Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli

We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.     

Rheology of binary colloidal structures assembled via specific biological cross-linking

The selectivity and range of energies offered by specific biological interactions serve as valuable tools for engineering the assembly of colloidal particles into novel materials. In this investigation, high affinity biological interactions between biotin-coated "A" particles (RA = 0.475 microm) and streptavidin-coated "B" particles (RB = 2.75 microm) drive the self-assembly of a series of binary colloidal structures, from colloidal micelles (a large B particle coated by smaller A particles) to elongated chain microstructures (alternating A and B particles), as the relative number of small (A) to large (B) particles (2 < or = NA/NB < or = 200) is decreased at a low total volume fraction (10(-4) < or = phiT < or = 10(-3)). At a significantly higher total volume fraction (phiT > or = 10(-1)) and a low number ratio (NA/NB = 2), the rheological behavior of volume-filling particle networks connected by streptavidin-biotin bonds is characterized. The apparent viscosity (eta) as a function of the shear rate gamma, measured for networks at phiT = 0.1 and 0.2, exhibits shear-rate-dependent flow behavior, and both the apparent viscosity and the extent of shear thinning increase upon an increase of a factor of 2 in the total volume fraction. Micrographs taken before and after shearing show a structural breakdown of the flocculated binary particle network into smaller flocs, and ultimately a fluidlike suspension, with increasing shear rate. Rheological measurements provide further proof that suspension microstructure is governed by specific biomolecular interactions, as control experiments in which the streptavidin molecules on particles were blocked displayed Newtonian flow behavior. This investigation represents the first attempt at measuring the rheology of colloidal suspensions where assembly is driven by biomolecular cross-linking.     

The rovibrational analysis of the high-resolution IR spectrum of CD2HF below 1200 cm: an interacting tetrad of vibrational levels

The spectrum of CD₂HF was measured by high-resolution interferometric Fourier-transform IR (FTIR) spectroscopy (apodised instrumental band with:0.004 cm⁻¹ fwhm) between 800 and 1200 cm⁻¹ covering the four lowest fundamentals. A complete rotational analysis using a semi-automatic assignment procedure yields accurate band centres (ν₉: 912.2028 cm⁻¹, ν₆:964.4994 cm⁻¹, ν₅: 1050.5104 cm⁻¹, ν₄: 1093.8632 cm⁻¹) and a complete set of first-order Coriolis coupling constants. The most important couplings occur between ν₉ and ν₆ (ξ_a= 1.069 cm⁻¹, ξ_c= −0.3535 cm⁻¹) and between ν₅ and ν₄ (ξ_b= −0.80606 cm⁻¹). The analysis was guided by and compared with results from our ab initio calculations for Coriolis constants and transition moments using CADPAC at TZP/MP2 level.     

The biosynthesis of quadrone and terrecyclic acid

Feedings of [1-¹³C]- and [1,2-¹³C₂]acetate Aspergillus terreus gave quadrone and terrecyclic acid which were analyzed by ¹³C NMR. The pattern of ¹³C-enrichments and couplings is consistent with the formation of 1 and 2 by cyclization of farnesyl pyrophosphate.     

Explorative data analysis of two-dimensional electrophoresis gels

Methods for classification of two-dimensional (2-DE) electrophoresis gels based on multivariate data analysis are demonstrated. Two-dimensional gels of ten wheat varieties are analyzed and it is demonstrated how to classify the wheat varieties in two qualities and a method for initial screening of gels is presented. First, an approach is demonstrated in which no prior knowledge of the separated proteins is used. Alignment of the gels followed by a simple transformation of data makes it possible to analyze the gels in an automated explorative manner by principal component analysis, to determine if the gels should be further analyzed. A more detailed approach is done by analyzing spot volume lists by principal components analysis and partial least square regression. The use of spot volume data offers a mean to investigate the spot pattern and link the classified protein patterns to distinct spots on the gels for further investigation. The explorative approach in analysis of 2-D gels makes it possible, in a fast and convenient way, to screen many gels in order to determine the protein patterns that form clusters and could be selected for further examination.     

The intramolecular asymmetric Pauson-Khand cyclization as a novel and general stereoselective route to benzindene prostacyclins: synthesis of UT-15 (treprostinil)

A general and novel solution to the synthesis of biologically important stable analogues of prostacyclin PGI(2), namely benzindene prostacyclins, has been achieved via the stereoselective intramolecular Pauson-Khand cyclization (PKC). This work illustrates for the first time the synthetic utility and reliability of the asymmetric PKC route for synthesis and subsequent manufacture of a complex drug substance on a multikilogram scale. The synthetic route surmounts issues of individual step stereoselectivity and scalability. The key step in the synthesis involves efficient stereoselection effected in the PKC of a benzoenyne under the agency of the benzylic OTBDMS group, which serves as a temporary stereodirecting group that is conveniently removed via benzylic hydrogenolysis concomitantly with the catalytic hydrogenation of the enone PKC product. Thus the benzylic chiral center dictates the subsequent stereochemistry of the stereogenic centers at three carbon atoms (C(3a), C(9a), and C(1)).     

Design, synthesis, and photophysical studies of a porphyrin-fullerene dyad with parachute topology; charge recombination in the marcus inverted region

As part of a continuing investigation of the topological control of intramolecular electron transfer (ET) in donor-acceptor systems, a symmetrical parachute-shaped octaethylporphyrin-fullerene dyad has been synthesized. A symmetrical strap, attached to ortho positions of phenyl groups at opposing meso positions of the porphyrin, was linked to [60]-fullerene in the final step of the synthesis. The dyad structures were confirmed by (1)H, (13)C, and (3)He NMR, and MALDI-TOF mass spectra. The free-base and Zn-containing dyads were subjected to extensive spectroscopic, electrochemical and photophysical studies. UV-vis spectra of the dyads are superimposable on the sum of the spectra of appropriate model systems, indicating that there is no significant ground-state electronic interaction between the component chromophores. Molecular modeling studies reveal that the lowest energy conformation of the dyad is not the C(2)(v)() symmetrical structure, but rather one in which the porphyrin moves over to the side of the fullerene sphere, bringing the two pi-systems into close proximity, which enhances van der Waals attractive forces. To account for the NMR data, it is proposed that the dyad is conformationally mobile at room temperature, with the porphyrin swinging back and forth from one side of the fullerene to the other. The extensive fluorescence quenching in both the free base and Zn dyads is associated with an extremely rapid photoinduced electron-transfer process, k(ET) approximately 10(11) s(-)(1), generating porphyrin radical cations and C(60) radical anions, detected by transient absorption spectroscopy. Back electron transfer (BET) is slower than charge separation by up to 2 orders of magnitude in these systems. The BET rate is slower in nonpolar than in polar solvents, indicating that BET occurs in the Marcus inverted region, where the rate decreases as the thermodynamic driving force for BET increases. Transient absorption and singlet molecular oxygen sensitization data show that fullerene triplets are formed only with the free base dyad in toluene, where triplet formation from the charge-separated state is competitive with decay to the ground state. The photophysical properties of the P-C(60) dyads with parachute topology are very similar to those of structurally related rigid pi-stacked P-C(60) dyads, with the exception that there is no detectable charge-transfer absorption in the parachute systems, attributed to their conformational flexibility. It is concluded that charge separation in these hybrid systems occurs through space in unsymmetrical conformations, where the center-to-center distance between the component pi-systems is minimized. Analysis of the BET data using Marcus theory gives reorganization energies for these systems between 0.6 and 0.8 eV and electronic coupling matrix elements between 4.8 and 5.6 cm(-)(1).     

Synthesis of the new metastable skutterudite compound NiSb(3) from modulated elemental reactants

A new metastable binary compound with the skutterudite crystal structure has been synthesized from modulated elemental reactants, through an amorphous intermediate, using a novel low-temperature synthesis technique. The amorphous reaction intermediate undergoes nucleation at 87 degrees C, an extremely low temperature for solid-state reactions. When heated above 350 degrees C, the metastable phase NiSb(3) disproportionates into the thermodynamically stable phases NiSb(2) and Sb. Also, if the sum of the individual elemental layer thicknesses is greater than 30 A, a mixture of different phases forms. Simulation of the high-angle powder X-ray diffraction spectrum confirms that NiSb(3) is isostructural with CoSb(3).     

Identification of components in waste streams by electrospray and tandem mass spectrometry

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.     

Capillary-channeled polymer fibers as stationary phases in liquid chromatography separations

A method utilizing capillary-channeled polymer (C-CP) fibers as stationary phases in high-performance liquid chromatographic separations has been investigated. Polymeric fibers of differing backbones (polypropylene and polyester) having nominal diameters of approximately 50 and approximately 35 microm and a channeled structure on their periphery were packed into stainless steel tubing (305 x 4.6 mm I.D.) for use in reversed-phase separations of various mixtures. The fibers have eight channels running continuously along the axis which exhibit very high surface activity. As such, solvent transport is affected through the channels through wicking action. Bundles of 1000-3000 fibers are loaded co-linearly into the tubing, providing flow channels extending the entire length of the columns. As a result, backing pressures are significantly lowered (approximately 50% reduction) in comparison to packed-sphere columns. In addition, the capital costs of the fiber material (< US$0.25 per column) are very attractive. Flow-rates of up to 5 ml/min can be used to achieve near baseline separation of related compounds in reasonable run times, indicating very fast mobile phase mass transfer (C-terms). The polymer stationary phases demonstrate high selectivity for a wide variety of analytes with gradient elution employed successfully in many instances. Specifically, separations of three polyaromatic hydrocarbons (benzo[a]pyrene, chrysene, pyrene), mixtures of both organic and inorganic lead compounds [chlorotriethyllead, chlorotriphenyllead, lead nitrate, lead(II) phthalocyanine], and a lipid standard of triglycerides were accomplished on the polymeric stationary phases. Other species of biological interest, including groups of aliphatic and aromatic amino acids have also been effectively separated. The reversed-phase nature of the fiber surfaces is supported through atomic force microscopy measurements using hydrophilic and hydrophobic functionalized polystyrene beads as the probe tips. Separations of the various analytes demonstrate the feasibility of utilizing C-CP fibers as stationary phases in reversed-phase LC. It is envisioned that columns of this nature would be particularly useful in prep-scale separations as well as for immobilization matrices for organic constituents in aqueous environments.