Matrix-assisted laser desorption/ionization mass spectrometry screening for pseudouridine in mixtures of small RNAs by chemical derivatization, RNase digestion and signature products

We have developed a method to screen for pseudouridines in complex mixtures of small RNAs using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). First, the unfractionated crude mixture of tRNAs is digested to completion with an endoribonuclease, such as RNase T1, and the digestion products are examined using MALDI-MS. Individual RNAs are identified by their signature digestion products, which arise through the detection of unique mass values after nuclease digestion. Next, the endonuclease digest is derivatized using N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), which selectively modifies all pseudouridine, thiouridine and 2-methylthio-6-isopentenyladenosine nucleosides. MALDI-MS determination of the CMCT-derivatized endonuclease digest reveals the presence of pseudouridine through a 252 Da mass increase over the underivatized digest. Proof-of-concept experiments were conducted using a mixture of Escherichia coli transfer RNAs and endoribonucleases T1 and A. More than 80% of the expected pseudouridines from this mixture were detected using this screening approach, even on an unfractionated sample of tRNAs. This approach should be particularly useful in the identification of putative pseudouridine synthases through detection of their target RNAs and can provide insight into specific small RNAs that may contain pseudouridine.     

Synthesis, characterization and crystal structure determination of zinc (II) and mercury (II) complexes with 2,2′-dimethyl-4,4′-bithiazole

The 2,2′-dimethyl-4,4′-bithiazole ligand (1), (dm4bt), and its Zn and Hg complexes have been prepared. A conformational property calculation at the DFT level for the ligand shows the anti conformation is energetically more stable by about 22.83 kJ/mol and the rotational barrier is about 32.01 kJ/mol for the anti → syn conversion, a phenomena happening during complex formation. The complexes [Zn(dm4bt)Cl₂] (2) and [Hg(dm4bt)Cl₂] (3) have spectral properties typical for d¹⁰ metal diimine systems. The structures of the ligand and the two complexes have been determined by the single crystal diffraction method. The X-ray structure determinations show that both complexes are four coordinated by two chloride atoms and one bidentate dm4bt. In the Hg complex one of the two chlorides is set at a semi-bridging position.     

The length of esterifying alcohol affects the aggregation properties of chlorosomal bacteriochlorophylls

Abstract Chlorosomes, the main light-harvesting complexes of green photosynthetic bacteria, contain bacteriochlorophyll (BChl) molecules in the form of self-assembling aggregates. To study the role of esterifying alcohols in BChl aggregation we have prepared a series of bacteriochlorophyllide c (BChlide c) derivatives differing in the length of the esterifying alcohol (C(1), C(4), C(8) and C(12)). Their aggregation behavior was studied both in polar (aqueous buffer) and nonpolar (hexane) environments and the esterifying alcohols were found to play an essential role. In aqueous buffer, hydrophobic interactions among esterifying alcohols drive BChlide c derivatives with longer chains into the formation of dimers, while this interaction is weak for BChlides with shorter esterifying alcohols and they remain mainly as monomers. All studied BChlide c derivatives form aggregates in hexane, but the process slows down with longer esterifying alcohols due to competing hydrophobic interactions with hexane molecules. In addition, the effect of the length of the solvent molecules (n-alkanes) was explored for BChl c aggregation. With an increasing length of n-alkane molecules, the hydrophobic interaction with the farnesyl chain becomes stronger, leading to a slower aggregation rate. The results show that the hydrophobic interaction is the driving force for the aggregation in an aqueous environment, while in nonpolar solvents it is the hydrophilic interaction.     

Conformational and self-assembly studies of helix forming hexapeptides containing two α-amino isobutyric acids

Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula Boc-Ile-Aib-Xx-Ile-Aib-Yy-OMe, where Xx and Yy are Leu in peptide I, Leu and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent conformations that can be described as mixed 3₁₀/α-helice with two 4→1 and two 5→1 intramolecular N-H?OC H-bonds. The peptides do not generate any helix-terminating Schellman motif despite having Aib at the penultimate position from C-terminus. In the crystalline state, the helices are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. The CD studies of the three hexapeptides in acetonitrile indicate that they are folded in well-developed 3₁₀-helical structures. NMR studies of peptide I in CDCl₃ also suggest the formation of a homogeneous 3₁₀-helical structure. The field emission scanning electron microscopic (FE-SEM) images of peptide II in the solid state reveal a non-twisted ribbon-like morphology, which is formed through lateral association of non-twisted filaments.     

Effect of pyrolysis temperature and operating temperature on the performance of nanoporous carbon membranes

Technology designed to capture and store carbon dioxide (CO₂) will play a significant role in the near-term reduction of CO₂ emissions and is considered necessary to slow global warming. Nanoporous carbon (NPC) membranes show promise as a new generation of gas separation membranes suitable for CO₂ capture.We have made supported NPC membranes from polyfurfuryl alcohol (PFA) at various pyrolysis temperatures. Positron annihilation lifetime spectrometry (PALS) and wide angle X-ray diffraction (WAXD) results indicate that the pore size decreases whilst the porosity increases with increasing pyrolysis temperature. The membrane performance results support these findings with a significant increase in permeance being seen with increasing pyrolysis temperature, which relates to the increase in porosity.Mixed gas performance measurements also show an increase in CH₄ permeance as the operating temperature is increased from 35 to 200 °C, which can be related to an increase in the rate of diffusion. However, the selectivity decreases with increasing operating temperature due to the smaller changes in the CO₂ permeance. These smaller changes in CO₂ permeance can be related to the stronger adsorption of this gas on the carbon surface at lower operating temperatures. Interestingly, regardless of the original pyrolysis temperature, the selectivity at higher operating temperatures is similar, whereas the permeance remains related to this pyrolysis temperature.     

Investigation of the synthetic and mechanistic aspects of the highly stereoselective transformation of alpha-thioamides to alpha-thio-beta-chloroacrylamides

Treatment of a series of alpha-thioamides with N-chlorosuccinimide results in efficient transformation to the analogous alpha-thio-beta-chloroacrylamides. The mechanistic pathway has been established through isolation and characterisation of intermediate compounds. The scope of the transformation has been explored-aryl and alkylthio substituents, primary, secondary and tertiary amides can be employed. In most instances, the chloroacrylamides are formed exclusively as the Z-stereoisomer; however, with tertiary propanamides or with amides derived from butanoic or pentanoic acid a mixture of E- and Z-stereoisomers is formed.     

Ruthenium(III)‐mediated oxidation of D‐mannitol by cerium(IV) in aqueous sulfuric acid medium: A kinetic and mechanistic approach

The oxidation of D ‐mannitol by cerium(IV) has been studied spectrophotometrically in aqueous sulfuric acid medium at 25°C at constant ionic strength of 1.60 mol dm^?3. A microamount of ruthenium(III) (10^?6 mol dm^?3) is sufficient to enhance the slow reaction between D ‐mannitol and cerium(IV). The oxidation products were identified by spot test, IR and GC‐MS spectra. The stoichiometry is 1:4, i.e., [D ‐mannitol]: [Ce(IV)] = 1:4. The reaction is first order in both cerium(IV) and ruthenium(III) concentrations. The order with respect to D ‐mannitol concentration varies from first order to zero order as the D ‐mannitol concentration increases. Increase in the sulfuric acid concentration decreases the reaction rate. The added sulfate and bisulfate decreases the rate of reaction. The active species of oxidant and catalyst are Ce(SO₄)₂ and [Ru(H₂O)₆]³⁺, respectively. A possible mechanism is proposed. The activation parameters are determined with respect to a slow step and reaction constants involved have been determined. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 440–452, 2010     

Diastereoselective sulfur oxidation of 2-thio-3-chloroacrylamides

Diastereoselective sulfur oxidation in 2-thio-3-chloroacrylamides is described. A range of chiral amine auxiliaries was incorporated in the β-chloroacrylamide, and the efficiency with which the stereochemistry was relayed to the sulfur centre during sulfoxidation was investigated. Diastereomeric ratios of up to 3.3:1 were achieved.