Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis

Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.     

Electrostatics of PEGylated micelles and liposomes containing charged and neutral lipopolymers

The electrostatics of large unilamellar vesicles (LUVs) of various lipid compositions were determined and correlated with steric stabilization. The compositional variables studied include (a) degree of saturation, comparing the unsaturated egg phosphatidylcholine (EPC) and the fully hydrogenated soy phosphatidylcholine (HSPC) as liposome-forming lipids; (b) the effect of 40 mol % cholesterol; (c) the effect of mole % of three methyl poly(ethylene glycol) (mPEG)-lipids (the negatively charged mPEG-distearoyl phosphoethanolamine (DSPE) and two uncharged lipopolymers, mPEG-distearoyl glycerol (DSG) and mPEG-oxycarbonyl-3-amino-1,2-propanediol distearoyl ester (DS)); and (d) the negatively charged phosphatidyl glycerol (PG). The lipid phases were as follows: liquid disordered (LD) for the EPC-containing LUV, solid ordered (SO) for the HSPC-containing LUV, and liquid ordered (LO) for either of those LUV with the addition of 40 mol % cholesterol. The LUV zeta potential and electrical surface potential (psi(0)) were determined. It was found that progressive addition of mPEG(2k)-DSPE to liposomes increases negative surface potential and reduces surface pH to a similar extent as the addition of PG. However, due to the "hidden charge effect", zeta potential was more negative for liposomes containing PG than for those containing mPEG(2k)-DSPE. Replacing mPEG-DSPE with mPEG(2k)-DS or mPEG-DSG had no effect on surface pH and surface potential, and zeta potential was approximately zero. Addition of low concentrations of cationic peptides (protamine sulfate and melittin) to PG- or mPEG-DSPE-containing liposomes neutralized the liposome negative surface potential to a similar extent. However, only in liposomes containing PG, did liposome aggregation occur. Replacing the negatively charged lipopolymer mPEG-DSPE with the neutral lipopolymers mPEG-DS or mPEG-DSG eliminates or reduces such interactions. The relevance of this effect to the liposome performance in vivo is discussed.     

Catalytic asymmetric vinylation of ketones

This communication describes the catalytic asymmetric 1,2-addition of vinylzinc reagents to aromatic, alpha,beta-unsaturated, and dialkyl ketones with enantioselectivities between 79 and 97% and with yields ranging from 84 to 98%. The products of these reactions are tertiary allylic alcohols with chiral quaternary centers that are useful in organic synthesis. The reaction involves hydrozirconation of a terminal alkyne, transmetalation to zinc, and addition to a ketone in the presence of a chiral titanium-based Lewis acid catalyst. The reactions proceed smoothly at room temperature in under 24 h.     

Surfactant control of gas uptake: effect of butanol films on HCl and HBr entry into supercooled sulfuric acid

The entry of HCl into 60-68 wt % D(2)SO(4) and HBr into 68 wt % acid containing 0-0.18 M 1-butanol was monitored by measuring the fractions of impinging HCl and HBr molecules that desorb as DCl and DBr after undergoing H --> D exchange within the deuterated acid. The addition of 0.18 M butanol to the acid creates butyl films that reach approximately 80% surface coverage at 213 K. Surprisingly, this butyl film does not impede exchange but instead enhances it: the HCl --> DCl exchange fractions increase from 0.52 to 0.74 for 60 wt % D(2)SO(4) and from 0.14 to 0.27 for 68 wt % D(2)SO(4). HBr --> DBr exchange increases even more sharply, rising from 0.22 to 0.65 for 68 wt % D(2)SO(4). We demonstrate that this enhanced exchange corresponds to enhanced uptake into the butyl-coated acid for HBr and infer this equivalence for HCl. In contrast, the entry probability of the basic molecule CF(3)CH(2)OH exceeds 0.85 at all acid concentrations and is only slightly diminished by the butyl film. The OD groups of surface butanol molecules may assist entry by providing extra interfacial protonation sites for HCl and HBr dissociation. The experiments suggest that short-chain surfactants in sulfuric acid aerosols do not hinder heterogeneous reactions of HCl or HBr with other solute species.     

Synthesis of 4-aryl-2,3,6,7-tetrahydro-1H-pyrimido[4,5-b][1,4]-diazepin-6-ones from 4,5-diamino-1H-pyrimidin-6-ones and 1-aryl-3-(dimethylamino)-1-propanones

The reaction of 1-aryl-3-(dimethylamino)-1-propanones 1 with one equivalent of 4,5-diamino-1H-pyrimidin-6-ones 2 , in acidic medium, leads to the formation of 4-aryl-2,3,6,7-tetrahydro-1H-pyrimido[4,5-b]-[1,4]diazepin-6-ones 3 . The structure elucidation of the products is based on detail nmr analysis of experiments such as ¹³C, ¹H and DEPT including selective ¹³C{¹H} decoupling experiments.     

1,4 polymerization of isoprene by titanate catalyst systems

Titanates are versatile in the 1,4 polymerization of isoprene. The (R′O)₄Ti/RAlCl₂ catalyst gives either cis- or trans-1,4-polyisoprene, depending on the nature of both the titanate and the solvent. Primary titanates give cis-1,4-polyisoprene in both aliphatic and aromatic solvents. Secondary titanates give cis-polyisoprene in aliphatic solvents, and trans-1,4-polyisoprene in aromatic solvents. Tertiary titanates give trans-polyisoprene in both aliphatic and aromatic solvents. A mechanism is postulated which takes into consideration the role of the solvent. ESR studies of the various titanate–RAlCl₂ catalysts were made; the paramagnetic structures are related to polymerization mechanisms.     

Nitrosation of Phenolic Compounds: Effects of Alkyl Substituents and Solvent

Summary.  Nitrosation reactions of phenol, o-cresol, 2,6-dimethylphenol, o-tert-butylphenol, 2-hydroxyacetophenone, and 2-allylphenol in water and water/acetonitrile were studied. Kinetic monitoring of the reactions was accomplished by spectrophotometric analysis of the nitrosated products at 345 nm. The dominant reaction was C-nitrosation via a mechanism consisting of an attack on the nitrosatable substrate by NO⁺/NO₂H₂ ⁺ followed by a slow proton transfer. The values of the rate constants of phenolic C-nitrosation were increased by electron donating substituents, and a good Hammett correlation was observed with ρ = −6.1. The results also revealed the strong effect of pH and the permitivity of the reaction medium on the rate constant, whose maximum values were observed for pH ≈ 3, decreasing strongly for higher pH values. The study in water/acetonitrile with up to 25% acetonitrile showed that it is possible to inhibit the reaction strongly by increasing the percentage of the organic component. The conclusions drawn show that (i) it is possible to predict the rate of nitrosation of phenolics as a function of the meta-substituents on the phenol ring and (ii) the nitrosation of phenolics can be strongly inhibited by increasing the pH of the reaction medium as well as by lowering its dielectric constant. Received July 13, 2001. Accepted (revised) September 18, 2001     

Time-resolved ultraviolet laser-induced breakdown spectroscopy for organic material analysis

Ultraviolet pulses (266 nm) delivered by a quadrupled Nd:YAG laser were used to analyze organic samples with laser-induced breakdown spectroscopy (LIBS). We present characteristics of the spectra obtained from organic samples with special attentions on the emissions of organic elements, O and N, and molecular bonds CN. The choice of these atomic or molecular species is justified on one hand, by the importance of these species to specify organic or biological materials; and on the other hand by the possible interferences with ambient air when laser ablation takes place in the atmosphere. Time-resolved LIBS was used to determine the time-evolution of line intensity emitted from these species. We demonstrate different kinetic behaviors corresponding to different origins of emitters: native atomic or molecular species directly vaporized from the sample or those generated through dissociation or recombination due to interaction between laser-induced plasma and air molecules. Our results show the ability of time-resolved UV-LIBS for detection and identification of native atomic or molecular species from an organic sample.     

Singlet fission in a hexacene dimer: energetics dictate dynamics

Singlet fission (SF) is an exciton multiplication process with the potential to raise the efficiency limit of single junction solar cells from 33% to up to 45%. Most chromophores generally undergo SF as solid-state crystals. However, when such molecules are covalently coupled, the dimers can be used as model systems to study fundamental photophysical dynamics where a singlet exciton splits into two triplet excitons within individual molecules. Here we report the synthesis and photophysical characterization of singlet fission of a hexacene dimer. Comparing the hexacene dimer to analogous tetracene and pentacene dimers reveals that excess exoergicity slows down singlet fission, similar to what is observed in molecular crystals. Conversely, the lower triplet energy of hexacene results in an increase in the rate of triplet pair recombination, following the energy gap law for radiationless transitions. These results point to design rules for singlet fission chromophores: the energy gap between singlet and triplet pair should be minimal, and the gap between triplet pair and ground state should be large.

We report the synthesis and photophysical characterization of highly exoergic singlet fission in a hexacene dimer revealing exciton dynamics that follow the energy gap law.     

H atom transfer along an ammonia chain: tunneling and mode selectivity in 7-hydroxyquinoline.(NH3)3

Excitation of the 7-hydroxyquinoline(NH(3))(3) [7HQ(NH(3))(3)] cluster to the S(1) (1)pi pi(*) state results in an O-H-->NH(3) hydrogen atom transfer (HAT) reaction. In order to investigate the entrance channel, the vibronic S(1)<-->S(0) spectra of the 7HQ.(NH(3))(3) and the d(2)-7DQ.(ND(3))(3) clusters have been studied by resonant two-photon ionization, UV-UV depletion and fluorescence techniques, and by ab initio calculations for the ground and excited states. For both isotopomers, the low-frequency part of the S(1)<--S(0) spectra is dominated by ammonia-wire deformation and stretching vibrations. Excitation of overtones or combinations of these modes above a threshold of 200-250 cm(-1) for 7HQ.(NH(3))(3) accelerates the HAT reaction by an order of magnitude or more. The d(2)-7DQ.(ND(3))(3) cluster exhibits a more gradual threshold from 300 to 650 cm(-1). For both isotopomers, intermolecular vibrational states above the threshold exhibit faster HAT rates than the intramolecular vibrations. The reactivity, isotope effects, and mode selectivity are interpreted in terms of H atom tunneling through a barrier along the O-H-->NH(3) coordinate. The barrier results from a conical intersection of the optically excited (1)pi pi(*) state with an optically dark (1)pi sigma(*) state. Excitation of the ammonia-wire stretching modes decreases both the quinoline-O-H...NH(3) distance and the energetic separation between the (1)pi pi(*) and (1)pi sigma(*) states, thereby increasing the H atom tunneling rate. The intramolecular vibrations change the H bond distance and modulate the (1)pi pi(*)<-->(1)pi sigma(*) interaction to a much smaller extent.