Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B,Cel9A,and Cel5A on BMCC and Avicel

In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonospora fusca E₃, E₄, and E₅), it was observed that binding capacities for these three cellulases were 18–30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate, accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding The processive endoglucanase, Cel9A, had four times the extent of conversion of the end endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.     

Relative reactivities of some dienes and acetylenes in copolymerization with sulfur dioxide

The reactivities of isoprene, piperylene,2,3-dimethylbutadiene, hex-1-yne, and phenylacetylene, at ?20°C, relative to that of cyclohexene, have been determined for the radical-initiated copolymerization with sulfur dioxide to form 1:1 polysulfones. The unsaturated hydrocarbons were copolymerized with sulfur dioxide in pairs and the composition of the terpolymers determined from the 100 MHz NMR spectra. The dienes react 11–15 times as fast as hex-1-ene, while hex-1-yne reacts 16 times more slowly. Phenylacetylene reacts 21 times as fast as hex-1-yne. The relative reactivities are interpreted mainly in terms of the effect of electron delocalization on the stability of the product radical.     

A new photochemically removable protecting group for amines

A new arylmethylsulfonyl chloride reacts with secondary or primary amines to give sulfonamides which can be photochemically cleaved, making it suitable for use as a photoremovable protecting group.     

The synthesis of poly(acrylic acid hydrazide) and poly(methylacrylic acid hydrazide) and their reaction products with ribonucleoside dialdehydes

Atactic and syndiotactic poly(acrylic acid hydrazide) and atactic, syndiotactic and isotactic poly(methylacrylic acid hydrazide) have been reacted with the di-aldehydes derived from adenosine, guanosine, inosine, cytidine and uridine to give polymers each containing a single type of base residue. Not all of the hydrazide residues of the polymeric hydrazides reacted; guanosine dialdehyde gave the most reaction and inosine dialdehyde the least. Isotactic poly(methylacrylic acid hydrazide) was much less reactive than the other polymeric hydrazides. The adenine-containing and the cytosine-containing polymers with atactic backbones had a low solubility in water whereas those with syndiotactic backbones had a relatively high solubility. For a given polymeric hydrazide backbone the adenine-containing polymers were always the least soluble in water. Most of the ribonucleoside dialdehyde-containing polymers, with the exception of those containing uridine dialdehyde, had only a low solubility in salt solution (0.3M sodium chloride, 0.03M trisodium citrate). No evidence could be obtained for any interaction of these polymers with polynucleotides.     

Identification of diastereomeric chlorophyll allomers by atmospheric pressure chemical ionisation liquid chromatography/tandem mass spectrometry

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry (APCI-LC/MS) has been used for identification of the epimers of hydroxy, methoxy and methoxylactone allomers of chlorophyll a (13(2)-HO-chl a, 13(2)-MeO-chl a and 15(1)-MeO-lact-chl a), the hydroxy allomer of bacteriochlorophyll a (13(2)-HO-bchl a) and the hydroxy and methoxylactone allomers of bacterioviridin a (13(2)-HO-bvir a and 15(1)-MeO-lact-bvir a). The APCI mass spectra show that facile fragmentations involve the methoxyl or hydroxyl groups at the C-13(2) or C-15(1) chiral centres. Losses involving the C-13(2) or C-15(1) hydroxyl or methoxyl groups occur more easily from the S-epimer than from the R-epimer due to the greater relief of the steric strain associated with interaction with the bulky C-17 substituent. The differences in mass spectrometric fragmentation can be used as a diagnostic tool for the assignment of the stereochemical configuration at the C-13(2) or C-15(1) chiral centres.     

Biochemical and biomedical applications of capillary column GLC using SIM

This report focuses on recent applications of capillary column GLC to the analysis of drugs and metabolites, other xenobiotics, natural products, and environmental contaminants in samples of biological origin. The increasing use of selected ion monitoring, combined with stable isotope methods as a means of GLC detection and quantification, is emphasized. Specific topics covered include the use of capillary column GLC for 1) determining the pharmacokinetics of timolol (BLOCADREN) in human volunteers by simultaneous oral and intravenous administration of this beta-blocker and its 13C3-labeled analog; 2) measuring residue levels of the forced molting agent, xylonidine, in yolk and albumen of chicken eggs; 3) monitoring animal plasma and tissues for the depletion of 5-hydroxy-1,3-dioxane and 4-hydroxymethyl-1,3-dioxolane (isomeric components of glycerol formal, an animal drug formulation component); 4) detecting 13C, 15N-methyltryptamine (a biosynthesis product from 13C, 15N-tryptamine) in human urine; and 5) measuring estradiol levels in biological samples.     

Pulsed EPR and NMR spectroscopy of paramagnetic iron porphyrinates and related iron macrocycles: how to understand patterns of spin delocalization and recognize macrocycle radicals

Pulsed EPR spectroscopic techniques, including ESEEM (electron spin echo envelope modulation) and pulsed ENDOR (electron-nuclear double resonance), are extremely useful for determining the magnitudes of the hyperfine couplings of macrocycle and axial ligand nuclei to the unpaired electron(s) on the metal as a function of magnetic field orientation relative to the complex. These data can frequently be used to determine the orientation of the g-tensor and the distribution of spin density over the macrocycle, and to determine the metal orbital(s) containing unpaired electrons and the macrocycle orbital(s) involved in spin delocalization. However, these studies cannot be carried out on metal complexes that do not have resolved EPR signals, as in the case of paramagnetic even-electron metal complexes. In addition, the signs of the hyperfine couplings, which are not determined directly in either ESEEM or pulsed ENDOR experiments, are often needed in order to translate hyperfine couplings into spin densities. In these cases, NMR isotropic (hyperfine) shifts are extremely useful in determining the amount and sign of the spin density at each nucleus probed. For metal complexes of aromatic macrocycles such as porphyrins, chlorins, or corroles, simple rules allow prediction of whether spin delocalization occurs through sigma or pi bonds, and whether spin density on the ligands is of the same or opposite sign as that on the metal. In cases where the amount of spin density on the macrocycle and axial ligands is found to be too large for simple metal-ligand spin delocalization, a macrocycle radical may be suspected. Large spin density on the macrocycle that is of the same sign as that on the metal provides clear evidence of either no coupling or weak ferromagnetic coupling of a macrocycle radical to the unpaired electron(s) on the metal, while large spin density on the macrocycle that is of opposite sign to that on the metal provides clear evidence of antiferromagnetic coupling. The latter is found in a few iron porphyrinates and in most iron corrolates that have been reported thus far. It is now clear that iron corrolates are remarkably noninnocent complexes, with both negative and positive spin density on the macrocycle: for all chloroiron corrolates reported thus far, the balance of positive and negative spin density yields -0.65 to -0.79 spin on the macrocycle. On the other hand, for phenyliron corrolates, the balance of spin density on the macrocycle is zero, to within the accuracy of the calculations (Zakharieva, O.; Schünemann, V.; Gerdan, M.; Licoccia, S.; Cai, S.; Walker, F. A.; Trautwein, A. X. J. Am. Chem. Soc. 2002, 124, 6636-6648), although both negative and positive spin densities are found on the individual atoms. DFT calculations are invaluable in providing calculated spin densities at positions that can be probed by (1)H NMR spectroscopy, and the good agreement between calculated spin densities and measured hyperfine shifts at these positions leads to increased confidence in the calculated spin densities at positions that cannot be directly probed by (1)H NMR spectroscopy. (13)C NMR spectroscopic investigations of these complexes should be carried out to probe experimentally the nonprotonated carbon spin densities.     

Models of the low-spin iron(III) hydroperoxide intermediate of heme oxygenase: magnetic resonance evidence for thermodynamic stabilization of the d(xy) electronic state at ambient temperatures

The (13)C pulsed ENDOR and NMR study of [meso-(13)C-TPPFe(OCH(3))(OO(t)Bu)](-) performed in this work shows that although the unpaired electron in low-spin ferrihemes containing a ROO(-) ligand resides in a d(pi) orbital at 8 K, the d(xy) electron configuration is favored at physiological temperatures. The variable temperature NMR spectra indicate a dynamic situation in which a heme with a d(pi) electron configuration and planar porphyrinate ring is in equilibrium with a d(xy) electron configuration that has a ruffled porphyrin ring. Because of the similarity in the EPR spectra of the hydroperoxide complexes of heme oxygenase, cytochrome P450, and the model heme complex reported herein, it is possible that these two electron configurations and ring conformations may also exist in equilibrium in the enzymatic systems. The ruffled porphyrinate ring would aid the attack of the terminal oxygen of the hydroperoxide intermediate of heme oxygenase (HO) on the meso-carbon, and the large spin density at the meso-carbons of a d(xy) electron configuration heme suggests the possibility of a radical mechanism for HO. The dynamic equilibrium between the ruffled (d(xy)) and planar (d(pi)) conformers observed in the model complexes also suggests that a flexible heme binding cavity may be an important structural motif for heme oxygenase activity.     

An investigation of enhanced secondary ion emission under Au n + (n=1–7) bombardment

We investigate the mechanism of the nonlinear secondary ion yield enhancement using Au(n)+ (n = 1, 2, 3, 5, 7) primary ions bombarding thin films of Irganox 1010, DL-phenylalanine and polystyrene on Si, Al, and Ag substrates. The largest differences in secondary ion yields are found using Au+, Au2+, and Au3+ primary ion beams. A smaller increase in secondary ion yield is observed using Au5+ and Au7+ primary ions. The yield enhancement is found to be larger on Si than on Al, while the ion yield is smaller using an Au+ beam on Si than on Al. Using Au(n)+ ion structures obtained from Density Functional Theory, we demonstrate that the secondary yield enhancement is not simply due to an increase in energy per area deposited into the surface (energy deposition density). Instead, based on simple mechanical arguments and molecular dynamics results from Medvedeva et al, we suggest a mechanism for nonlinear secondary ion yield enhancement wherein the action of multiple concerted Au impacts leads to efficient energy transfer to substrate atoms in the near surface region and an increase in the number of secondary ions ejected from the surface. Such concerted impacts involve one, two, or three Au atoms, which explains well the large nonlinear yield enhancements observed going from Au+ to Au2+ to Au3+ primary ions. This model is also able to explain the observed substrate effect. For an Au+ ion passing through the more open Si surface, it contacts fewer substrate atoms than in the more dense Al surface. Less energy is deposited in the Si surface region by the Au+ primary ion and the secondary ion yield will be lower for adsorbates on Si than on Al. In the case of Au(n)+ the greater density of Al leads to earlier break-up of the primary ion and a consequent reduction in energy transfer to the near-surface region when compared with Si. This results in higher secondary ion yields and yield enhancements on silicon than aluminum substrates.     

Isotopic scrambling in Di-13C-labeled 2-butyl cation: evidence for a protonated cyclopropane intermediate

The (13)C NMR spectrum of 2-butyl-1,2-(13)C(2) cation (1) is unchanged on heating the sample to -78 degrees C, indicating no isomerization to another isotopomer. In contrast, the spectrum of 2-butyl-2,3-(13)C(2) cation (2) shows rapid formation of all of the other isotopomers except 1. These results are consistent with a protonated cyclopropane intermediate in the rearrangement process. In this mechanism, either C(1) and C(2) or C(3) and C(4) interchange. Only the bond between C(2) and C(3) breaks.