A Mets motif peptide found in copper transport proteins selectively binds Cu(I) with methionine-only coordination

Mets motifs, which refer to methionine-rich sequences found in the high-affinity copper transporter Ctr1, also appear in other proteins involved in copper trafficking and homeostasis, including other Ctrs as well as Pco and Cop proteins isolated from copper-resistant bacteria. To understand the coordination chemistry utilized by these proteins, we studied the copper binding properties of a peptide labeled Mets7-PcoC with the sequence Met-Thr-Gly-Met-Lys-Gly-Met-Ser. By comparing this sequence to a series of mutants containing noncoordinating norleucine in place of methionine, we confirm that all three methionine residues are involved in a thioether-only binding site that is selective for Cu(I). Two independent methods, one based on mass spectrometry and one based on rate differences for the copper-catalyzed oxidation of ascorbic acid, provide an effective K(D) of approximately 2.5 microM at pH 4.5 for the 1:1 complex of Mets7-PcoC with Cu(I). These results establish that a relatively simple peptide containing an MX(2)MX(2)M motif is sufficient to bind Cu(I) with an affinity that corresponds well with its proposed biological function of extracellular copper acquisition.     

Suzuki-Miyaura cross-coupling of aryl and alkyl halides using palladium/imidazolium salt protocols

A simple new protocol for the high yielding Suzuki-Miyaura cross-couplings of aryl chlorides with aryl boronic acids using a palladium/imidazolium salt catalytic system is presented. The first examples of a palladium/imidazolium salt protocol for sp³-sp³ Suzuki-Miyaura couplings of alkyl halides are also disclosed.     

Nonlinear decrease of background fluorescence in polymer thin-films - a survey of materials and how they can complicate fluorescence detection in microTAS

Polymers and plastics are receiving increased attention as materials for microfluidics and microTAS applications. Given the ubiquity of fluorescence detection techniques in micro-analytical systems, the fluorescence properties of polymers and plastics should not be overlooked. We survey some commonly available polymer thin-films for their fluorescence behaviour under standardized conditions to determine which materials are most suitable for high-sensitivity fluorescence detection lab chips. The initial fluorescence intensities of some of the materials surveyed were significantly higher than glass and fused silica controls, and decreased over the three hour period with complex kinetics. We then discuss how this has confounded fluorescence detection in our analytical context, and possible mechanisms for the decrease.     

Quinazolines. VII. Synthesis of 1,3-Diamino-5,6-dihydrobenzo[f]quinazolines

Eighteen new 1,3-diamino-5,6-dihydrobenzo[f] quinazolines ( 6 , R. = alkyl, Cl, MeO) were synthesized via the condensation of appropriate 2-tetralones with cyanoguanidine under fusion conditions. Methods were developed for the preparation of a number of heretofore undescribed 2-tetralones as precursors. The final products can be viewed as conformationally rigid analogs of pyrimethamine ( 2 ), and are of interest as inhibitors of dihydrofolate reductase and as potential antimalarial and antitumor agents.     

Synthesis of the ABC ring system of manzamine A

A synthesis of the core ABC ring system of the manzamine alkaloids is described, starting from arecoline. The key steps involve a Claisen rearrangement to set up a 4-substituted-3-methylenepiperidine and a stereoselective azomethine ylide dipolar cycloaddition reaction. Condensation of the aldehyde 6 and sarcosine ethyl ester hydrochloride salt gives an intermediate azomethine ylide, which undergoes an intramolecular cycloaddition reaction to set up two new rings and three new chiral centers stereoselectively. The aldehyde 6 was not a suitable substrate for related azomethine ylide cycloaddition reactions with other amines. However, the related dimethyl acetal 26 could be condensed with a variety of amines to give the desired tricyclic products. The cycloaddition reaction with N-methyl or N-allyl glycine ethyl ester gave almost exclusively the exo adduct, whereas cycloaddition with glycine ethyl ester gave the endo adduct.     

Nucleophilic Addition of CH, NH, and OH Bonds to the Phosphadiazonium Cation and Interpretation of (31)P Chemical Shifts at Dicoordinate Phosphorus Centers

The phosphadiazonium cation [MesNP](+) reacts quantitatively with the fluorenylide anion, MesNH(2), and MesOH (Mes = 2,4,6-tri-tert-butylphenyl), resulting in formal insertion of the N-P moiety into the H-Y (Y = C, N, O) bonds. Specifically, reaction of MesNPCl with fluorenyllithium gives the aminofluorenylidenephosphine [crystal data: C(31)H(38)NP, monoclinic, P2(1)/c, a = 9.568(8) ?, b = 24.25(2) ?, c = 11.77(1) ?, beta = 101.38(8) degrees, Z = 4]. Similarly, reaction of [MesNP][GaCl(4)] with MesNH(2) gives the diaminophosphenium salt [MesN(H)PN(H)Mes][GaCl(4)] [crystal data: C(36)H(60)Cl(4)GaN(2)P, monoclinic, C2/c, a = 24.921(2) ?, b = 10.198(4) ?, c = 16.445(2) ?, beta = 93.32(1) degrees, Z = 4], and reaction with MesOH gives the first example of an aminooxyphosphenium salt [MesN(H)POMes][GaCl(4)]. It is proposed that the reactions involve nucleophilic attack at phosphorus followed by a 1,3-hydrogen migration from Y to N. Experimental evidence for the formation of sigma-complex intermediates is provided by the isolation of [MesNP-PPh(3)][SO(3)CF(3)] [crystal data: C(37)H(44)F(3)NO(3)P(2)S, triclinic, P&onemacr;, a = 10.663(1) ?, b = 19.439(1) ?, c = 10.502(1) ?, alpha = 103.100(7) degrees, beta = 113.311(7) degrees, gamma = 93.401(7) degrees, Z = 2]. As part of the unequivocal characterization of the aminooxyphosphenium salt, detailed solid-state (31)P NMR studies and GIAO calculations on the phosphenium cations have been performed. Contrary to popular belief, the phosphorus shielding in dicoordinate cations is not caused by the positive charge but results from efficient mixing between the phosphorus lone pair and pi orbitals.     

A novel oxygen and/or carbon dioxide-sensitive optical transducer

A novel oxygen (O(2)) and/or carbon dioxide (CO(2))-sensitive transducer for the measurement of both gaseous O(2) and CO(2) over the concentration ranges of O(2), 0-100% and CO(2), 0-10% has been described employing a solution of 10.6 muM fluorescein (FL) and 190 muM potassium hydroxide in a solvent mixtures of 1:1 (v/v) N,N'-diethylaniline (DEA) and N,N-dimethylformamide. Increasing O(2) concentrations cause the absorbance of the solution at a wavelength of 400 nm to increase owing to a contact charge transfer reaction existing between O(2) and DEA molecules, and increasing CO(2) concentrations produce a non-linear fall in absorbance at 520 nm as the colour of FL changes from its orange dianion form to the colourless neutral, lactonic form. Both processes are independent of each other and reversible. The response to changes in O(2) concentrations is in good agreement with Beer-Lambert's law and the response to changes in CO(2) concentrations in non-linear. A fibre optic sensing system based on this solvent-dye solution has been set up for continuous and reversible determination of both gaseous O(2) and CO(2). Possible applications include environmental and physiological monitoring of O(2) over the ranges of 0-100% and CO(2), 0-10%.     

Eliminating positively charged lysine epsilon-NH3+ groups on the surface of carbonic anhydrase has no significant influence on its folding from sodium dodecyl sulfate

This study compares the folding of two polypeptides--bovine carbonic anhydrase (BCA) and peracetylated BCA (BCA-Ac(18))--having the same sequence of amino acids but differing by 18 formal units of charge, from a solution containing denaturing concentrations of sodium dodecyl sulfate (SDS). Acetylation of BCA with acetic anhydride converts all 18 lysine-epsilon-NH(3)(+) groups to lysine-epsilon-NHCOCH(3) groups and generates BCA-Ac(18). Both BCA and BCA-Ac(18) are catalytically active, and circular dichroism spectroscopy (CD) suggests that they have similar secondary and tertiary structures. SDS at concentrations above approximately 10 mM denatured both proteins. When the SDS was removed by dialysis, both proteins were regenerated in native form. This study suggests that large differences in the net charge of the polypeptide have no significant influence on the structure, the ability to refold, or the rate of refolding of this protein from solutions containing SDS. This study reinforces the idea that charged residues on the surface of BCA do not guide protein folding and raises the broader question of why proteins have charged residues on their surface, outside of the region of the active site.     

Uranium analysis in urine by inductively coupled plasma dynamic reaction cell mass spectrometry

Urine uranium concentrations are the best biological indicator for identifying exposure to depleted uranium (DU). Internal exposure to DU causes an increased amount of urine uranium and a decreased ratio of ²³⁵U/²³⁸U in urine samples, resulting in measurements that vary between 0.00725 and 0.002 (i.e., natural and depleted uraniums ²³⁵U/²³⁸U ratios, respectively). A method based on inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS) was utilized to identify DU in urine by measuring the quantity of total U and the ²³⁵U/²³⁸U ratio. The quantitative analysis was achieved using ²³³U as an internal standard. The analysis was performed both with and without the reaction gas oxygen. The reaction gas converted ionized ²³⁵U⁺ and ²³⁸U⁺ into ²³⁵UO₂⁺ (m/z=267) and ²³⁸UO₂⁺ (m/z=270). This conversion was determined to be over 90% efficient. A polyatomic interference at m/z 234.8 was successfully removed from the ²³⁵U signal under either DRC operating conditions (with or without oxygen as a reaction gas). The method was validated with 15 urine samples of known uranium compositions. The method detection limit for quantification was determined to be 0.1 pg U mL^–1 urine and an average coefficient of variation (CV) of 1–2% within the sample measurements. The method detection limit for determining ²³⁵U/²³⁸U ratio was 3.0 pg U mL^–1 urine. An additional 21 patient samples were analyzed with no information about medical history. The measured ²³⁵U/²³⁸U ratio within the urine samples correctly identified the presence or absence of internal DU exposure in all 21 patients.The opinions and assertions expressed herein are those of the authors and are not to be construed as official or as representing the views of the Armed Forces Institute of Pathology, the Department of the Army, or the Department of Defense     

Magic angle spinning29Si and27Al NMR study of mordenite dealumination

Magic angle spinning²⁹Si NMR presents a rapid qualitative method of assessing the degree of dealumination of sodium mordenite modified by acid leaching and heat treatment. A quantitative determination of the²⁹Si coordination sphere is hampered by the overlap of chemical shift ranges for Si?O?Al and Si?O?H species. MAS²⁷Al NMR indicates the presence of octahedrally coordinated Al in interstitial sites in all treated samples. On treating mordenite samples at high temperatures (～700°C) much of the Al becomes invisible to the NMR experiment because of its location in sites of low symmetry and large quadrupole coupling constant.