Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.     

Gastric emptying, absorption, and carbohydrate oxidation during prolonged exercise.

This study was designed to examine aspects of digestive function that may limit assimilation of water and oxidation of orally ingested carbohydrate (CHO) during exercise. Eight males completed a crossover study in which each cycled on four occasions for 80 min at 70% maximal O2 consumption. Beverage was consumed at 0, 20, 40, and 60 min. Beverages were water, 4.5% glucose (4.5G), 17% glucose (17G), and 17% maltodextrin (17MD). CHO beverages contained 20 meq/l NaCl and were 13C enriched to measure exogenous CHO oxidation. Gastric (beverage) volume was measured at 80 min. Water uptake was estimated by including 2H2O in the beverage and measuring 2H accumulation in blood. Jejunal perfusion tests were conducted at rest with the same subjects and beverages. In 60 min, 1,294 +/- 31 (SE) ml were ingested; at 80 min, volumes emptied with H2O (1,257 +/- 32 ml) and 4.5G (1,223 +/- 32 ml) were greater than with 17G (781 +/- 56 ml) and 17MD (864 +/- 71 ml; P less than 0.05). Total CHO oxidized was similar with all beverages, but there was a greater increase in exogenous CHO oxidation over time with 17G and 17MD than with 4.5G; 54, 19, and 18% of the CHO ingested with 4.5G, 17G, and 17MD, respectively, was oxidized. This represents 57, 32, and 27%, respectively, of the CHO emptied from the stomach. 2H accumulation in the blood was more rapid with H2O and 4.5G than with 17G or 17MD. Net jejunal water absorption was greater from 4.5G than from water. Net water absorption was also observed from 17MD, whereas net secretion was observed with 17G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bile acids. LXVIII. Allylic and benzylic photo-chemical oxidation of steroids

To provide 7-oxocholesterol derivatives in yields superior to those obtained by chemical oxidation, the preparation of steroidal allylic or benzylic ketones was studied. Air-induced oxidation was investigated with a highly selective low energy UV lamp in the presence of mercuric bromide. With this procedure cholesteryl acetate, 5-cholestene-3β, 27-diol diacetate, 24(R)-24-methyl-5-cholesten-3β-yl acetate, 24(R)-24-ethyl -5-cholesten-3β-yl acetate and 24(R)-24-ethyl-(22E)-cholesta-5, 22-dien -3β-yl acetate were oxidized to the allylic keto-derivative in good yields; estradiol-17β diacetate was similarly converted to the 6-oxo-product in improved yield. This method can be very useful in the synthesis of 7-oxocholesteryl acetate and its analogs and 6-oxoestratrienes.     

New D-modified androstane derivatives as aromatase inhibitors.

Starting from a 16-oximino derivative of 5-androstene the newly-synthesized 16-oximino-17-hydroxy-17-substituted derivatives 2-4 gave by the Beckmann fragmentation reaction the corresponding D-seco derivatives 6-9. Besides, in the case of the 17-hydroxy-17-methyl-16-oximino derivative 2, as a result of the rearrangement, the hydrolysis product 5 of the 16-oximino group with the opposite configuration at the C-17 was obtained. By the Oppenauer oxidation and/or by dehydration of 7 with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), the corresponding derivatives 12, 13, and 14 were obtained. The structures of 6 and 12 were unambiguously proved by the appropriate X-ray structural analysis. Kinetic analysis for anti-aromatase activity showed that compound 12 expressed the highest inhibition in the denucleated rat ovarian fraction in comparison to other androstene derivatives (IC(50) was 0.42 microM). In comparison to aminoglutethimide (AG) activity, it was 3.5 times lower. The inhibition was competitive, with K(i) of 0.27 microM. Introduction of additional units of unsaturation (compounds 13 and 14) in D-seco derivatives did not increase anti-aromatase activity.     

Radioimmunoassay of contraceptive steroids. I. Synthesis of 6-oxomestranol 6-(O-carboxymethyl) oxime

A synthesis of 6-(O-carboxymethyl) oxime of 6-oxo-mestranol (3-methoxy-17-ethinyl-17β -hydroxy-1,3,5 (10)-estratrien-6-one) which was required for coupling with bovine serum albumin in order to produce a specific anti-sera for mestranol (3-methoxy-17-ethinyl-1,3,5 (10)-estratrien-17β-ol) has been described. 6-Oxoestradiol-17β 3-methyl ether was prepared from estradiol-17β 3,17-diacetate by chromic acid oxidation, followed by hydrolysis and methylation. It was converted to its O-carboxymethyloxime derivative which was smoothly oxidized by Jones reagent to the corresponding es estrone derivative. This was easily ethinylated with lithium acetylide-ethylenediamine complex to the desired compound. In an alternate approach to the desired compound, it was found that 6-oxoestradiol-17β 3- methyl ether could not be converted to its ketal under any of a variety of conditions. Ethinylation of 6-oxoestrone 3-methyl ether with limited amount of lithium acetylide reagent probably gave the 17α -ethinyl derivative as was indicated from IR and UV spectra, but its identity could not be further confirmed due to its extremely poor yield.     

Inhibition of mitochondrial palmitate oxidation by calmodulin antagonists

1. The effect of calmodulin antagonists on the rate of palmitate oxidation by isolated rat liver mitochondria was studied. 2. In the presence of 100 microM amitriptyline, chlorpromazine, prenylamine, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, palmitate oxidation was inhibited by 17, 34, 49, 31 and 37%, respectively. 3. The degree of inhibition of palmitate oxidation exerted by these chemical compounds did not appear to correlate appreciably with changes in mitochondrial membrane fluidity.     

Bioconversion of steroids by Cochliobolus lunatus--II. 11 beta-hydroxylation of 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate in dependence of the inducer structure.

The 11 beta-hydroxylase of the filamentous fungus Cochliobolus lunatus m 118 was induced with the substrate 17 alpha, 21-dihydroxypregna-1,4-diene-3,20-dione 17-acetate (11 beta-deoxyprednisolone 17-acetate) itself, substrate analogues, different pregnane compounds, sterols, intermediates of microbial sterol side-chain degradation or bile acids, together with 24 different steroids in a standardized test system. The resulting 11 beta-hydroxylation rate, leading to prednisolone 17-acetate and prednisolone, respectively, was determined and compared with the hydroxylation rate of non-induced cultures. The transformation yield strongly depended on the inducer structure. The microbial sterol side-chain degradation intermediates (20S)-20-hydroxymethylpregn-4-en-3-one and the corresponding pregna-1,4-diene compound caused the highest induction effects (induction factors 5.1 and 4.9, respectively). The metabolism of (20S)-20-hydroxymethylpregna-1,4-dien-3-one during the cultivation was elucidated. The induction effect decreased with the rising oxidation of the inducer. The significant increase of the 11 beta-hydroxylation rate of 1-dehydro-pregnane substrates by specific induction allows alternative pathways to glucocorticoid partial syntheses.     

Serum level of IgG autoantibodies against oxidized low density lipoproteins and lag-phase of serum oxidation in coronary heart disease--inverse correlation.

High affinity IgG autoantibodies (ABs) against oxLDLs and lag-phase of serum oxidation were tested in patients with coronary heart disease (CHD). Fifty one (37 M/14 F) patients with CHD defined as Q-wave myocardial infarction and/or stenosis of more than 50% and 51 (34 M/17 F) healthy blood donors as controls participated in this study. LDLs were isolated by gradient ultracentrifugation and oxidized with CuSO4. The modified LDLs (oxLDLs) or native LDLs (nLDLs) were used as antigens in an enzyme immunoassay (ELISA) to detect IgG ABs in both groups. The serum was oxidized by CuSO4 and the oxidation was monitored spectrophotometrically at lambda = 234 nm to follow the formation of conjugated diens. The lag-phase (in minutes) is the interval between the addition of CuSO4 to the serum and the beginning of extensive oxidation (increasing absorbance at 234 nm). The concentrations of total cholesterol, triglycerides, HDL-cholesterol, apo-A and apo-B were measured as well. The mean level of ABs against oxLDLs (expressed as optical density units) was 0.590 +/- 0.330 in CHD-patients vs 0.244 +/- 0.200 in controls (p < 0.001). The lag-phase in minutes was 47.00 +/- 27.19 in CHD-patients and 80.23 +/- 26.30 in controls (p < 0.001). A negative correlation between ABs levels and lag-phase was established in CHD-patients (r = -0.69, p < 0.001) and controls (r = -0.62, p < 0.001). A poor correlation was established between ABs levels or lag-phase, on one hand, and other measured parameters. In conclusion, the lag-phase of serum oxidation by Cu2+ could be informative for LDL susceptibility to modification and the extent of consequent humoral immune response.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.     

Synthesis of 19-oxygenated derivatives of the competitive inhibitor of aromatase, 5-androstene-4,17-dione.

19-Hydroxy- and 19-oxo-steroids 13 and 15, respectively, which are potential metabolites of the aromatase inhibitor 5-androstene-4,17-dione (3), were synthesized from 19-(tert-butyldimethylsilyloxy)androst-5-en-17-one (5) or 4beta-acetoxyandrost-5-en-17-one (16), respectively, through 5alpha-bromo-4beta-hydroxy-6beta,19-epoxyandrostan+ ++-17-one (10) as a key intermediate in each sequence. Reaction of the 19-siloxy compound 5 with Br2 gave 5alpha-bromo-6beta,19-epoxide 8, which was treated with N,N'-dimethylacetamide followed by reaction with N-bromoacetamide and 0.28 M HCIO4, to yield compound 10. On the other hand, treatment of the 4beta-acetoxy steroid 16 with N-bromoacetamide-HCI04 followed by oxidation with Pb (IV) acetic acid and I2 under irradiation and subsequent hydrolysis with K2CO3 also produced compound 10 and in better yield than that in the above synthesis. Jones oxidation of the 4beta-ol 10 followed by reductive debromination with zinc dust yielded the 19-ol 13 in low yield as well as 6beta,19-epoxy-4-one 12 as the major product. Furthermore, the major product 12 was converted into the 19-ol 13 in moderate yield from compound 12 through acetolysis and subsequent alkaline hydrolysis. The 19-oxo steroid 15 was obtained after treatment of compound 13 with pyridinium dichromate. Compounds 13 and 15 were analyzed as the methoxime-trimethylsilyl and methoxime-dimethylisopropylsilyl derivatives and the methoxime derivative, respectively, using gas chromatography-mass spectrometry.     

Synthesis of 17 beta-[(1S)-1-hydroxy-2-propynyl]- and 17 beta-[(1R)-1-hydroxy-2-propynyl]androst-4-en-3-one. Potential suicide substrates of 20 alpha- and 20 beta-hydroxysteroid dehydrogenases.

The title compounds have been synthesized for evaluation as potential suicide substrates of 20 alpha- and 20 beta-hydroxysteroid dehydrogenases. Synthesis was achieved by the following route. Acetylenedimagnesium bromide was reacted with 3 beta-hydroxyandrost-4-ene-17 beta-carboxaldehyde to give 17 beta-[(1R,S)-1-hydroxy-2-propynyl] androst-4-en-3 beta-ol. Separation of the R and S diols was achieved by HPLC (high pressure liquid chromatography). Selective oxidation of the 3 beta-hydroxyl group with Jones reagent at 0 degrees gave the title compounds. Further oxidation with Jones reagent converted each acetylenic alcohol to the conjugated acetylenic ketone, 17 beta-(1-oxo-2-propynyl)androst-4-en-3-one.     

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity.

The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.     

Studies of the synthesis of biomarkers. VII. Synthesis of 5 alpha-(17R,20R)-14,15-secocholestane

5 alpha-(17R,20R)-14,15-Secocholestane (12) was synthesized from cholesterol (1) in 12 steps. The key intermediate, 5 alpha-cholest-14-en-3 beta-yl acetate (4), underwent ozonization, reduction, hydrolysis, and oxidation to provide 5 alpha-14,15-secocholesta-3,14,15-trione (8). One of the Clemmensen reduction products of 8 is 5 alpha(17R,20R)-14,15-secocholest-15-ol (11); treatment of the alcohol (11) with tosyl chloride and subsequent reduction with lithium aluminum hydride yielded the target molecule (12).     

Microbiological degradation of bile acids. The preparation of hexahydroindane derivatives as substrates for studying cholic acid degradation.

Relatively large amounts of 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb), which is believed to be one of the intermediates involved in the degradation of cholic acid (I), were needed to identify is further degradation products. A simple method for the preparation of this compound was then investigated. Arthrobacter simplex could degrade-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIa) to 3-(1beta-carboxy-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVa) in good yield, the structure of which was established by partial synthesis. It was therefore expected that, if a similar degradation by this organism occurred with 17alpha-hydroxy-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIb), which is easily obtained by chemical oxidation of commercially availabe 17alpha-hydroxydeoxycorticosterone, the resulting product, 3-(1beta-carboxy-3aalpha-hexahydro-1alpha-hydroxy-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVb), could be readily converted chemically into the required dioxocarboxylic acid, (IIb). Exposure of compound (IIIb) to A. simplex produced, as expected, compound (IVb) which was then oxidized with NaBiO3 to give a reasonable yield of compound (IIb).     

Oxidation of methionine residues in antithrombin. Effects on biological activity and heparin binding

Commercially available human plasma-derived preparations of the serine protease inhibitor antithrombin (AT) were shown to contain low levels of oxidation, and we sought to determine whether oxidation might be a means of regulating the protein's inhibitory activity. A recombinant form of AT, with similarly low levels of oxidation as purified, was treated with hydrogen peroxide in order to study the effect of oxidation, specifically methionine oxidation, on the biochemical properties of this protein. AT contains two adjacent methionine residues near the reactive site loop cleaved by thrombin (Met314 and Met315) and two exposed methionines that border on the heparin binding region of AT (Met17 and Met20). In forced oxidations with hydrogen peroxide, the methionines at 314 and 315 were found to be the most susceptible to oxidation, but their oxidation did not affect either thrombin-inhibitory activity or heparin binding. Methionines at positions 17 and 20 were significantly oxidized only at higher concentrations of peroxide, at which point heparin affinity was decreased. However at saturating heparin concentrations, activity was only marginally decreased for these highly oxidized samples of AT. Structural studies indicate that highly oxidized AT is less able to undergo the complete conformational change induced by heparin, most probably due to oxidation of Met17. Since this does not occur in less oxidized, and presumably more physiologically relevant, forms of AT such as those found in plasma preparations, oxidation does not appear to be a means of controlling AT activity.     

Low concentrations of 17beta-estradiol reduce oxidative modification of low-density lipoproteins in the presence of vitamin C and vitamin E.

Micromolar concentrations of estradiol are required to inhibit the oxidation of low-density lipoproteins (LDL) in vitro. Recent evidence suggests that estradiol must be modified before it can become an effective antioxidant at physiological levels. Our aim was to determine other possible conditions under which low concentrations of 17beta-estradiol can reduce LDL oxidation. LDL susceptibility to oxidation was monitored by measurements of conjugated diene formation. High levels of 17beta-estradiol reduced oxidative modification of LDL. Vitamin C and vitamin E also increased LDL resistance to Cu2+-mediated oxidation. More importantly, 10 nM 17beta-estradiol, which on its own had no effect, exhibited significant antioxidant actions in the presence of either vitamins C or E. In conclusion, supraphysiological concentrations of 17beta-estradiol are required to exert antioxidant effects directly in vitro. However, in the presence of vitamins C and E, concentrations of 17beta-estradiol close to physiological levels can also protect LDL from oxidation.     

Energy-conserving reactions in phosphorylating electron-transport particles from Nitrobacter winogradskyi. Activation of nitrite oxidation by the electrical component of the protonmotive force.

1. In electron-transport particles (ET particles) prepared from Nitrobacter winogradskyi, the uncoupling agent carbonyl cyanide phenylhydrazone increased the rate of NADH oxidation but decreased the rate of oxidation of NO2-. Its effectiveness in stimulating NADH oxidation closely paralleled its effectiveness in inhibiting NO2- oxidation. 2. In the presence of ADP and phosphate the oxidation of NADH was stimulated, whereas the oxidation of NO2- was inhibited. In the presence of excess of Pi the concentration dependence with respect to ADP was the same for acceleration of NADH oxidation and inhibition of NO2- oxidation. 3. Oligomycin inhibited NADH oxidation and stimulated the oxidation of NO2-. The concentration of oligomycin required to produce half-maximal effect in both systems was the same. 4. The apparent Km for NO2- was not affected by ADP together with Pi, by uncoupling agent or by oligomycin. 5. With NADH as substrate, classical respiratory control was observed. With NO2- as substrate the respiratory-control ratio was less than unity. 6. A reversible uptake of H+ accompanied the oxidation of NO2- by ET particles. 7. In the presence of NH4Cl or cyclohexylamine hydrochloride, H+ uptake was abolished and increased rates of NO2- oxidation were observed. When valinomycin was present in the reaction medium, low concentrations of NH4Cl inhibited NO2- oxidation. 8. Pretreatment of ET particles with oligomycin enhanced the stimulation of NO2- oxidation induced by NH4Cl or by cyclohexylamine hydrochloride. Pretreatment with the uncoupler carbonyl cyanide phenylhydrazone prevented these stimulations. 9. In the presence of dianemycin together with K+, the uptake of H+ was abolished and the rate of NO2- oxidation was increased. In contrast, in the presence of valinomycin together with K+, the uptake of H+ was increased, and the rate of NO2- oxidation decreased. 10. Sodium tetraphenylboron was found to be an inhibitor of NO2- oxidation, but caused a stimulation of NADH oxidation which was dependent on the presence of NH4Cl or cyclohexylamine hydrochloride. 11. It is concluded that the enhanced rate of NO2- oxidation observed in the absence of energy-dissipating processes clearly relates to some state before the involvement of adenine nucleotides, and it is suggested that the oxidation of NO2- generates a protonmotive force, the electrical component of which controls the rate of NO2- oxidation.     

Synthesis of 7-(2,4-dichlorophenyl)-D-erythro-3-hydroxy-5-heptanolide, 6-(2,4-dichlorophenyl)-D-erythro-2,4-dihydroxyhexane-1-sulfonic acid, and 6-(2,4-dichlorophenyl)-D-erythro-2,4-dihydroxyhexylphosphonic acid.

6-(2,4-Dichlorophenyl)-D-erythro-1,2,4-hexanetriol, synthesised from D-glucose, was partially silylated, then reacted with 2-methoxypropene to afford 1-O-tert-butyldimethylsilyl-6-(2,4- dichlorophenyl)-2,4-O-isopropylidene-D-erythro-1,2,4-hexanetriol (17). Desilylation of 17 gave 6-(2,4-dichlorophenyl)-2,4-O-isopropylidene-D- erythro-1,2,4-hexanetriol, which was converted into the 1-tosylate 18 and the 1-bromo derivative 19. Reaction of 18 with potassium thiolbenzoate gave, after debenzoylation, oxidation, and deprotection, 6-(2,4-dichlorophenyl)-D-erythro-2,4-dihydroxyhexane-1-sulfonic acid (4). Reaction of 18 or 19 with triethyl phosphite gave, after deprotection, 6-(2,4-dichlorophenyl)-D-erythro-2,4-dihydroxyhexyl-phosphonic acid (5), and reaction of 19 with potassium cyanide gave, after subsequent hydrolysis and deprotection, 7-(2,4-dichlorophenyl)-D-erythro-3-hydroxy-5-heptanolide (3).