Reactive Ketone Research Articles

Flexible composite of PEEK and liquid crystalline polymer in presence of polyphosphazene

AbstractBinary blends of a reactive ketone based polymer with liquid crystalline polymer (LCPA‐950) were studied. The main properties required are flexibility and thermal resistance of material in presence of polyphosphazene, which acts as a compatibilizer. It has been observed that with the addition of LCP the composites showed reduced viscosity during blending and changes in the crystallization of the LCP phase. FTIR study showed that there was a partial interaction between the PEEK and LCP in presence of polyphosphazene. Polyphosphazene is miscible with both PEEK and LCP, which was evident from DMA results. The thermal stability of the composites has been studied by DTA/TGA. The thermal analysis of the blends showed that the degradation process is accelerated by blending, but with the addition of polyphosphazene the onset temperature of first degradation slightly shifted towards higher side than the PEEK/LCP blend. Measurement of the tensile properties showed an increase in the elongation as well as enhanced modulus and strength. From SEM micrographs of tensile fractured surfaces, it was revealed that there is good adhesion between the matrix and dispersed phase upon addition of polyphosphazene to ketone based polymer (PEEK) with LCP. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 3758–3765, 2007

Asymmetric Catalysis of Ene Reactions with Trifluoropyruvate Catalyzed by Dicationic Palladium(II) Complexes.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Asymmetric catalysis of ene reactions with trifluoropyruvate catalyzed by dicationic palladium(II) complexes

Chiral dicationic SEGPHOS–Pd(II) complex achieves a high chemical yield, ( E)-olefin selectivity, anti-diastereoselectivity, along with high enantioselectivity even with less reactive mono- and 1,2-disubstituted olefins in this much less reactive ketone–ene reactions. The high levels of enantioselectivity stem from the effective shielding with diphenyl groups on phosphines caused by the narrow dihedral angle of metal complexes with SEGPHOS.

A Cyclodextrin‐Modified Ketoester for Stereoselective Epoxidation of Alkenes.

AbstractFor Abstract see ChemInform Abstract in Full Text.

A new approach to the chemical synthesis of keto-proteins.

To increase the versatility of protein-conjugation, an orthogonal protection strategy is described, which enables the efficient synthesis of keto-proteins bearing a reactive ketone functionality using Boc, Fmoc, and chemical ligation methodologies. A 1,3-dithiolane group was used to protect the ketone function of levulinate- and pyruvate-derivatized peptides during solid-phase synthesis, acidolytic cleavage, and purification. When required, the 1,3-dithiolane group could be cleanly removed using aqueous silver or mercuric solutions to regenerate the reactive keto-protein at ambient temperature. The liberated keto-protein was chemoselectively conjugated in situ to an aminooxy-derivatized monodisperse polymer.

A cyclodextrin-modified ketoester for stereoselective epoxidation of alkenes.

A beta-cyclodextrin-modified ketoester 2 was prepared by covalent attachment of a reactive ketone moiety to beta-cyclodextrin. Treatment of 2 with Oxone as terminal oxidant would produce CD-substituted dioxirane, which can effect stereoselective alkene epoxidation. The 2-mediated (S)-alpha-terpineol epoxidations proceeded to give terpineol oxides in high yields, and the stereoselectivities (i.e., cis-/trans-epoxide ratio) decreased from 2.5:1 to 1:1.2 with increasing steric bulkiness of the terpenes. This steric-dependent stereoselectivity can be understood based on different binding geometries of the 2/terpene inclusion complexes according to the (1)H NMR titration and 2D ROESY experiments. Enantioselective epoxidation of styrenes has also been achieved with 2 as catalyst (20-50 mol %) in aqueous acetonitrile solution, and up to 40% ee was obtained in 4-chlorostyrene epoxidation at 0 degrees C. Similar enantioselectivities were also obtained for the 2-mediated epoxidation of 1,2-dihydronaphthalene (37% ee), 4-chlorostyrene (36% ee), and trans-stilbene (31% ee).

Surface engineering of living myoblasts via selective periodate oxidation

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.

Glutathione and NADH, but not Ascorbate, Protect Lens Proteins from Modification by UV Filters

Age-dependent human lens colouration and fluorescence may stem primarily from the covalent binding of UV filters to crystallins. The tendency of the kynurenine (Kyn) UV filters to deaminate at neutral pH, with the generation of reactive α,β-ketoalkenes, underlies this phenomenon. In this study the authors examined the ability of small molecular weight antioxidants, which are known to be present in the lens, to inhibit this process. Crystallins were incubated with Kyn at pH 7 in the presence of glutathione (GSH), ascorbate or NADH. Ascorbate, even at high (15mM ) levels, was not found to significantly retard the time-dependent covalent binding of Kyn to the proteins. GSH, and to a lesser extent NADH, however, had a major impact in preventing this modification. The increase in protein UV absorbance and fluorescence was inhibited by GSH intercepting the reactive ketone intermediate, to form a GSH–Kyn adduct. NADH seemed to protect by both reduction of the reactive ketone intermediate and by competing with Kyn for presumably hydrophobic sites on the crystallins. This may indicate that the covalent attachment of aromatic Kyn molecules could be facilitated by initial hydrophobic interactions. Since GSH is present at far greater concentrations than NADH, these results show that in primate lenses, GSH is the key agent responsible for protecting the crystallins from covalent modification.

Biochemical engineering of the N-acyl side chain of sialic acid: biological implications.

N-Acetylneuraminic acid is the most prominent sialic acid in eukaryotes. The structural diversity of sialic acid is exploited by viruses, bacteria, and toxins and by the sialoglycoproteins and sialoglycolipids involved in cell-cell recognition in their highly specific recognition and binding to cellular receptors. The physiological precursor of all sialic acids is N-acetyl D-mannosamine (ManNAc). By recent findings it could be shown that synthetic N-acyl-modified D-mannosamines can be taken up by cells and efficiently metabolized to the respective N-acyl-modified neuraminic acids in vitro and in vivo. Successfully employed D-mannosamines with modified N-acyl side chains include N-propanoyl- (ManNProp), N-butanoyl- (ManNBut)-, N-pentanoyl- (ManNPent), N-hexanoyl- (ManNHex), N-crotonoyl- (ManNCrot), N-levulinoyl- (ManNLev), N-glycolyl- (ManNGc), and N-azidoacetyl D-mannosamine (ManNAc-azido). All of these compounds are metabolized by the promiscuous sialic acid biosynthetic pathway and are incorporated into cell surface sialoglycoconjugates replacing in a cell type-specific manner 10-85% of normal sialic acids. Application of these compounds to different biological systems has revealed important and unexpected functions of the N-acyl side chain of sialic acids, including its crucial role for the interaction of different viruses with their sialylated host cell receptors. Also, treatment with ManNProp, which contains only one additional methylene group compared to the physiological precursor ManNAc, induced proliferation of astrocytes, microglia, and peripheral T-lymphocytes. Unique, chemically reactive ketone and azido groups can be introduced biosynthetically into cell surface sialoglycans using N-acyl-modified sialic acid precursors, a process offering a variety of applications including the generation of artificial cellular receptors for viral gene delivery. This group of novel sialic acid precursors enabled studies on sialic acid modifications on the surface of living cells and has improved our understanding of carbohydrate receptors in their native environment. The biochemical engineering of the side chain of sialic acid offers new tools to study its biological relevance and to exploit it as a tag for therapeutic and diagnostic applications.

Beta-Scission of C-3 (beta-carbon) alkoxyl radicals on peptides and proteins: a novel pathway which results in the formation of alpha-carbon radicals and the loss of amino acid side chains.

Exposure of proteins to radicals in the presence of O(2) brings about multiple changes in the target molecules. These alterations include oxidation of side chains, fragmentation, cross-linking, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes, and formation of new reactive groups, including hydroperoxides. These processes can result in the loss of structural or enzymatic activity. Backbone fragmentation is known to occur via a number of mechanisms, most of which involve hydrogen abstraction from the alpha-carbon site on the backbone. In this study, we demonstrate that initial attack at a side chain site, the beta-position (C-3), can give rise to formation of alpha-carbon radicals, and hence backbone cleavage, via the formation and subsequent beta-scission of C-3 alkoxyl radicals. This beta-scission reaction is rapid (k estimated to be >10(7) s(-)(1)) even with primary alkoxyl radicals derived from Ala residues, and occurs when the alkoxyl radicals are generated from a variety of precursors, including hydroperoxides and nitrate esters. These reactions release the former side chain as a reactive aldehyde or ketone; thus, Ala peptides release high yields of methanal (formaldehyde). This product has been quantified with a number of oxidized peptides and proteins, and can account for up to 64% of the initial attacking radicals with some Ala peptides. When quantified together with the hydroperoxide precursors, these species account for up to 80% of the initial radicals, confirming that this is a major process. Methanal causes cell toxicity and DNA damage and is an animal carcinogen and a genotoxic agent in human cells. Thus, the formation and subsequent reaction of alkoxyl radicals formed at the C-3 position on aliphatic amino acid side chains on peptides and proteins can give rise to both backbone fragmentation and the release of further reactive species which can cause cell toxicity and mutagenicity.

Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), in rats. Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne.

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3, 4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with (13)C stable isotope labels. Rats were orally administered [(13)C]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the (13)C labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of (14)C-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.

Immunochemical detection of oxalate monoalkylamide, an ascorbate-derived Maillard reaction product in the human lens

Carbohydrates with reactive aldehyde and ketone groups can undergo Maillard reactions with proteins to form advanced glycation end products. Oxalate monoalkylamide was identified as one of the advanced glycation end products formed from the Maillard reaction of ascorbate with proteins. In these experiments, we have analyzed human lens proteins immunochemically for the presence of oxalate monoalkylamide. Oxalate monoalkylamide was absent in most of the very young lenses but was present in old and cataractous lenses. The highest levels were found in senile brunescent lenses. Incubation experiments using bovine lens proteins revealed that oxalate monoalkylamide could form from the ascorbate degradation products, 2,3-diketogulonate and L-threose. These data provide the first evidence for oxalate monoalkylamide in vivo and suggest that ascorbate degradation and its binding to proteins are enhanced during lens aging and cataract formation.

Metabolic Delivery of Ketone Groups to Sialic Acid Residues: APPLICATION TO CELL SURFACE GLYCOFORM ENGINEERING

The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125-1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.

The Synthesis of (±)-Wilforonide

The first synthesis of (±)-wilforonide has been completed in ten steps from commercially available starting materials. A high-yielding monomethylation of a reactive ketone enolate, a regioselective α-annulation of an unactivated α-substituted cyclohexanone, and a selective hydrogenation of the resulting unsaturated keto ester were key steps in the synthesis.

Molecular handles open the door to novel biomaterials and cancer therapies

In a marriage of chemical engineering and cell biology, a chemist in the United States has installed what amounts to molecular handles on the surface of cells. Carolyn Bertozzi (University of California, Berkeley, CA, USA) has engineered the surfaces of human cells to contain a reactive ketone group, making the cells amenable to being covalently bound to synthetic materials, such as ceramics, plastics and metals. She foresees applications not only in bioreactors and biosensors, but also in targeting toxins to tumor cells.

Synthesis of Some amino‐4,5‐dihydropyrazolo[3,4‐a]acridines as potential cholinesterase inhibitors

AbstractA synthesis of the 4,5‐dihydro derivatives of the previously known pyrazolo[3,4‐a]acridine ring system is described. The reaction of a 3,4‐dihydroacridin‐1(2H)‐one with N,N‐dimethylformamide dimethyl acetal gave a reactive enamino ketone, which yielded the desired heterocycle upon reaction with hydrazine. Using this chemistry, 11‐amino‐4,5‐dihydro‐2H‐pyrazolo[3,4‐a)acridine (3) and a number of its 2‐substituted derivatives 4a‐k were synthesized and evaluated as acetylcholinesterase inhibitors, based on their relationship to 1,2,3,4‐tetrahydro‐9‐acridinamine (THA). 1‐Amino‐4,5‐dihydro‐1H‐pyrazolo[3,4‐a]acridine (11a) and 2‐amino‐4,5‐dihydro‐1H‐pyrazolo[3,4‐a]acridine (11b) were also synthesized and investigated as potential cholinesterase inhibitors.

Photochemistry in cyclodextrins

The photochemistry of two ketones with contrasting excited state reactivity (benzophenone and 4-phenylbenzophenone) incorporated into β-cyclodextrin (β-CD) was investigated. The formation of a 1:1 benzophenone-β-CD complex was demonstrated by UV spectroscopy and the association constant K was determined to be 670 ± 60 mol −1 dm 3. However, the reactive ketone triplet state was quenched by the carbohydrate, preventing the observation of room temperature phosphorescence (RTP). The possibility that the 3n,π* state was self-quenched in a 2:1 complex was ruled out on the basis of the observed photochemistry and the temperature dependence of the quenching. In contrast, the RTP of the related compound, 4-phenylbenzophenone, in β-CD solution was demonstrated for the first time.

ChemInform Abstract: Analogues of the Cytostatic and Antimitogenic Agents Chlamydocin and HC‐Toxin: Synthesis and Biological Activity of Chloromethyl Ketone and Diazomethyl Ketone Functionalized Cyclic Tetrapeptides.

The synthesis and biological activity of four novel analogues of the cytostatic and antimitogenic agents chlamydocin and HC-toxin are reported in which the natural products' reactive epoxy ketone side-chain moiety is replaced by a chloromethyl or a diazomethyl ketone functionality, but the respective 12-membered cyclic tetrapeptide ring systems are retained. Syntheses of the linear tetrapeptide sequences were, in each case, achieved by conventional methodology and designed such that cyclization would be onto proline. The use of suitably protected L-2-aminosuberic acid (Asu) enabled the ready assimilation of the desired chloromethyl and diazomethyl ketone functionalities after cyclization. Cyclization was accomplished by using bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl). Yields of cyclic product were comparable to or, in the case of the HC-toxin ring system, better than those previously reported. Liberation of the Asu-side-chain acid and manipulation to the required functionalities via mixed anhydride to the diazomethyl ketone and quenching with HCl to yield the chloromethyl ketone was achieved in excellent yield for the HC-toxin analogues but in only moderate yield for the chlamydocin analogue. The antimitogenic activities of HC-toxin chloromethyl ketone (IC50 = 30-40 ng/mL) and chlamydocin chloromethyl ketone (IC50 = 3-10 ng/mL) were found to be 3-4-fold lower than those of the natural products themselves. The diazomethyl ketone analogue of HC-toxin was found to be inactive (IC50 greater than 2000 ng/mL). A modification of the HC-toxin peptide ring system, [L-Phe]3-HC-toxin chloromethyl ketone was found not to be a more active analogue (IC50 = 40-100 ng/mL). The nature of the putative target molecule, the binding interactions of the various analogues and the contribution of rate of inhibition toward activity are briefly discussed. The chloromethyl ketones herein reported constitute the most potent synthetic antimitogenic cyclic tetrapeptide analogues yet designed.

The selective protection of the 3-ketone functions of steroids as heptafluoro-p-tolyl enol ethers

Conjugated and unconjugated 3-ketone functions in steroids react with octafluorotoluene at above 100 °C in the presence of caesium fluoride to give heptafluoro-p-tolyl enol ethers. The related but unusually reactive Wieland–Miescher ketone (11) reacted at room temperature in the presence of tetra-n-butylammonium fluoride. Enones were regenerated from their derivatives by acidic hydrolysis. Hydrolysis of the derivative (10) of 4,5α-dihydrotestosterone was sluggish but sodium methoxide regenerated the parent steroid. The methods have been applied in a synthesis of deuterium-labelled testosterone.

Inactivation of Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase by an enzyme-generated ethoxyacetylenic ketone in the presence of a thiol.

Replacement of the 21-methyl group of 20 beta-hydroxypregn-4-en-3-one with an ethoxyacetylene group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 microM, Vmax = 4.6 nmol min-1 micrograms-1) for the Streptomyces hydrogenans NAD(H)-dependent 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 X 10(4) L mol-1 s-1 and 6.94 X 10(4) L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 microM alcohol and 5 microM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.