Absolute Quantification of Noncoding RNA by Microscale Thermophoresis

Accurate quantification of the copy numbers of noncoding RNA has recently emerged as an urgent problem, with impact on fields such as RNA modification research, tissue differentiation, and others. Herein, we present a hybridization‐based approach that uses microscale thermophoresis (MST) as a very fast and highly precise readout to quantify, for example, single tRNA species with a turnaround time of about one hour. We developed MST to quantify the effect of tRNA toxins and of heat stress and RNA modification on single tRNA species. A comparative analysis also revealed significant differences to RNA‐Seq‐based quantification approaches, strongly suggesting a bias due to tRNA modifications in the latter. Further applications include the quantification of rRNA as well as of polyA levels in cellular RNA.     

Generation of Stoichiometric Ethylene and Isotopic Derivatives and Application in Transition‐Metal‐Catalyzed Vinylation and Enyne Metathesis

Ethylene is one of the most important building blocks in industry for the production of polymers and commodity chemicals. ¹³C‐ and D‐isotope‐labeled ethylenes are also valuable reagents with applications ranging from polymer‐structure determination, reaction‐mechanism elucidation to the preparation of more complex isotopically labeled compounds. However, these isotopic derivatives are expensive, and are flammable gases, which are difficult to handle. We have developed a method for the controlled generation of ethylene and its isotopic variants including, for the first time, fully isotopically labeled ethylene, from simple alkene precursors by using Ru catalysis. Applying a two‐chamber reactor allows both the synthesis of ethylene and its immediate consumption in a chemical transformation permitting reactions to be performed with only stoichiometric amounts of this two carbon olefin. This was demonstrated in the Ni‐catalyzed Heck reaction with aryl triflates and benzyl chlorides, as well as Ru‐mediated enyne metathesis.     

Synthesis of a New Series of N‐Mannich Bases and Polyhydroxy Mannich Bases of Pharmaceutical Interest Related to Isatin and Its Schiff Bases

The reaction of isatin 1 with benzaldehyde and a sec‐amine or the appropriate aldimine afforded the N‐Mannich bases 2 – 3 and the bis‐base 4 . Treatment of 1 with glutaric dialdehyde and morpholine gave the bis‐base 5 . Mannich reaction of the Schiff bases 6a – f derived from 1 , led to the new Mannich bases and bis‐bases 7 – 9 . The use of N‐methyl‐D‐glucamine as the amine component in the Mannich reaction with 6b – f led to the polyhydroxy Mannich bases 11 – 13 .     

Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC–MS/MS

A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography–MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-¹³C₂,¹⁵N) was established using H₂O₂ as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25–30 % (coverage factor?=?2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.     

Polymeric Schiff Bases. III. Some Prototype Exchange Reactions of Schiff Bases

The amine moiety in Schiff bases can be exchanged quantitatively by another amine to yield new Schiff bases if the volatility of the replacing amine is lower than the derived amine, thereby allowing the latter to distilled from the reaction mass. This amine exchange was shown to be quantitative also for diamines and di-Schiff bases. Similarly, quantitative conversions were found for aldehydes and acetal exchanges with Schiff bases for both monofunctional and difunctional reactants. The bis exchange, involving two complementary Schiff bases, was quantitative also when the reactants were so selected that one of the new derived Schiff bases could be removed by distillation. The bis exchange was demonstrated with mono and di-Schiff bases.

Mechanisms are suggested for these Schiff base exchange reactions: attempts to isolate the proposed intermediates physically were unsuccessful; however spectroscopic evidence indicates the formation of intermediate compounds.

The Schiff base exchanges involving polyfunctional reactants are of interest in the synthesis of polymers.     

SYNTHESIS AND DETERMINATION OF THE BIOLOGICAL ACTIVITIES OF YEAST ALANINE tRNA ANALOGUES

The role of base modification in yeast tRNAAl(?) function in protein synthesis was examined by the use of unmodified tRNA analogues. Unmodified full length tRNAs, 5'-half tRNAs (nucleotides 1-35) and 3'-half tRNAs (nucleotides 37-75) were transcribed in vitro using T7-RNA polymerase. Unmodified tRNA half molecules were joined to normally modified half molecules (5'-half, nucleotides 1-36; 3'-half, nucleotides 36-75) by T4-RNA ligase. Using this method, we synthesized three analogues of yeast tRNAAl(?): (i) tRNAAl(?) which lacks base modifications in the 5'-half of the molecule; (ii) tRNAAl(?) which lacks base modifications in the 3'-half of the molecule; and (iii) tRNAAla completely lacking base modifications. We determined the biological activities of these analogues. In rat aminoacyl-tRNA synthetase reactions, the alanine acceptance activity decreased by 52%, 79% and 85% when modified bases were absent from the 5'-half molecule, the 3'-half molecule or the total molecule, respectively. In rabbit retic     

High resolution mass spectrometry in molecular structure studies—VI: The fragmentation of amaryllis alkaloids in the crinine series. Compounds bearing a hydroxyl substituent at C-11 (and some 11-oxo derivatives)

High resolution mass spectral fragmentation patterns of Amaryllidaceae alkaloids in the crinine series bearing a substituent at C-11 have been investigated, as well as certain isotopically labeled derivatives. The substituent at C-11 governs almost entirely the fragmentation pattern of these bases due to the ability of the hydroxylic hydrogen atom to rearrange. In contrast to the bases lacking this hydrozyl substituent, stereochemistry exerts a profound influence on the fragmentation.     

Synthesis of [1-C] and stereo-specifically [1-H] labeled fluorinated substrate analogues of IspH enzyme in the deoxyxylulose phosphate pathway

IspH in the deoxyxylulose phosphate (DXP) pathway catalyzes the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are the starting materials for the synthesis of thousands of isoprenoids. Several models have been proposed in the literature to account for this unique transformation, and most of them involve the formation of an allylic radical intermediate. To facilitate trapping and characterizing the proposed intermediates in the IspH-catalyzed reactions, in the present work, we report the synthesis of four isotopically labeled IspH substrate analogues. These isotopically labeled mechanistic probes will be utilized in the future for characterizing the proposed IspH reaction intermediates by the combination of bioorganic and biophysical approaches.     

A Convenient Method of Synthesis of Bis-Indolylmethanes: Indium Trichloride Catalyzed Reactions of Indole with Aldehydes and Schiff's Bases

Anhydrous indium trichloride (InCl₃) is found to catalyze the electrophilic substitution reaction of indole with aromatic aldehydes and Schiff's bases which resulted in an efficient synthesis of bis-indolylmethanes in good to excellent yield.     

Green and Catalyst-Free One-Pot Synthesis of Anthranilamide Schiff Bases: An Approach Toward Sirtinol

A novel and simple method for the green one-pot synthesis of anthranilamide Schiff bases is described. The reported Schiff bases are obtained via the reaction of isatoic anhydride, amines, and aromatic aldehydes in water at room temperature, without using any catalysts. No cyclization toward 2,3-dihydro-4(1H)-quinazolinones occurred in this method and anthranilamide Schiff bases were produced exclusively. This approach offers a green method to prepare the medicinally important Schiff base sirtinol and other bioactive anthranilamide Schiff bases.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]     

Selective Enzymatic Demethylation of N2,N2‐Dimethylguanosine in RNA and Its Application in High‐Throughput tRNA Sequencing

The abundant Watson–Crick face methylations in biological RNAs such as N¹‐methyladenosine (m¹A), N¹‐methylguanosine (m¹G), N³‐methylcytosine (m³C), and N²,N²‐dimethylguanosine (m²₂G) cause significant obstacles for high‐throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild‐type E. coli AlkB and its D135S mutant can remove most of m¹A, m¹G, m³C modifications in transfer RNA (tRNA), but they work poorly on m²₂G. Here we report the design and evaluation of a series of AlkB mutants against m²₂G‐containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m²₂G modification to N²‐methylguanosine (m²G). We also show that this new enzyme improves the efficiency of tRNA sequencing.     

Synthesis and Reactivity of Mannich Bases. XVIII. Reaction of Dithiocarbamic Acid Salts With Mannich Bases Derived from Ortho-Hydroxyacetophenones

The reaction of Mannich bases derived from ortho-hydroxyacetophenones with N,N-dialkyldithiocarbamic acid salts result in the formation of the corresponding dithiocarbamic acid esters via an amine moiety replacement when the process is conducted in cold water. Attempts to carry out the synthesis in refluxing ethanol-water mixture led to the insertion of carbon sulfide at the C—N bond in Mannich bases. N-Aryldithiocarbamic acid salts afforded on reaction with the above-mentioned Mannich bases only bis-(2-(2-hydroxybenzoyl)ethyl)thioethers.     

Synthesis of Mono- and Di-Deuterated (2S, 3S)-3-Methylaspartic Acids to Facilitate Measurement of Intrinsic Kinetic Isotope Effects in Enzymes

Kinetic isotope effects provide a powerful method to investigate the mechanisms of enzyme-catalyzed reactions, but often other slow steps in the reaction such as substrate binding or product release suppress the isotopically sensitive step. For reactions at methyl groups, this limitation may be overcomed by measuring the isotope effect by an intra-molecular competition experiment. This requires the synthesis of substrates containing regio-specifically mono- or di-deuterated methyl groups. To facilitate the mechanistic investigations of the adenosylcobalamin-dependent enzyme, glutamate mutase, we have developed a synthesis of mono- and di-deuterated (2S,3S)-3-methylaspartic acids. Key intermediates are the correspondingly labeled mesaconic acids and their dimethyl esters that potentially provide starting materials for a variety of isotopically labeled molecules.     

Nitrogen Heterocyclic Bases as Catalysts for the Selective Synthesis of Symmetric P,P′-Disubstituted Bisphosphonate Esters

Abstract

Nitrogen heterocyclic bases were investigated as potential catalysts for the selective synthesis of symmetric P,P′-disubstituted partial esters of methylenebisphosphonic acid. The size, presence of oxygen or sulfur donor atoms as well as position and number of nitrogen atoms in the ring were varied systematically to determine the effect of these structural modifications on catalyst selectivity. 1-Methylimidazole and 1H-1,2,3-triazole, which yield exclusively the desired diester product, were shown viable alternatives to 1H-tetrazole for catalyzing the selective coupling of methylenebis- (phosphonic dichloride) with 2 equiv. of alcohol.     

Time-resolved events on the reaction pathway of transcript initiation by a single-subunit RNA polymerase: Raman crystallographic evidence

The nucleotidyl transfer reaction leading to formation of the first phosphodiester bond has been followed in real time by Raman microscopy, as it proceeds in single crystals of the N4 phage virion RNA polymerase (RNAP). The reaction is initiated by soaking nucleoside triphosphate (NTP) substrates and divalent cations into the RNAP and promoter DNA complex crystal, where the phosphodiester bond formation is completed in about 40 min. This slow reaction allowed us to monitor the changes of the RNAP and DNA conformations as well as bindings of substrate and metal through Raman spectra taken every 5 min. Recently published snapshot X-ray crystal structures along the same reaction pathway assisted the spectroscopic assignments of changes in the enzyme and DNA, while isotopically labeled NTP substrates allowed differentiation of the Raman spectra of bases in substrates and DNA. We observed that substrates are bound at 2-7 min after soaking is commenced, the O-helix completes its conformational change, and binding of both divalent metals required for catalysis in the active site changes the conformation of the ribose triphosphate at position +1. These are followed by a slower decrease of NTP triphosphate groups due to phosphodiester bond formation that reaches completion at about 15 min and even slower complete release of the divalent metals at about 40 min. We have also shown that the O-helix movement can be driven by substrate binding only. The kinetics of the in crystallo nucleotidyl transfer reaction revealed in this study suggest that soaking the substrate and metal into the RNAP-DNA complex crystal for a few minutes generates novel and uncharacterized intermediates for future X-ray and spectroscopic analysis.     

A Vastly Increased Chemical Variety of RNA Modifications Containing a Thioacetal Structure

Recently discovered new chemical entities in RNA modifications have involved surprising functional groups that enlarge the chemical space of RNA. Using LC‐MS, we found over 100 signals of RNA constituents that contained a ribose moiety in tRNAs from E. coli. Feeding experiments with variegated stable isotope labeled compounds identified 37 compounds that are new structures of RNA modifications. One structure was elucidated by deuterium exchange and high‐resolution mass spectrometry. The structure of msms²i⁶A (2‐methylthiomethylenethio‐N6‐isopentenyl‐adenosine) was confirmed by methione‐D3 feeding experiments and by synthesis of the nucleobase. The msms²i⁶A contains a thioacetal, shown in vitro to be biosynthetically derived from ms²i⁶A by the radical‐SAM enzyme MiaB. This enzyme performs thiomethylation, forming ms²i⁶A from i⁶A in a first turnover. The new thioacetal is formed by a second turnover. Along with the pool of 36 new modifications, this work describes a new layer of RNA modification chemistry.     

Utility of Ketonic Mannich Bases in Synthesis of Some New Functionalized 2‐Pyrazolines

The styryl ketonic Mannich base 2 has been used as a precursor in the synthesis of 2‐pyrazolines having a basic side chain at C‐3 and a phenolic Mannich base at C‐5. Treatment of the bis(styryl ketonic bases) 6a and 8a with phenylhydrazine affords the bis(3‐functionalized 2‐pyrazolines) 7 and 9 . The transamination between the styryl keto base 10 and 4‐aminoantipyrine leads to 12 , which reacts with piperazine to give 13 . N‐Nitrosation of the sec‐Mannich bases 15a – d followed by reductive cyclization affords 2‐pyrazolines 17a – d . The keto base 14b has been used for the synthesis of 2‐pyrazolines having a phenolic Mannich base at C‐3 and its reaction with 3,5‐dimethyl‐1H‐pyrazole affords 23 . The alkylation of 3‐methyl‐1‐phenyl‐2‐pyrazolin‐5‐one with the bis(Mannich base) 25 was investigated.     

Polymeric Schiff Bases. VI. The Direct Syntheses of Poly-Schiff Bases

The synthesis of black, high molecular weight polymeric Schiff bases directly from aryldiamines and aryldialdehydes by two methods is described. The first method utilizes benzylideneaniline as the reaction medium. The second method is a direct one-step synthesis involving the reaction of the aryldiamine, aryldialdehyde, aniline, and benzaldehyde. The thermogravimetric analyses of the polymers thus prepared compared favorably with similar polymers prepared by the bis-Schiff base exchange method.