Solid phase synthesis of porcine LH-releasing hormone with hydrogen chloride in formic acid as the deblocking reagent.

The decapeptide corresponding to the amino acid sequence of porcine luteinizing hormone-releasing hormone (LH-RH) which involves 1 mol of tryptophan was synthesized via solid phase synthesis with two different deblocking procedures which used hydrogen chloride in formic acid and hydrogen chloride in acetic acid containing 1% 2-mercaptoethanol. After some fundamental studies on the former reagent with respect to deblocking efficiency toward the Boc group, 0.5 M hydrogen chloride (a 10-fold molar excess with respect to the N-terminal Boc group) in formic acid was used in the present synthesis. The two synthetic products exhibited the same chemical and biological properties as an authentic LH-RH. Hydrogen chloride in formic acid has proved effective without a scavenger although loss of peptide from the resin occurred to a somewhat greater extent than that with hydrogen chloride in acetic acid. A derivative of the synthetic LH-RH formylated at the indole nitrogen had a greatly diminished biological activity, indicating that the intact indole side chain is essential for the activity.     

Bromine derivatives of amino acids as intermediates in the peroxidase-catalyzed formation of singlet oxygen

Recently, J. R. Kanofsky et al. (1988, J. Biol. Chem. 263, 9692-9696) reported that human eosinophils generated modest amounts of singlet oxygen. In the mechanism proposed, hypobromous acid (made from the peroxidase-catalyzed oxidation of bromide ion) reacted with hydrogen peroxide to form singlet oxygen. In contrast, human neutrophils, which generate both hypochlorous acid and hydrogen peroxide, do not make singlet oxygen. The failure of human neutrophils to generate singlet oxygen is due in part to the trapping of hypochlorous acid by endogenous amines. In this paper, I show that amino acids are much more effective traps for hypochlorous acid than for hypobromous acid. Glycine totally inhibits singlet oxygen generation from a model enzyme system composed of chloroperoxidase, hydrogen peroxide, and chloride ion, but causes only a 35% reduction in singlet oxygen generation from an analogous enzyme system containing bromide ion instead of chloride ion. The products of the reaction of hypobromous and glycine (presumably an equilibrium mixture of N-bromoglycine, N,N-dibromoglycine, and hypobromous acid) retain the ability to react with hydrogen peroxide to form singlet oxygen. In contrast, the products of the reaction of hypochlorous acid and glycine do not react with hydrogen peroxide to produce singlet oxygen. Similar results were obtained for L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cystine, L-glutamic acid, L-glutamine, L-histidine, L-lysine, L-phenylalanine, L-proline, L-serine, and L-tyrosine. Thus, bromine derivatives of amino acids may act as intermediates in the peroxidase-catalyzed generation of singlet oxygen.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Lipase-catalyzed conversions of trimethylsilyl ethers: deprotection, acetylation, epoxidation and one-pot-multi-step reactions

Unsaturated trimethylsilyl ethers are converted by lipase-catalyzed hydrolysis of ethyl acetate directly to alkenol acetates; the hydrolysis of diethyl carbonate yields unstable carbonic acid monoethylester, which deprotects trimethylsilyl ethers under mild conditions and without remaining acid. By the analogous lipase-catalyzed perhydrolysis of these esters with hydrogen peroxide, epoxyalkanol acetates and epoxyalkanols are obtained in one-pot reactions with selectivities of 90–97%. Using longer chain peroxy fatty acids, generated in-situ by lipase-catalyzed reaction of fatty acid and hydrogen peroxide, trimethylsilyl ethers are selectively (83–95%) epoxidized without removal of the protecting trimethylsilyl group.     

Oxidative cleavage of carboxylic esters by cytochrome P-450

Cytochrome P-450 was demonstrated to catalyze the oxidative cleavage of carboxylic acid esters to the corresponding carboxylic acids. 2,6-Dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester and related dialkyl esters were shown to serve as substrates in NADPH-fortified rat liver microsomes and reconstituted systems containing purified cytochrome P-450 enzymes. The ethyl group gave rise to acetaldehyde. The reactions proceed with large kinetic deuterium isotope effects, consistent with the view that P-450 abstracts a hydrogen atom in the mechanism. Oxygen rebound to the radical site is then postulated to complete the reaction and lead to a hemiacetal-like structure which collapses to give the products. Rate studies with differing alkyl substituents showed that the reaction was more rapid with removal of an ethyl than a methyl or isopropyl group, consistent with the view that the ethyl optimizes steric and inductive effects. Oxidative cleavage of carboxylic acid esters has little biochemical precedent, due to the difficult character of the reaction, and should be considered as an alternative to direct hydrolysis.     

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

A convenient method for the synthesis of C-protected esters of BOC-/Z-alpha,alpha- dialkylamino acids by the mixed carboxylic carbonic anhydride method

The synthesis of C-protected esters of Boc-/Z-alpha,alpha-dialkylamino acids is accomplished by using alkyl/aryl chloroformate in presence of DMAP as a catalyst. The reaction proceeds through mixed carboxylic carbonic anhydride, which was monitored by IR. The reaction was clean and complete in about 2 hr. All the esters prepared have been obtained in good yield and are fully characterized.     

IDENTIFICATION OF SOME CYTOCHEMICAL REACTION PRODUCTS BY HISTORADIOGRAPHY

The reaction products of cytochemical methods for acid phosphatase, carbonic anhydrase succinate dehydrogenase, and thiolacetic acid esterase have been identified by historadiographic methods in sections of kidney. The absorption of x-rays by these products gives information on their localisation when they cannot be seen readily in the light microscope. The requirements for successful demonstration of such reaction products and the possibley value of the technique are discussed.     

Experiments towards the synthesis of the ergot alkaloids and related structures. Part 8. The total synthesis of 4-methyl-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carbonitrile hydrochloride hemihydrate,an immediate precursor of the despyrrole analogues of lysergic acid and its amides

Exposure of the N-methoxycarbonyl-bicyclic-keto-acid 5 (improved preparation) to the Barnick beta-keto-acid synthesis yielded an aqueous solution of the sodium salts of the beta-keto-acids 26 and 27 which on heating at 60-65 degrees C furnished the N-methoxycarbonyl-tricyclic-ketone 9 (55%) plus the hydroxy-ketone 28 which on acid treatment raised the yield of 9 to 68%. Reduction (NaBH4) of 9 yielded the alcohol 32 (94%) which was treated with thionyl chloride followed by copper (I) cyanide and sodium iodide in acetonitrile to give the tricyclic-N-methoxycarbonyl nitrile 35 whose relative configuration was obtained by X-ray analysis. Attempts to remove the N-methoxycarbonyl group from 35 were unsuccessful. Conversion of the alcohol 32 to its methoxypropyl ether 41 followed by reaction with ethereal MeLi-LiBr yielded the amino-alcohol 39 (75%) converted to the N-formyl-tricyclic alcohol 42 with formic-acetic anhydride (70%). The alcohol 42 was then converted into the N-formyl nitrile 44 via the chloride 43 as employed in the earlier synthesis of the nitrile 35. Removal of the N-formyl group from the nitrile 44 was achieved by refluxing methanolic hydrochloric acid to give the required amino-nitrile hydrochloride 46 (91%) whose structure was confirmed by X-ray analysis. Reaction of the free base with methyl iodide in ethyl acetate in the presence of calcium carbonate furnished the N-methyl base 48 isolated as its hydrochloride, hemihydrate 49 (59%). The overall yield of 49 via this eleven-step synthesis was 3.4%.     

Preparation of cyclic 2',3'-carbamate derivatives of erythromycin macrolide antibiotics

Tricarbonylation of clarithromycin has been effected in a one-pot reaction with phosgene. The 11,12-diol moiety was closed into a cyclic carbonate, while the dimethylamino alcohol of the desosamine sugar was cyclised with loss of a methyl group to form a cyclic 2',3'-carbamate. The 4' hydroxyl group in clarithromycin was converted into a chloroformate group and subsequently to an allyl carbonate which on Pd-catalysis furnished a novel N-demethylclarithromycin 2',3'-carbamate-11,12-carbonate. Hydrolytic removal of the cladinose sugar and a subsequent oxidation furnished the corresponding ketolide. The 11,12-cyclic carbonate moiety was cleaved by sodium azide to the 10,11-anhydro-9-ketone. 11-N-Arylated cyclic 11,12:2',3'-dicarbamate derivatives were prepared in a copper(I) chloride aided reaction between aryl isocyanates and 10,11-anhydro 9-ketones. The products are novel N-arylated-N'-demethylated 11,12:2',3'-dicarbamate ketolides derived from clarithromycin.     

Synthesis of short chain alkyl esters using cutinase from Burkholderia cepacia NRRL B2320

Short chain alkyl esters are well appreciated for fruity flavors they provide. These are mainly applied to the fruit-flavored products like jam, jelly, beverages, wine and dairy. Cutinase from Burkholderia cepacia NRRL B 2320 was found to be active in catalyzing the synthesis of alkyl esters in organic solvent. The optimal temperature range for the enzyme catalyzed synthesis was found to be from 35 °C to 40 °C. The maximum conversion (%) during synthesis of ester was obtained for butyric acid (C4) and valeric acid (C5) with butanol reflecting the specificity of the enzyme for short-chain length fatty acids. In case of alcohol specificity, butanol was found to be most preferred substrate by the enzyme and conversion (%) decreased with increasing carbon chain length of alcohol used in the esterification reaction. The kinetic analysis for the synthesis of butyl butyrate by varying concentration of one substrate at a time (butanol or butyric acid), showed that Ping–Pong Bi Bi model with acid inhibition and influence of initial water is most suitable model for the prediction of the reaction kinetics.     

Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (EC 1.1.1.95) has been shown to be A site specific in its hydrogen transfer capacity unlike other dehydrogenases which use phosphorylated substrates. The experiments have been carried out using a coupled assay system with yeast alcohol dehydrogenase. The specific activity measurements of the reaction products indicate the possible influence of an isotope effect on this system.     

Investigation of the reaction of peroxynitrite with propofol at acid pH: predominant production of oxidized, nitrated, and halogenated derivatives.

We reported here the reaction, in acidic conditions, of peroxynitrite (ONOO(-)) with the anaesthetic agent propofol (2,6-diisopropylphenol, PPF). The most interesting finding is that peroxynitrite is able to nitrate and to oxidize propofol leading to 4-nitropropofol, quinone, and diphenylquinone as the major products. More surprisingly, we also found that peroxynitrite is capable of halogenating propofol in the presence of halide ions, suggesting the formation of nitrosyl chloride (NOCl) or nitryl chloride (NO(2)Cl) from the reaction of peroxynitrite with chloride ions. A significant enhancement of the halogenation yield is observed with a simultaneous decrease of the yields of the other products in the presence of methanol or hydrogen peroxide. Increased halogenation of PPF probably results from the formation of peroxynitrate (O(2)NOO(-)), that further oxidizes chloride ions in hypochlorous acid (HOCl) or molecular chlorine (Cl(2)). Spontaneous decay of peroxynitrate is relatively slow in acidic medium, thus explaining the decrease of the yields of the other products. By direct EPR techniques, we also observed that this reaction occurs via a radical pathway.     

Methanolysis of sphingomyelin. Toward an epimerization-free methodology for the preparation of D-erythro-sphingosylphosphocholine

It is well known that acid hydrolysis of natural sphingomyelin in aqueous methanol or 1-butanol at refluxing temperature is accompanied by epimerization at the C-3 position of the long-chain base. An improved procedure for the hydrolysis of commercially available, naturally occurring sphingomyelin is described. Prolonged exposure (3;-4 days) of sphingomyelin to freshly prepared 0.5 M anhydrous methanolic hydrogen chloride (generated by trapping the gas evolved from the reaction of concentrated sulfuric acid with solid sodium chloride in anhydrous methanol) at 50 degrees C resulted in cleavage of the amide side chain. The extent of epimerization of the allylic alcohol stereocenter was quantified by integration of the C-5 signal of the (13)C nuclear magnetic resonance spectrum of lysosphingomyelin.The method described here is superior to the traditional acid hydrolysis methods because it provides the product as a approximately 10:1 ratio of d-erythro/l-threo epimers; in contrast, a ratio of approximately 1. 3:1 was obtained by the previous methods. We also report that use of dichloromethane as a cosolvent with N,N-dimethylformamide in the reaction of lysosphingomyelin with an activated fatty acid reduced the time required for completion of the N-acylation reaction.     

Carbonic acid from decarboxylation by "malic" enzyme in lactic acid bacteria

Carbonic anhydrase studies were used to determine the primary form of carbonic acid produced from decarboxylation of l-malic acid by "malic" enzyme in malolactic strains of five different species of lactic acid bacteria. Addition of carbonic anhydrase to the reaction mixture containing crude bacterial extract and l-malic acid, at pH 7, in all five cases resulted in an increase (13 to 23%) in the rate of carbon dioxide evolution over the control. The results indicated that the primary form of carbonic acid released from "malic" enzyme was not anhydrous carbon dioxide as previously supposed and as has been shown for other decarboxylating enzymes. The standard free-energy changes of the malo-lactic reaction with the various forms of carbonic acid as the primary decarboxylation product were calculated. The reaction is less exergonic when carbonic acid, bicarbonate ion, or carbonate ion is the primary decarboxylation product compared to anhydrous carbon dioxide. The free-energy of the reaction is not biologically available to the bacteria; with carbon dioxide not the primary decarboxylation product, the potential energy lost in a malo-lactic fermentation is not as great as previously considered. Endogenous carbonic anhydrase activity was not found.     

Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis. Crystallographic,spectroscopic, and mutational studies

Cytochrome P450 isolated from Bacillus subtilis (P450(BSbeta); molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha- and beta-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450(BSbeta) in the substrate-bound form, determined at a resolution of 2.1 A. P450(BSbeta) exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg(242) guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the alpha- and beta-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg(242) in the same fashion as has been reported for the active site of chloroperoxidase, His(105)-Glu(183), which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450(BSbeta) is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.     

Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Picolinyl esters for the structural determination of fatty acids by GC/MS

The position of unsaturation, chain branching, and other structural features of fatty acids are not often apparent from the mass spectra of common derivatives such as methyl esters because of factors such as charge location at the carboxy termiunus and migration of double bonds. The spectra of picolinyl esters, on the other hand, contain fragment ions that provide this information. The esters are synthesized by reaction of the acids with thionyl chloride to form the acid chloride that is reacted with 3-pyridylcarbinol to give the ester. Under electron impact conditions in the mass spectrometer, an electron is removed from the nitrogen of the pyridine ring and a hydrogen atom is abstracted from the alkyl chain to this electron-deficient site. This process produces a radical site in the chain that initiates chain cleavage. Hydrogen atoms can be removed from any position of the chain with varying probability, depending on the chain structure. Thus, diagnostic ions are produced from each type of fatty acid whose masses and relative abundances reflect the structure of the alkyl chain and any substituents. Patterns of fragmentation for straight-chain, branched-chain, unsaturated and cyclic fatty acids are described together with those containing hydroxy-, epoxy-, keto-, and ether groups.     

Synthesis of methyl ω-deuterated tetradecanoate and hexadecanoate

Methyl ω-deuterated tetradecanoates have been prepared in high purity by two synthetic routes from methyl hydrogen tetradecanedioate. One method utilizes the selective reducing properties of sodium borodeuteride and sodium cyanoborodeuteride towards, acid chloride, ester, tosyloxy, and iodo groups to introduce the deuterium label at only the carboxyl group of methyl hydrogen tetradecanedioate. The second procedure utilizes a coupling reaction between an organic halide and lithium di-(trideuteriomethyl) cuprate (I). Corresponding ω-deuterated derivatives of methyl hexadecanoate may be prepared by the same methods from methyl hydrogen hexadecanedioate. The two methods should be generally applicable in the synthesis of ω-deuterated alkanoic acids.     

Experiments Towards the Synthesis of the Ergot Alkaloids and Related Structures. Part 8.17 The Total Synthesis of 4-Methyl-2,3,4,4a,5,6-hexahydro-benzo[f]quinoline-2-carbonitrile Hydrochloride Hemihydrate,an Immediate Precursor of the Despyrrole Analogues of Lysergic Acid and Its Amides

Exposure of the N -methoxycarbonyl-bicyclic-keto-acid 5 (improved preparation) to the Barnick β -keto-acid synthesis 1 yielded an aqueous solution of the sodium salts of the β -keto-acids 26 and 27 which on heating at 60-65°C furnished the N -methoxycarbonyl-tricyclic-ketone 9 (55%) plus the hydroxy-ketone 28 which on acid treatment raised the yield of 9 to 68%. Reduction (NaBH 4) of 9 yielded the alcohol 32 (94%) which was treated with thionyl chloride followed by copper (I) cyanide and sodium iodide in acetonitrile to give the tricyclic- N -methoxycarbonyl nitrile 35 whose relative configuration was obtained by X-ray analysis. Attempts to remove the N -methoxycarbonyl group from 35 were unsuccessful. Conversion of the alcohol 32 to its methoxypropyl ether 41 followed by reaction with ethereal MeLi-LiBr yielded the amino-alcohol 39 (75%) converted to the N -formyl-tricyclic alcohol 42 with formic-acetic anhydride (70%). The alcohol 42 was then converted into the N -formyl nitrile 44 via the chloride 43 as employed in the earlier synthesis of the nitrile 35. Removal of the N -formyl group from the nitrile 44 was achieved by refluxing methanolic hydrochloric acid to give the required amino-nitrile hydrochloride 46 (91%) whose structure was confirmed by X-ray analysis. Reaction of the free base with methyl iodide in ethyl acetate in the presence of calcium carbonate furnished the N -methyl base 48 isolated as its hydrochloride, hemihydrate 49 (59%). The overall yield of 49 via this eleven-step synthesis was 3.4%.