Brian G. Sayer

The Biogenetic Anatomy of Vitamin B6 A 13C NMR INVESTIGATION OF THE BIOSYNTHESIS OF PYRIDOXOL IN ESCHERICHIA COLI

It is shown by incorporation experiments with 13C bond-labeled substrates, followed by analysis by means of 13C NMR spectroscopy, that two compounds, 1-deoxy-D-xylulose (12) and 4-hydroxy-L-threonine (13), serve as precursors of pyridoxol (vitamin B6) (1) in Escherichia coli. Together, these two compounds account for the entire C8N skeleton of the vitamin. 1-Deoxy-D-xylulose supplies the intact C5 unit, C-2′,2,3,4,4′ of pyridoxol. 4-Hydroxy-L-threonine undergoes decarboxylation in supplying the intact C3N unit, N-1,C-6,5,5′. Both precursors are ultimately derived from glucose. The C5 unit of pyridoxol that is derived from 1-deoxy-D-xylulose originates by union of a triose phosphate (yielding C-3,4,4′) with pyruvic acid (which decarboxylates to yield C-2′,2). D-Erythroate (11) enters the C3 unit, C-6,5,5′, and is therefore an intermediate on the route from glucose into 4-hydroxy-L-threonine.

Studies of phospholipid hydration by high-resolution magic-angle spinning nuclear magnetic resonance.

A sample preparation method using spherical glass ampoules has been used to achieve 1.5-Hz resolution in 1H magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of aqueous multilamellar dispersions of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), serving to differentiate between slowly exchanging interlamellar and bulk water and to reveal new molecular-level information about hydration phenomena in these model biological membranes. The average numbers of interlamellar water molecules in multilamellar vesicles (MLVs) of DOPC and POPC were found to be 37.5 +/- 1 and 37.2 +/- 1, respectively, at a spinning speed of 3 kHz. Even at speeds as high as 9 kHz, the number of interlamellar waters remained as high as 31, arguing against dehydration effects for DOPC and POPC. Both homonuclear and heteronuclear nuclear Overhauser enhancement spectroscopy (NOESY and HOESY) were used to establish the location of water near the headgroup of a PC bilayer. 1H NMR comparisons of DOPC with a lipid that can hydrogen bond (monomethyldioleoylphosphatidylethanolamine, MeDOPE) showed the following trends: 1) the interlamellar water resonance was shifted to lower frequency for DOPC but to higher frequency for MeDOPE, 2) the chemical shift variation with temperature for interlamellar water was less than that of bulk water for MeDOPE MLVs, 3) water exchange between the two lipids was rapid on the NMR time scale if they were mixed in the same bilayer, 4) water exchange was slow if they were present in separate MLVs, and 5) exchange between bulk and interlamellar water was found by two-dimensional exchange experiments to be slow, and the exchange rate should be less than 157 Hz. These results illustrate the utility of ultra-high-resolution 1H MAS NMR for determining the nature and extent of lipid hydration as well as the arrangement of nuclei at the membrane/water interface.

Chemical applications of /sup 99/Tc NMR spectroscopy: preparation of novel Tc(VII) species and their characterization by multinuclear NMR spectroscopy

The /sup 99/Tc NMR parameters of a number of Tc(VII) and one Tc(V) species have been determined. The anion TcO/sub 4//sup -/ (..xi.. = 22.508 311 MHz in H/sub 2/O) was chosen as the standard for /sup 99/Tc NMR spectroscopy. A /sup 99/Tc-/sup 17/O coupling constant of 131.4 Hz was obtained from a /sup 17/O- and /sup 18/O-enriched sample of TcO/sub 4//sup -/ whose /sup 99/Tc spectrum also showed an isotopic shift of 0.22 ppm/mass number arising from a statistical distribution of /sup 16/O//sup 17/O//sup 18/O isotopic isomers. Technetium-99 and proton NMR provided definitive proof for the existence of the stereochemically nonrigid TcH/sub 9//sup 2 -/ anion. Both TcO/sub 3/F and the novel TcO/sub 3//sup +/ cation were synthesized and characterized by /sup 99/Tc, /sup 17/O, and /sup 19/F NMR spectroscopy. Preliminary results on two new technetium(VII) oxyfluorides tentatively identified as F/sub 2/O/sub 2/TcOTcO/sub 2/F/sub 2/ and TcO/sub 2/F/sub 3/ are also reported. The diagmagnetic d/sup 2/ anion, TcO/sub 2/(CN)/sub 4//sup 3 -/, represents the most deshielded /sup 99/Tc environment encountered in the present study.

Titanium-47 and -49 nuclear magnetic resonance spectroscopy: Chemical applications

Abstract Direct observation of 47,49 Ti resonances in a series of TiX 4 (X = Cl, Br, I, OPr i , NEt 2 ), (C 5 H 5 ) 2 TiX 2 (X = F, Cl, Br, I, N 3 , NCS), and TiX 6 2− (X = F, Br) complexes is reported. 47,49 Ti nuclei are equally sensitive to changes of halogen ligands in the (C 5 H 5 ) 2 MX 2 series as the 91 Zr nucleus. The inverse halogen dependence of δ( 47,49 Ti) is extended and placed on a more quantitative basis in the (C 5 H 5 ) 2 TiX 2 systems. The TiF 6 2− ion reveals septets for both the 47 Ti and 49 Ti resonances, with 1 J 47 Ti 19 F = 1 J 49 T 19 F = 34.0 Hz. Spin-lattice relaxation times are reported for TiCl 4 and TiF 6 2− . A detailed study of TiCl 4 reveals the dominance of a quadrupolar relaxation mechanism. Correlation times calculated from the Gierer and Wirtz relationship combined with observed T 1 results allow the quadrupole coupling constants for 47,49 Ti nuclei to be estimated as 2.8 and 2.4 MHz, respectively.

15N NMR Study of the Ionization Properties of the Influenza Virus Fusion Peptide in Zwitterionic Phospholipid Dispersions

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.

Properties of Mixtures of Cholesterol with Phosphatidylcholine or with Phosphatidylserine Studied by 13 C Magic Angle Spinning Nuclear Magnetic Resonance

The behavior of cholesterol is different in mixtures with phosphatidylcholine as compared with phosphatidylserine. In (13)C cross polarization/magic angle spinning nuclear magnetic resonance spectra, resonance peaks of the vinylic carbons of cholesterol are a doublet in samples containing 0.3 or 0.5 mol fraction cholesterol with 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) or in cholesterol monohydrate crystals, but a singlet with mixtures of cholesterol and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC). At these molar fractions of cholesterol with POPS, resonances of the C-18 of cholesterol appear at the same chemical shifts as in pure cholesterol monohydrate crystals. These resonances do not appear in samples of POPS with 0.2 mol fraction cholesterol or with POPC up to 0.5 mol fraction cholesterol. In addition, there is another resonance from the cholesterol C18 that appears in all of the mixtures of phospholipid and cholesterol but not in pure cholesterol monohydrate crystals. Using direct polarization, the fraction of cholesterol present as crystallites in POPS with 0.5 mol fraction cholesterol is found to be 80%, whereas with the same mol fraction of cholesterol and POPC none of the cholesterol is crystalline. After many hours of incubation, cholesterol monohydrate crystals in POPS undergo a change that results in an increase in the intensity of certain resonances of cholesterol monohydrate in (13)C cross polarization/magic angle spinning nuclear magnetic resonance, indicating a rigidification of the C and D rings of cholesterol but not other regions of the molecule.

Long chain branching in ethylene polymerization using constrained geometry metallocene catalyst

We report an experimental investigation on long chain branching (LCB) in ethylene polymerization with the Dow Chemicals constrained geometry catalyst system, CGC-Ti/TPFPB/MMAO, using a continuous stirred-tank reactor (CSTR) at 140°C, 3.45 × 10 3 kPa, and a mean residence time (τ) of 4 min. The effects of the catalyst (CGC-Ti) and co-catalyst (TPFPB and MMAO) concentrations on the catalyst activity, polymer molecular weight, and shear thinning were systematically examined. The boron cocatalyst had a great influence on the CGC activity. Increasing the ratio TPFPB/CGC-Ti from 0.66 to 5 gave ethylene propagation rates from 1.65 × 10 3 to 1.36 × 10 4 L mol -1 . s -1 . The addition of MMAO appeared to be essential, most likely acting as an impurity scavenger. The LCB polyethylenes showed enhanced shear thinning properties. The melt flow index ratios I 10 /I 2 were in the range of 6.96 to 23.4, with the I 2 of 0.172 to 0.681 g/10 min. The weight-average molecular weight M w was correlated to I 2 using a power equation within narrow I 10 /I 2 ranges. The exponential factors were in the range of 4.24 to 6.31. The experimental and calculated M w s were in a good agreement.

Reaction of [η5-C5H5)2ZrH(μ-H)]2 with diphenylacetylene: mechanistic and theoretical considerations

The polymeric (Cp2ZrH2)x molecule is shown by 1H NMR studies to adopt a dimeric structure with bridging and terminal hydride ligands in both benzene-d6 and toluene-d8 solution. The reaction of the zirconium dihydride with diphenylacetylene has been re-investigated and produces a zirconacyclopentadiene complex 7, contrary to literature suggestions of a dimetallic zirconabenzene structure. A qualitative molecular orbital analysis of the [Cp2ZrH(μ-H)]2 dimer lends support to a suggested mechanism involving reaction with diphenylacetylene via a dimeric species.

Environmental variability in the reactivity of freshwater dissolved organic carbon to UV-B

Reductions in the global stratospheric ozone layer arethought to be increasing the amount of ultraviolet B (UV-B) radiationreaching the planet’s surface and may be affecting the chemistry ofdissolved organic carbon (DOC) in surface waters. We studied theabundance of chromophores in DOC collected in four different aquaticenvironments in southwestern Nova Scotia using 13C nuclearmagnetic resonance (13C NMR) spectroscopy. We showed a clearseasonal pattern in the distribution of structural carbon related tolight-sensitive chromophores. There seemed to be little variation in theUV-B related chemical structure of DOC between lakes and streams, thoughwater from a bog pool showed large differences from the other samples. Thesepatterns of potential UV-B reactivity tend to be dampened however, byvariations in DOC concentrations which also occur seasonally.

Biosynthesis of vitamins B1 and B6 in Escherichia coli: concurrent incorporation of 1-deoxy-D-xylulose into thiamin (B1) and pyridoxol (B6)

It is shown by 13C NMR spectroscopy that, in Escherichia coli mutant WG2, the C-2,-3 bond of [2,3-13C2]-1-deoxy-D-xylulose 2 enters C-4,-5 of the thiazole unit of thiamin (B1) 1 and C-2,-3 of pyridoxol (B6) 3, providing the first direct evidence that the intact C5 chain of 1-deoxy-D-xylulose is incorporated concurrently into each of the two B Vitamins.