Allylic Sites Research Articles

A Novel Bifunctional Addition-Fragmentation Agent for Photoinitiated Cationic Polymerization

A novel addition-fragmentation agent (AFA), namely, 2-benzyl-2-(N,N-dimethyl-2-ethoxycarbonyl-1-propenyl) ammonium hexafluoroantimonate-1-(4-morpholinophenyl)-butane-1-one, for photoinitiated cationic polymerization was synthesized and characterized. With this compound, it is possible to initiate the cationic polymerization of cyclohexene oxide (CHO), butyl vinyl ether (BVE) and N-vinylcarbazole (NVC) photochemically at λ = 380 nm. The mechanism involves the formation of radicals from the alkylphenone moiety and the addition of these radicals to the allylic site and fragmentation of the AFA.

Hydroxylation of benzylic and allylic sites by plant cultured suspension cells

A novel transformation of α-substituted benzylcyanides by hydroxylation of benzyl sites using cultured cells of cotton, Gossypium hirsutum, producing alkano- or benzophenones via presumably α-cyanohydrin is demonstrated. Also regioselective hydroxylation of 4 S-(−)-perillyl alcohol and 3 S-(−)-citronellol by cultured suspension cells of Catharanthus roseus is reported.

Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity.

Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation of the C(5) substrate dimethylallyl diphosphate (DMAPP) to the C(15) product farnesyl diphosphate (FPP) by addition of two molecules of isopentenyl diphosphate (IPP). The synthesis of FPP proceeds in two steps, where the C(10) product of the first addition, geranyl diphosphate (GPP), is the substrate for the second addition. The product selectivity of avian FPPase was altered to favor synthesis of GPP by site-directed mutagenesis of residues that form the binding pocket for the hydrocarbon residue of the allylic substrate. Amino acid substitutions that reduced the size of the binding pocket were identified by molecular modeling. FPPase mutants containing seven promising modifications were constructed. Initial screens using DMAPP and GPP as substrates indicated that two of the substitutions, A116W and N144'W, strongly discriminated against binding of GPP to the allylic site. These observations were confirmed by an analysis of the products from reactions with DMAPP in the presence of excess IPP and by comparing the steady-state kinetic constants for the wild-type enzyme and the A116W and N114W mutants.

Acetonitrile chemical ionization tandem mass spectrometry to locate double bonds in polyunsaturated fatty acid methyl esters.

A rapid method is presented for determining the location of double bonds in polyunsaturated fatty acid methyl esters (FAME) using an ion-trap mass spectrometer. The mass spectrum of the chemical ionization reagent acetonitrile in an ion trap includes a m/z 54 ion, identified previously as 1-methyleneimino-1-ethenylium ion. We show that it reacts with double bonds of polyunsaturated FAME to yield a series of covalent product ions all appearing at (M + 54)+. Collisional dissociation of these ions yields diagnostic fragments, permitting unambiguous localization of double bonds. For methylene-interrupted and conjugated FAME, one of these fragments results from loss of the hydrocarbon end of the chain, while the other involves loss of the methyl ester. Major diagnostic-fragment ions for monoene and diene FAME occur as a result of cleavage adjacent to either allylic sites or double bonds in the original analyte and appear at one mass unit above the mass expected for homolytic cleavage. Fragmentation of polyene FAME yields major diagnostic ions resulting from cleavage between double bonds that appear one mass unit lower. The method is shown to produce highly characteristic spectra for FAME with 1 to 6 double bonds. Identification of double-bond position in highly unsaturated fatty acids is demonstrated in a mixture of unknown polyunsaturated FAME from an extract of cultured Y79 human retinoblastoma cells.

Oxidation in fish lipids during thermal stress as studied by 13C nuclear magnetic resonance spectroscopy

Abstract13C Nuclear magnetic resonance spectroscopy has been applied to elucidate the mechanism of lipid oxidation occurring during thermal treatment of fish. Effects of temperature and time of processing have been studied by means of a model system of lipids, extracted from salmon (Salmo salar) muscle, to simulate industrial conditions of canning. Unsaturated fatty acids located at the sn‐2 position of the glycerol moiety were the most prone to oxidative damage. Regarding the mechanism of the reaction, results inferred from olefinic and methylenic resonances indicated a higher susceptibility of the allylic sites closest to the carbonyl group, followed by those placed near the methyl terminal group. Unsaturations located in the middle of the carbon chain did not show much damage. The glyceryl region provided an unusual resonance at 53.4 ppm, which could be assigned to a hydroxylic compound formed during process.

Desorption chemical ionization tandem mass spectrometry of polyprenyl and dolichyl phosphates

Negative-ion desorption chemical ionization (DCI) tandem mass spectrometry was applied to the analysis of nanomole quantities of semisynthetic polyisoprenyl phosphates, the chain length of which ranged from 7 to 20 isoprene units. The DCI spectrum of all the compounds tested show the presence of independently generated ions [M-HPO3-H](-), [M-H3PO2-H](-) and [M-H3PO4-H](-) resulting from the loss of a part of or the entire phosphate group of a polyisoprenyl-P. In tandem mass spectrometry, the [M-H3PO4-H](-) fragment produces series of ions 68 mass units apart, indicative of the polyisoprenoid nature of a compound. Studies with deuterated and α-saturated polyisoprenyl phosphates demonstrated that fragmentations of the [M-H3PO4-H](-) ion proceed from both ends (α and ω) of a polyisoprenoid chain and may occur at either allylic (A) or vinylic (V) sites. Fragments of masses equal to [n×68 - 1] and [n×68 - 13] (where n is the number of isoprene units and 3≤n is less than the total number of isoprene residues within a polyisoprenoid chain) comprise the αA and ωV series, respectively, and represent the most abundant ions in tandem mass spectra of the [M-H3PO4-H](-) fragment of polyprenyl phosphates, α-Saturated dolichyl phosphates can be distinguished easily from corresponding polyprenyl phosphates not only on the basis of a 2-u shift of the [M-H3PO4-H](-) ion and the α series of fragments, but also because of the presence of an additional (A+14) series of ions 14 u heavier than fragments resulting from the allylic cleavages of an α-saturated polyisoprenoid chain. Possible mechanisms of the collision-induced dissociation reactions of polyprenyl phosphates are discussed.

Fundamental studies of grafting reactions in free radical copolymerization. IV. Grafting of styrene, acrylate, and methacrylate monomers onto vinyl‐polybutadiene using benzoyl peroxide and AIBN initiators in solution polymerization.

AbstractVinyl‐1,2 polybutadiene (vinyl‐PBD) was used as the backbone polymer for the grafting of styrene, methacrylate, and acrylate monomers using both benzoyl peroxide and AIBN initiators. Radical attack on the backbone can occur through the pendant vinyl group or at the tertiary, allylic hydrogen site. Effective graft sites are formed via double bond addition of either primary (initiator) or polymer radicals. The production of tertiary allylic radicals on the backbone chain also occurs and results in moderate to dramatic reaction rate re‐tardation in every monomer system. The type of initiator is only important when the polymer radicals are not very reactive, as in the case of styrene, and to a lesser extent for methacrylate monomer. Graft efficiencies are generally higher when using vinyl‐PBD than when using cis‐PBD. © 1995 John Wiley & Sons, Inc.

Chemical and enzymatic synthesis of glycoconjugates 1. Enzymatic galactosylation of conduritol B

(−) Conduritol B in a racemic mixture was galactosylated enantioselectively at the allylic site by a β1,4-galactosyltransferase catalyzed reaction. Enantioselectivity of the reaction supports a binding model of the galactosyltransferase for modified acceptor substrates.

Identification of the active site of vertebrate oxidosqualene cyclase.

Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane-bound enzyme, was carried out using the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently-modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp-rich motif may act as a point charge to stabilize incipient cationic charge.

Active site mapping of affinity-labeled rat oxidosqualene cyclase.

Rat liver oxidosqualene cyclase (OSC), a 78-kDa membrane-bound enzyme, was purified and labeled with the mechanism-based irreversible inhibitor, [3H]29-methylidene-2,3-oxidosqualene (Abe, I., Bai, M., Xiao, X.-Y., and Prestwich, G. D. (1992) Biochem. Biophys. Res. Commun. 187, 32-38). A 6-kDa CNBr peptide was separated by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to a polyvinylidene difluoride membrane. The sequence of the first 30 amino acids of this peptide were determined by Edman degradation and showed unexpectedly high similarity to the fungal OSC from Candida albicans (50% identity with Arg413-Val442) and to the bacterial squalene cyclase from Alicyclobacillus (formerly Bacillus) acidocaldarius (37% identity with Lys356-Leu385). Further, radioanalysis clearly established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala or DDTAEA) were equally labeled by the irreversible inhibitor. This result provides the first information on the structural details of the active site of OSC and shows for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of sesquiterpene cyclases and prenyl transferases.

Synthesis and photochemistry of (2S,4R)‐ and (2S,4R)‐1,2,3,4‐tetrahydro‐4‐ethyl‐1,1,4‐trimethyl‐3Z‐ethylidene‐2‐naphthalenols

AbstractThe synthesis and photochemistry of optically pure (2S,4R)‐ and (2R,4R)‐1,2,3,4‐tetrahydro‐4‐ethyl‐1,1,4‐trimethyl‐3Z‐ethylidene‐2‐naphthalenols 10a and 11a are described. Naphthalenols 10a and 11a were prepared in seven steps from 2‐phenylbutanenitrile. Synthesis is mainly based upon acylation of isobutene by the optically active 2‐methyl‐2‐phenylbutanoyl chloride (5), and a Wittig reaction, while the ethylidene naphthalenones 8a and 9a are separated by HPLC. Under inert conditions, irradiation of both alcohols gives rise to E‐Z isomerization, followed by photodegradation reactions, leading to the formation of indane 12 by the elimination of formaldehyde. Irradiation of oxygenated solutions of 10a reveals a rapid photoaddition of oxygen to the double bond, leading to the stereo‐ and regioselective formation of hydroperoxide 13 and epoxy alcohol 15a, which hydrolyses on standing to triol 16a. The observed stereochemistry is explained by the stereoselective coordination of the hydroxyl group at the chiral allylic site with the attacking electrophilic oxygen. In the case of 11a a more complicated reaction mixture was obtained in which, analogously, the epoxy alcohol 15b and triol 16b were present. Semi‐empirical MNDO, AM1 and PM3 calculations revealed that, in the preferential ground state conformation, the hydroxyl group occupies an orthogonal position with respect to the exocyclic double bond. Fluorescence measurements revealed that the reactive state in the photochemistry of these naphthalenol systems under inert conditions is the triplet state. In the photoreactions of optically pure compounds 10a and 11a, we were unable to identify products analogous to 2, which are claimed to originate from the racemic precursors 1 via a planar 1,3 OH shift from a singlet (in the double bond) excited state. Clean photochemical reactions were observed only when 10a and 11a were irradiated in oxygenated solutions, resulting in stereo‐ and regiospecific photoproducts.

Self‐crosslinkable plastic‐rubber blend system based on poly (vinyl chloride) and acrylonitrile‐co‐butadiene rubber

AbstractThat the melt‐mixed blend of poly (vinyl chloride) and acrylonitrile‐co‐butadiene rubber becomes crosslinked during high‐temperature molding is evident from Monsanto rheometric, solvent swelling, and infrared spectroscopic studies. Dynamic mechanical analysis shows that such a self‐crosslinkable plastic‐rubber blend is miscible in different blend ratios. The degree of crosslinking depends on the blend ratios and the molding conditions. The cross‐linking reaction involves the allylic chlorine sites in poly (vinyl chloride) and the CN group in the nitrile rubber. © 1993 John Wiley & Sons, Inc.

COQ2 is a candidate for the structural gene encoding para-hydroxybenzoate:polyprenyltransferase.

Coenzyme Q functions as a lipid-soluble electron carrier in eukaryotes. In Saccharomyces cerevisiae, the enzymes responsible for the assembly of the polyisoprenoid side chain and subsequent transfer to para-hydroxybenzoate (PHB) are encoded by the nuclear genes COQ1 and COQ2, respectively. Yeast mutants defective in coenzyme Q biosynthesis are respiratory defective and provide a useful tool to study this non-sterol branch of the isoprenoid biosynthetic pathway. We isolated a 5.5-kilobase genomic DNA fragment that was able to functionally complement a coq2 strain. Additional complementation analyses located the COQ2 gene within a 2.1-kilobase HindIII-BglII restriction fragment. Sequence analyses revealed the presence of a 1,116-base pair open reading frame coding for a predicted protein of 372 amino acids and a molecular mass of 41,001 daltons. The amino acid sequence exhibits a typical amino-terminal mitochondrial leader sequence and six potential membrane-spanning domains. Primer extension and Northern analyses indicate the gene is transcriptionally active. Transformation of a coq2 strain with the 2.1-kilobase HindIII-BglII genomic restriction fragment on a multicopy plasmid restores PHB:polyprenyltransferase activity to wild-type levels. Disruption of the chromosomal COQ2 gene indicates the gene is not essential for viability, yet is required for PHB:polyprenyltransferase activity and respiratory function. In addition, the deduced amino acid sequence of PHB:polyprenyltransferase contains a putative allylic polyprenyl diphosphate-binding site. The presence of this aspartate-rich domain in a number of functionally distinct proteins which utilize polyprenyl diphosphate substrates is reported.

Graft copolymers of polytetrahydrofuran and butyl rubber, 1. Cationic copolymerisation of tetrahydrofuran with bromobutyl rubber

AbstractGraft copolymers of butyl rubber (BR) with polytetrahydrofuran (PTHF) were prepared from bromobutyl rubber by a cationic “grafting‐from” reaction initiated by silver perchlorate at allylic bromine sites on the rubber. By studying the influence of variables, such as temperature etc., it was shown that initiation at −20°C for 30 min, followed by polymerisation at 0°C with a [AgClO4]/[Br] ratio of ca. unity, produced near optimum reaction conditions with grafting efficiencies >90%. Copolymers containing upto 40% of grafted PTHF were obtained by this means. The BR‐g‐PTHF copolymers have a two‐phase morphology comprising amorphous BR with crystalline domains of PTHF which function as points of reinforcement in the rubber upto ca. 300 K.

Allylic oxidation of olefins by cobalt (III), manganese(III), and cerium (IV) acetates in acetic acid in the presence of sodium bromide

The effect of added sodium bromide on the allylic oxidation of olefins by cobalt(III), manganese(III), and cerium(IV) acetates has been studied. Sodium bromide strongly accelerated the oxidation of cyclohexene by these oxidants to give cyclohex-2-enyl acetate in good yield. Only one product was obtained in the oxidation of cycloalkenes, but two allylic acetates were obtained in the oxidations of methyl-substituted cyclohexenes, which have two kinds of allylic hydrogen. A mechanism involving hydrogen abstraction from allylic sites by bromine radicals is suggested.

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue.

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [3H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cation. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [14C]isopentenyl pyrophosphate, and MgCl2, the labeled polypeptide gave a ratio of 14C/3H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cation are present.

Reaction of organolithium reagents with α-chlorovinyltrimethylsilanes

The reaction of (CH 3) 3SiC(Cl)=CRR′ (R = R′ = H; R = H, R′ = CH 3; R = R′ = CH 3; R = R′ = C 6H 5) with organolithium, reagents was examined. Alkenylsilanes of structure (CH 3) 3SiCHCH-alkyl were obtained from (CH 3) 3SiC(Cl)CH 2 and alkyllithium reagents. Substrates with R or R′ ≠ H inhibited addition of the organolithium species to the double bond and led to products derived from chlorinelithium exchange (R = R′ = C 6H 5) or proton abstraction from an allylic methyl site (R = H, R′ = CH 3; R = R′ = CH 3).

Heat Degradation of PVC Stabilized by Treatment with Alkylaluminum Compounds

Abstract Thermal degradation of PVC treated with alkylaluminum compounds has been studied. Four PVC samples of different molecular weights have been treated with Me3Al, and Et3A1, and the dehydrochlorination rates of the polymers were determined at 190 and 220°C under a nitrogen atmosphere. The alkylaluminum-treated low molecular weight samples show marked increase in thermal stability, i. e., slower rate of dehydrochlorination right from the beginning of degradation, whereas with the higher molecular weight samples stabilization becomes pronounced only after a few percent of dehydrochlorination. The color of R3Al-treated samples was much lighter (yellowish) than those of controls (dark brown) at 1% HCl loss. The average polyene sequence lengths formed during the early stages of dehydrochlorination are found to be much shorter with RsAl-treated PVC than with virgin samples. It appears as though polyene sequences which arose by zipping- initiation from allylic and/or tertiary chlorine sites are longer than...

PULSE RADIOLYSIS STUDIES IN MODEL LIPID SYSTEMS: FORMATION AND BEHAVIOR OF PEROXY RADICALS IN FATTY ACIDS

Abstract— Radiolytic formation and peroxidation of fatty acid radicals have been investigated by pulse radiolysis techniques in oleate, linoleate, linolenate and arachidonate systems. A strong absorption band at 280 nm associated with conjugated radicals, Rconj, formed in polyunsaturated fatty acid moieties has been used as a probe for kinetic processes occurring at doubly allylic sites in the hydrocarbon chain. Formation of Rconj by O‐ has been found to be more efficient than the less selective OH radical. Peroxidation of Rconj is shown to be somewhat slower, (kR+ O2˜ 3 × 108M‐1 s‐1), than O2 reactions with radicals in oleate (kR+ O2= 1 × 109M‐1 s‐1). Peroxy radicals generated in these reactions disappear slowly by essentially second order processes (2kRO1˜ 107M‐1 s‐1). The superoxide radical, O‐2, shows little if any reactivity towards 0.01 M linolenate or arachidonate over periods of 20 s.

Nuclear magnetic resonance studies. XXXII. Rearrangement, isomerization, and deuterium exchange via β-enolization in bicyclooctenones. A 2H nuclear magnetic resonance study

The equilibration of the α,α -diniethyl derivatives of bicyclo[2.2.2]octenone, bicyclo[3.2.1]-oct-2-en-6-one, and its Δ3 isomer has been examined at 155 and 185 °C in tert-BuO−/tert-BuOH. Using 2Hmr spectroscopy the rates and stereoselectivity of hydrogen–deuterium exchange in each of these ketones have been determined for equilibrations with tert-BuOD as solvent. The relative reactivities for exchange at the allylic and vinylic sites compared to the analogous results for the corresponding olefins reveal a significant activating effect of the carbonyl group. Comparison of the data with those for the corresponding saturated ketones shows that the double bond enhances the rate of isomerization appreciably and increases the bias of the equilibrium toward the [3.2.1] skeleton.