New reactive oxidizing species causes formation of carbon-centered radical adducts in organic extracts of blood following liver transplantation

-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) radical adducts from Folch (chloroform:methanol) extraction of blood of transplanted livers exhibited a large 6-line electron paramagnetic resonance (EPR) spectrum. Slow EPR sample preparation involving freezing and thawing prior to extraction over 15 min yielded a spectrum assigned as a lipid-derived free radical species, whereas rapid (< min) extraction without a freeze-thaw cycle yielded a mixture of radicals, one with coupling constants similar to the -hydroxymethyl-4-POBN adduct (4-POBN/^.CH₂OH). Extraction with purified chloroform, however, yielded a much weaker, probably lipid-derived signal. Use of ¹³C-methanol in the Folch extracting solution yielded a 12-line EPR spectrum, indicating that a new, highly reactive oxidant species from blood following liver transplantation can convert organic solvents used in tissue extractions to free radicals. This hypothesis was supported by simulation of EPR spectra of free radicals extracted rapidly with Folch, which indicated that the spectrum contained two carbon-centered species, one with hyperfine coupling constants similar to the -methylhydroxyl-4-POBN adduct, the other probably lipid-derived. Because the former originates from methanol in the Folch, extraction of samples with alcohol-free organic solvent is most likely superior when the potential for formation of stable oxidant species exists, such as after liver transplantation.     

The reactions of zirconium(IV) bromide and zirconium(III) bromide and chloride with ammonia and zirconium(IV) bromide with ammonium cyanide

Unlike ZrCl₄, ZrBr₄ is not ammonolysed in liquid ammonia at temperatures up to −33 °C. The existence of ammoniates ZrBr₄nH₃ (n = 17, 12 and 9) at −36 °C has been established; at room temperature, the hexammine ZrBr₄ · 6NH₃ is the stable species which becomes ZrBr₄ · 2NH₃ at 200 °C. When treated with an excess of NH₄CN in liquid ammonia, complete replacement of bromide ions by cyanide occurs to give an inseparable mixture of Zr(CN)₄ · 2NH₃ and NH₄Br. The chloride and bromide of zirconium(III) also undergo no ammonolysis in liquid ammonia; the ammoniates stable at room temperature are ZrCl₃ · 2.5NH₃ and ZrBr₃ · 6NH₃.     

Triphenylbismuth seven-coordinate complexes of molybdenum(II) and tungsten(II)

The seven-coordinate complexes [MI₂(CO)₃(NCMe)₂] (M = Mo and W) react with one equivalent of BiPh₃ in CH₂Cl₂ at room temperature to give the monoacetonitrile complexes [MI₂(CO)₃(NCMe)(BiPh₃)]. The molybdenum complex [MoI₂(CO)₃(NCMe)(BiPh₃)] after stirring in CH₂Cl₂ at room temperature for 5 h affords the iodide-bridged dimer [Mo(μ-I)I(CO)₃(BiPh₃)]₂, whereas the tungsten complex [WI₂(CO)₃(NCMe)(BiPh₃)] does not appear to dimerise even after stirring for 48 h in CH₂Cl₂ at room temperature. Reaction of [MI₂(CO)₃(NCMe)₂] with two equivalents of BiPh₃ gives the bistriphenylbismuth compounds [MI₂(CO)₃(BiPh₃)₂] in good yield. The new mixed ligand complexes [MI₂(CO)₃L(BiPh₃)] were prepared either by reaction of [MI₂(CO)₃(NCMe)(BiPh₃)]in situ with one equivalent of L(L = P(OPh)₃), or an in situ reaction of [MI₂(CO)₃(NCMe)L] (L = PPh₃ and SbPh₃; and L = AsPh₃ and PPh₂Cy (for M = Mo only) with an equimolar quantity of BiPh₃. Reaction of [MoI₂(CO)₃(NCMe)(BiPh₃)] with one equivalent of 2,2′-bipyridyl (bipy) in CH₂Cl₂ at room temperature afforded the cationic complexes [MoI(CO)₃(bipy)(BiPh₃)]I in good yield. The complex [WI₂(CO)₃(NCMe)(BiPh₃)] (prepared in situ) reacts with two equivalents of NaS₂CNMe₂·2H₂O to eventually give the non-triphenylbismuth containing product [W(CO)₃(S₂CNMe₂)₂] in high yield.     

Reactivities of hydroxylamine and sodium bisulphite with carbonyl-containing haems with the prosthetic groups of the erythrocyte green haemoproteins

The reactivities of alkaline NH(2)OH and neutral NaHSO(3) with carbonyl and olefinic groups conjugated with the tetrapyrrole nucleus of haems were studied. The reactions were carried out with 2-3nmol of haem a, spirographis haem, isospirographis haem, 2,4-diacetyldeuterohaem and protohaem. Vinyl side chains were found to be insensitive to the chemical action of both alkaline NH(2)OH and neutral NaHSO(3). The formyl-containing haems reacted rapidly with both reagents at room temperature, as evidenced by sizable hypsochromic shifts of the reduced pyridine haemochrome spectrum. In less alkaline solution, the reactions of these formyl-containing haems with NH(2)OH were much slower. 2,4-Diacetyldeuterohaem reacted with alkaline NH(2)OH, but not with neutral NaHSO(3). These rapid, simple and straightforward tests are readily usable in differentiating among formyl, acetyl and other electron-withdrawing side chains conjugated with the tetrapyrrole ring of haems. We applied these observations to an investigation of the two unique prosthetic groups of the bovine erythrocyte green haemoproteins. The prosthetic groups of these two proteins were isolated and spectrally characterized. Under the conditions used, the haems did not react with either NH(2)OH or NaHSO(3), but were altered by dithionite, suggesting that the previous interpretation that a formyl group was present [Hultquist, Dean & Reed (1976) J. Biol. Chem.251, 3927-3932] may have been premature. These studies also provide evidence that the alpha-hydroxyfarnesylethyl side chain of haem a affects the alpha-band maximum, but not the beta- or Soret bands of the reduced pyridine haemochrome spectrum of haem a.     

The application of the AMB protective group in the solid-phase synthesis of methylphosphonate DNA analogues.

Partially methylphosphonate-modified oligodeoxynucleotides were synthesized on solid-phase by employing the easily removable 2-(acetoxymethyl)benzoyl (AMB) group as base-protecting group. Although a rapid AMB deprotection can be accomplished in methanolic potassium carbonate, the lability of the methylphosphonate linkage towards potassium carbonate/methanol excludes the use of this deprotection reagent. Thus, saturated ammonia solution in methanol was investigated as an alternative reagent for AMB removal. It is demonstrated that the combination of the AMB protective group and ammonia/methanol as deprotection reagent significantly improves the synthesis of methylphosphonate-modified DNA fragments. A mild overnight treatment at room temperature is sufficient for complete removal of the AMB group, whereas deprotection of conventionally protected oligonucleotides requires much longer exposure to basic conditions at elevated temperatures.     

Reactivity of ubiquinones and ubiquinols with free radicals

The reactivity of quinones 1-4 and of the corresponding quinols 5-8 towards carbon- and oxygen-centred radicals were studied. All quinones bearing at least one nuclear position free, readily react with alkyl and phenyl radicals to afford the alkylated quinones 12-24; however, quinones 1 and 3 reacted with 2-cyano-2-propyl radical to yield products (the mono- and di-ethers 9-11) derived from the attack on the carbonylic oxygen. The reactions carried out on quinones with the benzoyloxy radical led to no reaction products and in the case of Q₁₀, the isoprenic chain also remained unchanged. Quinols 5-8 reacted only with oxygencentred radicals (benzoyloxy and 2-cyano-2-propylperoxy radicals) to give the corresponding quinones. The isoprenic chain of Q₁₀ did not undergo attack even with peroxy radicals. Carbon-centred radicals resulted unable to abstract hydrogen from the studied quinols.     

Chlorination studies II. The reaction of aqueous hypochlorous acid with α-amino acids and dipeptides

HOCl by oxidative decarboxylation converts several α-amino acids into a mixture of the corresponding nitriles (major) and aldehydes (minor product). In addition, chlorination of the ring of tyrosine was observed. Cysteine when reacted with HOCl yielded cystine and cysteic acid, while with cystine, cysteic acid was the only product identified. The nitrogen bond of several dipeptides was found to be resistant to aqueous HOCl at room temperature. Chlorination of these compounds gave the corresponding N,N-dichlorodipeptide.     

On the mechanism of silylformylation catalyzed by Rh-Co mixed metal complexes

Possible mechanisms for the silylformylation of 1-alkynes catalyzed by Rh₂Co₂(CO)₁₂ are investigated. Novel Rh-Co mixed metal complexes, (PhMe₂Si)₂Rh(CO)nCo(CO)₄ (n = 2 or 3) (3) and RhCo(HC≡CBuⁿ)(CO)₅ (5), are found to play important roles in this catalysis. The reaction of 3 with 1-hexyne and HSiMe₂Ph at ambient temperature and pressure of CO gives n-BuC(CHO)=CHSiMe₂Ph (1a, Z/E = 95/5), (PhMe₂Si)₂Rh(CO)₃Co(CO)₄ (3-B) and an Rh-Co mixed metal butterfly complex, h₂Co₂(HC≡CBuⁿ)(CO)₁₀ (4). The reaction of 5 with 1-hexyne and HSiMe₂Ph under the same ambient conditions affords 1a (100% Z) very cleanly as the sole reaction product. The crossover experiments u sing RhCo(DC≡CBuⁿ)(CO)₅(5-d), 1-hexyne-1d and DSiMe₂Ph strongly support the mixed metal bimetallic catalysis and involvement of bis(alkyne)-Rh-Co species. The most plausible catalytic cycle of silylformylation which can accommodate all the observed results is proposed.     

Preparation of sugarcane bagasse hemicellulosic succinates using NBS as a catalyst

The chemical modification of native sugarcane bagasse hemicelluloses with succinic anhydride using N-bromosuccinimide as a catalyst and N,N-dimethylacetamide/lithium chloride system as solvent was studied. The parameters optimised included succinic anhydride concentration by the molar ratio of succinic anhydride/anhydroxylose units in native hemicelluloses from 1:1 to 9:1, reaction time 0.5–6 h, NBS concentration 0.5–3.0%, and reaction temperature 25–85 °C required in the process. Results were also compared with other catalysts such as pyridine, DMAP, H₂SO₄, and other two tertiary amine catalysts, N-methyl pyrrolidine, and N-methyl pyrrolidinone. The degree of substitution of succinylated hemicelluloses ranged between 0.19 and 1.39, depending on the experimental conditions. FT-IR and ¹H and ¹³C NMR spectroscopic characterization of the esterified polymers indicated a monoester substitution. The thermal stability of the succinylated hemicelluloses decreased upon chemical modification.     

Reaction of sulfur diimides with organoboranes — studied by multinuclear magnetic resonance in solution

Reactions between sulfur diimides R(NSN)R′ (R=R′=^tBu (1a), SiMe₃ (1b), SnMe₃ (1c); R=^tBu, R′=SnMe₃ (1d); R=SiMez₃, R′=SnMe₃ (1e)) and various organoboranes were studied, and the products were characterized by multinuclear magnetic resonance data (¹H, ¹¹B, ¹³C, ¹⁵N, ²⁹Si and ¹¹⁹Sn NMR). Tetraalkyldiboranes(6) (Et₂BH₂BEt₂ (2), dimeric 9-borobicyclo[3,3.1]nonane (3)) react with 1a and 1b by 1,3-hydroboration to give the N-sulfanyl-dialkylaminoboranes 4 and 5 which are instable with respect to eliminatio of short-lived [R---NS]. Trialkylboranes (Et₃B (8)) react only sluggishly with 1a, but more readily with 1b mainly via S-ethylation, formally a 1,2-ethyloboration, to give the diethylborylamido-imino-ethanesulfinic acid 9b decomposes slowly at room temperature via ethene elimination to give 4b, followed by further decomposition via [R---NS] elimination. The compounds 9 can be prepared independently from the reaction between the N-lithio-imino-ethanesulfinic acid amide 10 and diorganoboron halides. The molecular structure of the lithium amide 10a (R=R′=tBu) was determined by X-ray analysis as a dimer in which the four nitrogen, two sulfur and two lithium atoms adopt a boat conformation, in contrast with other known derivatives of this type. If the sulfur diimides bear at least one trimethylstannyl group (1c-e), their reactions with Et₃B (8), iPr₃B (12) or 9-iso-butyl-9-borabicyclo[3,3,1]nonane (13) lead to the novel aminoboranes 14–16. These are products of a 1,1,-organoboration, since the Me₃Sn group moves from one nitrogen atom to the other, and both the boryl and an alkyl group end up at the same nitrogen atom.     

Enantioselectivity of Epoxide Formation from Halohydrins by Means of Flavobacterium Rigense

The formation of epoxides from several halohydrins was achieved using resting cells from Flavobacterium rigense. The reaction showed a high substrate specificity for halohydrins with a terminal halogen atom but only low enantioselectivity (12-58% e.e.). The epoxides always had the (S)-configuration. Substrates which in the halogen atom was replaced by another leaving group (-O-SO₂CH₃, -O-Tos, -N₃) were not accepted. An attempt to improve the enantioselectivity by using a two phase system consisting of an aqueous and an organic solvent phase was not successful.     

Acid-catalysed Reactions of Methyl β-(3,4-Dimethoxyphenyl) glycidate

Methyl trans-β-(3,4-dimethoxyphenyl)glycidate was found to rearrange to methyl 3,4-dimethoxyphenylpyruvate in high yield in DMSO, DMF or HMPT solution in the presence of boron trifluoride etherate or sulfuric acid at room temperature. It was also revealed that the glycidate, when treated with boron trifluoride in methanol at room temperature, opened at the β-position to give methyl β-(3,4-dimethoxyphenyl)-α-hydroxy-β-methoxy-propionate in 94% yield, which was a mixture of erythro and threo isomers in the ratio of 1 to 2.     

Chiral N-phosphonyl imine chemistry: Asymmetric synthesis of α,β-diamino esters by reacting phosphonyl imines with glycine enolates

Chiral phosphonyl imines attached with N-isopropyl protection group were found to react with lithium glycine enolates under convenient conditions to give α,β-diamino esters. Thirteen examples have been examined in good to excellent chemical yields (85–97%) diastereoselectivity (up to 99% de). By treating with HBr at room temperature, the chiral auxiliary can be readily removed and recycled. The absolute structure has been unambiguously determined by converting a product to a known sample.     

On the reaction of N-acetylchondrosine, N-acetylchondrosine 6-sulfate,chondroitin 6-sulfate,and heparin with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

N-Acetylchondrosine was activated at pH 4.75 with excess 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to give an O-acylisourea that consists of equimolar amounts of N-acetylchondrosine and 1-(3-dimethylaminopropyl)-3-ethylurea, with concomitant uptake of 0.94 mol of hydrogen ion per mol of N-acetylchondrosine. The product was treated with sodium borohydride to give a carboxyl-reduced disaccharide, but it did not react with a nucleophile reagent, such as glycine ethyl ester, over the pH range of 4.75–11.0. The O-acylisourea was hydrolyzed mostly into N-acetylchondrosine and 1-(3-dimethylaminopropyl)-3-ethylurea with 0.1m sodium carbonate overnight at room temperature, but a small proportion was transformed into the N-acylurea. N-Acetylchondrosine 6-sulfate, chondroitin 6-sulfate, and heparin were also activated at pH 4.75 with excess 1-(3-dimehtylaminopropyl)-3-ethylcarbodiimide hydrochloride to give the corresponding O-acylisoureas containing one mol of 1-(3-dimethylaminopropyl)-3-ethylurea moiety per mol of uronic acid residue, respectively.     

New synthesis of 2β-hydroxy-19-oxoandrost-4-ene-3,17-dione and its 2β-O analog

Treatment of 19-[oxygenated]-androst-4-ene-3,17-dione with Mn(AcO)₃ and ClCH₂COOH in benzene gave epimeric mixtures of the corresponding 2ξ-chloroacetates and 2ξ-acetates. The products were processed to give the title compound. For the synthesis of the 2-¹⁸O analog, ClCH₂C¹⁸OOH was used, which was prepared from ClCH₂COCl.     

Kinetic cyclohexylidenation and isopropylidenation of aldose diethyl dithioacetals

Aldose diethyl dithioacetals react with 1.2 equivalents of 1-ethoxycyclohexene or 2-methoxypropene in N,N-dimethylformamide at 0° with p-toluenesulfonic acid as catalyst to give the five-membered ring acetal attached to the two terminal oxygen atoms as the major product in every case. In most instances, a small proportion of the terminal, six-membered ring acetal was also obtained, and in a few cases, terminal seven-membered ring acetals were also isolated. Cyclohexylidenation at room temperature gave the same products, but isopropyl-idenation at room temperature resulted in certain cases in partial rearrangement. Cyclohexylidenation reactions gave smaller proportions of the minor six- and seven-membered ring products. Structures were established from ¹³C-n.m.r. and mass spectra. The ¹³C-n.m.r. spectra of model cyclohexylidene derivatives were found very similar to those of isopropylidene derivatives previously studied. Two new features useful for structure determination were noted when the spectra of the precursor diols were compared with those of both types of derived acetals; the chemical shift of C-2 of a 1,3-propanediol derivative was shifted upfield by 6–9 p.p.m. on acetalation and the shifts of the diol carbon atoms attached to oxygen were affected according to the type of acetal and ring-size formed. Similar observations were made for methylene acetals.     

Studies on asymmetric induction associated with the coupling of N-acylamino acids and N-benzyloxycarbonyldipeptides.

The asymmetric induction occurring during aminolysis by an amino acid benzyl ester of the 5(4H)-oxazolones obtained from N-acyl-DL-valine for acyl = formyl, acetyl, benzoyl, trifluoroacetyl and N-benzyloxycarbonyl-Xaa where Xaa = Gly, Ala and Leu in dichloromethane and dimethylformamide at +5 degrees and -5 degrees was determined by analysis of the epimeric products by high-performance liquid chromatography after removal of protecting groups by hydrogenolysis. The influence of the side-chain of the activated residue on induction was assessed by examining aminolysis of the 5(4H)-oxazolones from N-benzyloxycarbonyl glycyl-Xaa-OH for Xaa = Ala, Leu, Val, and Phe. The contribution of induction to the epimeric content of products obtained from couplings mediated by N,N'-dicyclohexylcarbodiimide in the presence and absence of l-hydroxybenzotriazole, and by the mixed-anhydride method, were calculated. The induction was affected at varying levels by the nature of the N-acyl group, the side-chain of Xaa, the nature of the aminolyzing nucleophile, the nature of the solvent, and the temperature, with diastereomeric excesses reaching -32 and +53. The influence of the side-chain of Xaa on the induction was different in the two solvents. For the N-acyl series, the epimeric content of products did not always correctly reflect the relative tendencies of the derivatives to racemize. The order for epimeric content of the products also depended on the method of coupling.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^-1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^-1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

A reaction for the simple sensitive fluorimetric assay of heparin and 2-amino sugars

1. 3,5-Diaminobenzoic acid reacted rapidly with the product from HNO(2) deamination of heparin, heparan sulphate and 2-amino-2-deoxyhexoses under very mild conditions (pH3.0 and 37 degrees C) to give stable fluorescent derivatives. 2. The fluorescence yield was rectilinearly related to the concentration of heparin etc. Less than 0.1mug of 2-amino-2-deoxyhexose was easily measurable in standard cuvettes. 3. The deamination products of glucosamine and (particularly) galactosamine were labile in the HNO(2) reagent, with half-lives of 20-40min at room temperature. At 0 degrees C they were much more stable. The analogous product from heparin was not so labile. 4. Under the standard conditions, and at room temperature, relative fluorescence yields (d-glucosamine=1.0) were: d-galactosamine, 0.75; d-gulosamine, 0.38; d-mannosamine, approx. 0.20. 5. Neutral sugars, chondroitin sulphates, DNA and N-acetylneuraminic acids did not react, nor did N-acetylamino sugars or non-deaminated hexosamines. 6. It is suggested that the Dische-Borenfreund [Dische & Borenfreund (1950) J. Biol. Chem.184, 517-522] indole method, the Kissane-Robins [Kissane & Robins (1962) J. Biol. Chem.233, 184-188] DNA assay and the proposed amino sugar method are all examples of simple aldehyde reactions. The specificity of the proposed method is considerably greater than that of the Dische-Borenfreund procedure, partly because of the much milder reaction conditions. 7. The proposed method is very reproducible, about 50-100 times as sensitive as the Elson-Morgan reaction, and 10-50 times as sensitive as the Dische-Borenfreund procedures. It is also convenient; acid hydrolysates of amino sugar-containing compounds can be directly neutralized with sodium acetate solution.     

Epimerization at C2 of Methyl 5-O-Benzyl-2-deoxy-2-fluoro-α-D-pentofuranosides upon Oxidation

Abstract

Oxidation of 1 with DMSO-acetic anhydride resulted in the formation of a mixture of epimeric ketones 2 and 3 in the ratio of ?3:1 in high combined yield. Acetolysis of methyl glycoside 5 afforded 1-O-acetyl-3,5-di-O-benzoyl-2-deoxy-2-fluoro-β-D-ribofuranoside (6)(83%). The latter was reacted with silylated N⁶-benzoyladenine to give α- and β-ribosides (1:3.7; 61%, combined).