End-labeled free-solution electrophoresis of DNA

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.     

Applying ZEKE-spectroscopy to clusters

In this paper we describe the application of a special form of electron photodetachment spectroscopy, zero electron kinetic energy (ZEKE) spectroscopy, to the study of cold metal cluster anions. From this study we have obtained vibrational and thus structural information of several neutral and charged clusters, e.g. Au ₂ ^?/0 , Ag ₃ ^?/0 , Sn ₂ ^?/0 and Al ₃ ^?/0 . In addition, in separate measurements of the relative photodetachment cross section of Au ₂ ^? , we have discovered sharp resonances which are due to electronic autodetachment from a short lived excited state.     

Quantitative analysis of mutagenic heterocyclic aromatic amines in cooked meat using liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry

Five mutagenic heterocyclic aromatic amines (HAAs) were quantified from meat extracts, and grilled and pan fried bacon samples using stable isotopically labeled internal standards. These compounds were isolated from the matrices by a tandem solid-phase extraction procedure, followed by separation on reversed-phase liquid chromatography (HPLC) and quantified by atmospheric pressure chemical ionization tandem mass spectrometry (APCIMS-MS). Tandem mass spectrometry (MS-MS) acquisition was done in selected reaction monitoring (SRM) mode to provide a high degree of sensitivity and selectivity for accurate quantification of HAAs. The detection and quantification limits of these HAAs approached 0.015 and 0.045 microg/kg (part-per-billion), respectively, with only 4 g of meat. The HAA levels ranged widely from 0.045 to 45.500 microg/kg, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was the predominant HAA found in these samples. The amount of HAAs formed was highly dependent upon the type of meat and method of preparation. An intralaboratory comparison of the extraction procedure showed that estimates of these HAAs obtained by three different individuals at HAA levels below 2 microg/kg were within 5% with coefficients of variation below 19%, indicating the robustness of the analytical method. Moreover, because all of these HAAs from this class of molecules undergo facile cleavage at the N-methylimidazole moiety under collision-induced dissociation (CID) conditions, MS-MS analysis in the constant neutral loss mode of [M+H]+-15 enabled the identification of two other HAAs, 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) and 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), which have rarely been reported in cooked meats.     

Diastereoselective synthesis of cyclopropane amino acids using diazo compounds generated in situ

A simple and high-yielding method for the preparation of cyclopropane amino acids is described. The novel method involves the one-pot cyclopropanation of readily available dehydroamino acids using aryl and unsaturated diazo compounds generated in situ from the corresponding tosylhydrazone salts. It was found that thermal 1,3-dipolar cycloaddition followed by nitrogen extrusion gave the cyclopropane amino acid derivatives with good E selectivity, while reactions in the presence of meso-tetraphenylporphyrin iron chloride gave predominantly the corresponding Z isomers. The synthetic utility of this process was demonstrated in the synthesis of (+/-)-(Z)-2,3-methanophenylalanine [(+/-)-(Z)-1], the anti-Parkinson (+/-)-(E)-2,3-methano-m-tyrosine [(+/-)-(E)-2], and the natural product (+/-)-coronamic acid [(+/-)-3].     

X-ray Crystal Structure, ab Initio Calculations, and Reactivity of 1,3,2lambda(5)-Diazaphosphetes: A New Type of 4-pi-Electron 4-Membered Heterocycle

The structure of P,P-bis(diisopropylamino)-4-phenyl-1,3,2lambda(5)-diazaphosphete, 1a, has been determined by a single-crystal X-ray diffraction study (C(19)H(33)N(4)P, monoclinic system, space group P2(1), a = 9.482(1) ?, b = 11.374(3) ?, c = 9.668(2) ?, beta = 97.16(1) degrees, Z = 2). According to quantum chemical calculations at an RHF level of optimization utilizing the 6-31g(d,p) basis set, 1a has a zwitterionic structure with the negative charge delocalized on the NCN allylic fragment and the positive charge localized at the phosphorus. Heterocycle 1a reacts with water and benzaldehyde affording N-phosphoranylbenzamidine 3 (95% yield) and the expected aza-Wittig adduct 4 (85% yield), respectively. Addition of 1 equiv of methyl trifluoromethanesulfonate and of 2 equiv of BH(3).THF to 1a affords cyclic phosphonium salt 5 (94% yield) and the bis(borane) adduct 6a (90% yield), respectively. Dimethyl acetylenedicarboxylate slowly reacts with 1a giving rise to 1,3,4lambda(5)-diazaphosphinine, 9, in 70% yield. The X-ray crystal structures of products 2,3, and 6a are reported (2: C(26)H(38)N(5)P, monoclinic system, space group C2/c, a = 16.337(8) ?, b = 19.810(2) ?, c = 8.800(2) ?, beta = 117.68(2) degrees, Z = 4. 3: C(19)H(35)N(4)OP, orthorhombic system, space group P2(1)2(1)2(1), a = 9.090(1) ?, b = 12.955(2) ?, c = 17.860(3) ?, Z = 4. 6a: C(19)H(39)B(2)N(4)P, orthorhombic system, space group P2(1)2(1)2(1), a = 10.340(1) ?, b = 13.247(1) ?, c = 16.996(1) ?, Z = 4).     

Self-assembled monolayer gold electrode for surfactant analysis

A gold electrode coated with a self-assembled monolayer of octane-thiol (SAM/Au) has been used as an amperometric detector for the determination of surfactants. This detector operated in the presence of a high percentage of organic solvent and was adapted to an HPLC System. At the SAM/Au, the electrochemical response of an electroactive tracer (potassium ferricyanide) was completely inhibited, but, in the presence of a cationic surfactant, the electrochemical reduction was progressively restored. In flow injection analysis, using the SAM/Au in an amperometric flow-through detector polarised at 0.0 V vs Ag/AgCl, a linear response (i=f{[surfactant]}) was observed for cationic surfactants e.g. cetylpyridinium chloride in the concentration range 2 × 10⁻⁶–1 × 10⁻³ M. The electrochemical data along with the determination of the ion pair stoichiometry between the redox tracer and the surfactant suggest an electrochemical response related to ion pair formation and governed by electron transfer by tunneling effect. Received: 28 January 1997 / Accepted: 7 March 1997     

Chemical ionization desorption mass spectrometry of some quaternary ammonium salts

The ammonia chemical ionization desorption spectra of N,N-dimethyl quaternary ammonium iodides in addition to high protonated molecular ion [M + H]⁺ intensity, show signals for an ion radical composed of N-methyl abstracted salt cation and ammonia [C + NH₃? CH₃]^+˙. These ions corresponding to the cation +2 show increased importance in the chemical ionization mode, using the same reagent gas. The technique of chemical ionization desorption appears suitable for the analysis of salts, and thus for the determination of the molecular weight of both anion and cation.     

Direct coupling of polymer-based microchip electrophoresis to online MALDI-MS using a rotating ball inlet

We report on the coupling of a polymer-based microfluidic chip to a MALDI-TOF MS using a rotating ball interface. The microfluidic chips were fabricated by micromilling a mold insert into a brass plate, which was then used for replicating polymer microparts via hot embossing. Assembly of the chip was accomplished by thermally annealing a cover slip to the embossed substrate to enclose the channels. The linear separation channel was 50 microm wide, 100 microm deep, and possessed an 8 cm effective length separation channel with a double-T injector (V(inj) = 10 nL). The exit of the separation channel was machined to allow direct contact deposition of effluent onto a specially constructed rotating ball inlet to the mass spectrometer. Matrix addition was accomplished in-line on the surface of the ball. The coupling utilized the ball as the cathode transfer electrode to transport sample into the vacuum for desorption with a 355 nm Nd:YAG laser and analyzed on a TOF mass spectrometer. The ball was cleaned online after every rotation. The ability to couple poly(methylmethacrylate) microchip electrophoresis devices for the separation of peptides and peptide fragments produced from a protein digest with subsequent online MALDI MS detection was demonstrated.     

EFFECT OF CHLORIDE AND BENZOATE ANIONS ON THE DELAYED LIGHT EMISSION IN DCMU-TREATED SPINACH CHLOROPLASTS

Abstract —Chloride anions, when added to DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]-treated spinach chloroplasts, change the rate of decay of the delayed light emission in the seconds region but do not change the shape or the temperature dependence of the decay. Benzoate anions, on the other hand, change both the rate and the shape of the decay of the delayed light emission. These results are consistent with a model in which the membrane potential and the structure of the reaction center affect the decay kinetics of the delayed light emission in the seconds region.     

Persistent (amino)(silyl)carbenes

(Amino)(silyl)carbenes, prepared by substitution reactions at a carbene center, can survive several days at 0 degrees C. These species are not push-pull carbenes as their phosphino analogues and therefore are excellent ligands for transition-metal centers.