‐glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O‐glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O‐glycosylation, i.e. O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O‐glycosylation. For the large‐scale analysis of mucin‐type glycosylation, the glycan simplification strategies including the ‘‘SimpleCell’’ technology were introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation.     

Halide‐Anion Binding by Singly and Doubly N‐Confused Porphyrins

The halide‐binding properties of N‐confused porphyrin (NCP, 1 ) and doubly N‐confused porphyrins (trans‐N₂CP ( 2 ), cis‐N₂CP ( 3 )) were examined in CH₂Cl₂. In the free‐base forms, cis‐N₂CP ( 3 ) showed the highest affinity to each anion (Cl^?, Br^?, I^?) with association constants K_a=7.8×10³, 1.9×10³, and 5.8×10² M ^?1, respectively. As metal complexes, on the other hand, trans‐N₂CP 2–Cu exhibited the highest affinity to Cl^?, Br^?, and I^? with K_a=9.0×10⁴, 2.7×10⁴, and 1.9×10³ M ^?1, respectively. The corresponding K_a values for cis‐N₂CP 3–Cu and NCP 1–Cu were about 1/10 and 1/2, respectively, of those of 2–Cu . With the help of density functional theory (DFT) calculations and complementary affinity measurements of a series of trisubstituted N‐confused porphyrins, the efficient anion binding of NCPs was attributed to strong hydrogen bonding at the highly polarized NH moieties owing to the electron‐deficient C₆F₅ groups at meso positions as well as the ideally oriented dipole moments and large molecular polarizability. The orientation and magnitude of the dipole moments in NCPs were suggested to be important factors in the differentiation of the affinity for anions.     

The Impact of Amino Acid Side Chain Mutations in Conformational Design of Peptides and Proteins

Local energetic effects of amino acid replacements are often considered to have only a moderate influence on the backbone conformation of proteins or peptides. As these effects are difficult to determine experimentally, no comparison has yet been performed. However, knowledge of the influence of side chain mutations is essential in protein homology modeling and in optimizing biologically active peptide ligands in medicinal chemistry. Furthermore, the tool of N‐methylation of peptides is of increasing importance for the design of peptidic drugs to gain oral availability or receptor selectivity. However, N‐methylation is often accompanied by considerable population of cis‐peptide bond structures, resulting in completely different conformations compared with the parent peptide. To retain a favored structure, it might be important to understand the effect of different side chains on the backbone conformation and to enable the introduction of an N‐methylation at the right position without disturbing a biologically active conformation. In order to detect even small energetic effects due to side chain mutations, we employed a trick to investigate the structural equilibrium of a selected cyclic pentapeptide in which two conformations are equally populated. Very small energetic differences between both conformations could easily be determined experimentally by identifying shifts in the population of both isomers.     

Synthesis of 4‐Arylpiperazine Derivatives of Moclobemide: Potential Antidepressants with a Dual Mode of Action

Abstract

We report here the synthesis of substituted 4‐chloro‐N‐[3‐oxo‐3‐(4‐aryl‐1‐piperazinyl)‐propyl] benzamides (5–9), as potential new antidepressants, incorporating in a single molecule structural moieties related to a dual pharmacological profile: MAO‐A inhibitor and 5‐HT_1A receptor affinity.     

Effect of methylation on relative energies of tautomers and on the intramolecular proton transfer barriers of protonated nitrosamine: A MR‐CISD study

MR‐CISD, MR‐CISD+Q, and MR‐AQCC calculations have been performed on the minima and transition states (corresponding to intramolecular proton transfer between the protonation sites) of the ground state of protonated nitrosamine and N,N‐dimethylnitrosamine. Our highest level results (MR‐AQCC/cc‐pVTZ) for the smaller system indicate that protonation on the N amino ( 2a ) is practically as favorable as the most favorable protonation on the O atom ( 1a ). They also suggest that protonation on the nitroso N atom ( 2c ) is ～14.5 kcal/mol less favorable than 1a . Results obtained at the MR‐CISD+Q/cc‐pVTZ level indicate that the effect of methylation on the relative energies of the tautomers is, in order of importance, 2a > 2c and increases their energies by ～17.5 and 4.8 kcal/mol, respectively. They also indicate that methylation alters significantly the intramolecular proton transfer barriers. The largest differences between the common geometric parameters of both systems have been found for 2a . © 2015 Wiley Periodicals, Inc.