BioCD-2008: Synchrotron Radiation Circular Dichroism Spectroscopy of Proteins and Other Biomolecules

A weeklong workshop focusing on circular dichroism (CD) spectroscopy using both conventional and synchrotron sources (SRCD) was presented at the National Synchrotron Light Source at Brookhaven National Laboratory from June 23–27, 2008.     

Droplet deformation and alignment for high-efficiency polarization-dependent holographic polymer-dispersed liquid-crystal reflection gratings

Droplet deformation and alignment are achieved in holographic polymer-dispersed liquid-crystal reflection gratings by applying an in situ shear during recording. High diffraction efficiency (99%) is obtained for light polarized parallel to the shear, with nearly zero efficiency for perpendicular polarization, and no increase of incoherent scattering. Permanent polarization dependence is related to stress-induced morphology changes of liquid-crystal droplets that are frozen by polymerization. The system is studied by electron microscopy and modeled by anisotropic coupled-wave and scattering theory. The morphology is consistent with the theory of small deformations of liquid droplets in fluid flow. Diffraction efficiency measurements are in agreement with theory incorporating this morphology as well as concomitant orientation and alignment of liquid-crystal molecules.     

Paper‐Origami‐Based Multiplexed Malaria Diagnostics from Whole Blood

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper‐folding technique of origami to enable the sequential steps of DNA extraction, loop‐mediated isothermal amplification (LAMP), and array‐based fluorescence detection. A low‐cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a “sample‐to‐answer” diagnosis from a finger‐prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold‐standard polymerase chain reaction (PCR) assay in an operator‐blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.     

Determination of ultra trace levels of 1,2-dichloroethane in air by sample enrichment micromachined gas chromatography-differential mobility detection

A novel analytical procedure has been developed for the analysis of ultra trace levels of 1,2-dichloroethane (EDC) in air using sample enrichment in combination with micromachined gas chromatography (GC) and differential mobility detection (DMD). When compared to other contemporary GC techniques, such as GC-flame ionization detection, GC-electron capture detection, or GC-electrolytic conductivity detection, the employment of a DMD in combination with a preconcentrator provided better sensitivity and markedly improved selectivity. The increase in sensitivity reduces false-negative results, while the improvement in selectivity decreases the potential for false-positive results. Using the technique described, a complete analysis can be conducted in less than 10 min, with a detection limit of 0.7 ppb (v/v) of EDC and a short term precision of less than 6%. A correlation coefficient of 0.9988 was obtained over an EDC concentration range from 0.7 ppb to 36.4 ppb (v/v). The analytical system also has an on-board microTCD in series with the DMD, allowing both detector outputs to be monitored simultaneously. With the pre-concentration technique, the microTCD can detect EDC as low as 15 ppb (v/v) with a substantially enhanced linear dynamic range in addition to providing a confirmation means for the presence of EDC at the level cited.     

Spiral coils for counter-current chromatography using aqueous polymer two-phase systems

Retention properties of polyethylene glycol-phosphate aqueous two-phase systems in a spiral coil (5 mm I.D.) on Type-J synchronous counter-current chromatographic devices have been compared for the elution mode where the lower phase is the mobile phase and flows from the inside head terminal. This was achieved with the aid of digital imaging under stroboscopic illumination, an image analysis and measurement of the displaced volume of the stationary phase. For the spiral coil, high and stable stationary phase retention at mobile phase flow rates up to 64 ml/min has been obtained. Wave-like disturbance of the interface near the proximal point was observed and analyses have been made for possible use in protein separation.     

Derivatisation using m-(trifluoromethyl)phenyltrimethylammonium hydroxide of organic materials in artworks for analysis by gas chromatography-mass spectrometry: unusual reaction products with alcohols

The quaternary ammonium reagent m-(trifluoromethyl)phenyltrimethylammonium hydroxide (TMTFTH) has gained widespread use for the derivatisation for GCMS analysis of organic materials found in artists' materials, such as oils, plant resins and waxes. This paper discusses products formed from reactions of TMTFTH with alcohols--including fatty alcohols, hydroxyacids, terpenes and glycerol--that have diagnostic value in the analysis of samples from artworks. In addition to methyl ethers, (trifluoromethyl)phenyl ethers were formed to variable degrees from the different types of alcohol. The products were identified from their EI mass spectra. An understanding of these multiple reaction products is important for the interpretation of GCMS data from complex samples, especially in the case of polyfunctional alcohols such as glycerol, which forms a number of methyl, (trifluoromethyl)phenyl and mixed ethers. The significance of the reaction products, and the relative advantages of TMTFTH as compared to alternative ammonium and sulfonium methylating reagents, are discussed.     

Synthesis of some heterocyclic skeletons via organoiron complexes. Crystal and molecular structure of (5a,6,7,8,9,9a-η6-1,4-benzoxathiino[3,2-b]pyridine)(η5-cyclopentadienyl)iron hexafluorophosphate

Synthesis of the heterocyclic skeletons of some biologically active compounds from (η⁶-o-dichlorobenzene)(η⁵-cyclopentadienyrl)iron hexafluorophosphate in a two step procedure is described. Cyclopentadienyliron hexafluorophosphate complexes of 1,4-benzodioxino[2,3-b]pyridine, 1,4-benzoxathiino[3,2-b]pyridine, 10H-pyrido[3,2-b]benzoxazine, benzo[b]naphtho[2,3-e][1,4]dioxin, 4-methylbenzo[b]benzopyran-2-one[7,6-e][1,4]dioxin and benzo[b]anthracen-9,10-diono[1,2-e][1,4]dioxin were isolated and characterized. Upon pyrolytic sublimation of these complexes the free heterocycles were obtained and characterized. (η⁶-1,4-Benzoxathiino[3,2-b]pyridine)(η⁵-cyclopentadienyl)iron hexafluorophosphate crystalizes in the orthothombic system, space group Pbca; the dihedral angle between the planes of outer rings was found to be 176.8 (1).     

IRRADIATION OF CIRCULAR DNA WITH 254 nm RADIATION OR SENSITIZATION IN THE PRESENCE OF Ag+ EVIDENCE FOR UNWINDING BY PHOTOPRODUCTS OTHER THAN PYRIMIDINE DIMERS

Abstract— Structural alterations of DNA irradiated with UV light were analyzed by the agarose gel technique. Relaxed, circular pAT 153 DNA molecules were sensitized by broad band radiation with a maximum at 313 nm in the presence of silver ions or irradiated with 254 nm light in buffer only. In both cases the electrophoretic mobility of DNA topoisomers was altered as a linear function of UV exposure. For DNA irradiated in the sensitized reaction the unwinding angle per site sensitive to Micrococcus luteus pyrimidine dimer endonuclease was found tobe–11.4°. This value is significantly smaller thanthe–14.3° already known for DNA topoisomers irradiated with 254 nm light. The irradiated DNAs were a very good substrate for the Escherichia coli photoreactivating enzyme (PRE). However, the photoenzymic removal of all sites sensitive to the endonuclease specific for pyrimidine dimers was not coupled to a full restoration of the original electrophoretic mobility. Thirty and 23% of the unwinding were still present in the photoreactivated topoisomers and the unwinding angles per pyrimidine dimer were then recalculatedas–10.1°and–8.7° for DNAs irradiated with 254 nm and sensitized, respectively. The limited difference between these two values could result from the different base composition of the pyrimidine dimers generated in the conditions of irradiation used. These results show that the tertiary structure of DNA is measureably altered by UV photodamages other than pyrimidine dimers.     

Optimising resolution for a preparative separation of Chinese herbal medicine using a surrogate model sample system

This paper builds on previous modelling research with short single layer columns to develop rapid methods for optimising high-performance counter-current chromatography at constant stationary phase retention. Benzyl alcohol and p-cresol are used as model compounds to rapidly optimise first flow and then rotational speed operating conditions at a preparative scale with long columns for a given phase system using a Dynamic Extractions Midi-DE centrifuge. The transfer to a high value extract such as the crude ethanol extract of Chinese herbal medicine Millettia pachycarpa Benth. is then demonstrated and validated using the same phase system. The results show that constant stationary phase modelling of flow and speed with long multilayer columns works well as a cheap, quick and effective method of optimising operating conditions for the chosen phase system—hexane–ethyl acetate–methanol–water (1:0.8:1:0.6, v/v). Optimum conditions for resolution were a flow of 20 ml/min and speed of 1200 rpm, but for throughput were 80 ml/min at the same speed. The results show that 80 ml/min gave the best throughputs for tephrosin (518 mg/h), pyranoisoflavone (47.2 mg/h) and dehydrodeguelin (10.4 mg/h), whereas for deguelin (100.5 mg/h), the best flow rate was 40 ml/min.     

The synthesis of anthracyclinones-ii : The synthesis of 7,9-bisdeoxycarminomycinone and -daunomycinone

7,9-Bisdeoxycarminomycinone has been synthesised from 5-hydroxyquinizarin by two annelation routes involving nitronate addition to C-2 and aldol condensation at C-3. 4-Demethoxy-7,9-bisdeoxydaunomycinone has been synthesised efficiently by similar methods.