On the stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile).

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies. Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S13) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.     

Physicochemical properties of flavodoxin from Desulfovibrio vulgaris

Various physicochemical and biochemical properties of the most potent uncoupler of oxidative phosphorylation known to date 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847), such as pH dependence of the uncoupling activity and binding to mitochondria, spectral properties in the presence of different types of liposomes, biopolymers and mitochondria, and effects on model membrane systems have been investigated. From the results, it is concluded that the uncoupler most likely is localized in the phospholipid part of the membrane.     

Radical-scavenging activity of butylated hydroxytoluene (BHT) and its metabolites

To clarify the radical-scavenging activity of butylated hydroxytoluene (BHT), a food additive, stoichiometric factors (n) and inhibition rate constants (k_inh) were determined for 2,6-di-tert-butyl-4-methylphenol (BHT) and its metabolites 2,6-di-tert-butyl-p-benzoquinone (BHT-Q), 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHA-CHO) and 3,5-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadiene-1-one (BHT-OOH). Values of n and k_inh were determined from differential scanning calorimetry (DSC) monitoring of the polymerization of methyl methacrylate (MMA) initiated by 2,2′-azobis(isobutyronitrile) (AIBN) or benzoyl peroxide (BPO) at 70 °C in the presence or absence of antioxidants (BHT-related compounds). The n values declined in the order BHT (1–2) > BHT-CHO, BHT-OOH (0.1–0.3) > BHT-Q (0). The n value for BHT with AIBN was approximately 1.0, suggesting dimerization of BHT. The k_inh values declined in the order BHT-Q ((3.5–4.6)×10⁴ M⁻¹ s⁻¹) > BHT-OOH (0.7–1.9×10⁴ M⁻¹ s⁻¹) > BHT-CHO ((0.4–1.7)×10⁴ M⁻¹ s⁻¹) > BHT ((0.1–0.2)×10⁴ M⁻¹ s⁻¹). The k_inh for metabolites was greater than that for the parent BHT. Growing MMA radicals initiated by BPO were suppressed much more efficiently by BHT or BHT-Q compared with those initiated by AIBN. BHT was effective as a chain-breaking antioxidant.     

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

Antioxidant properties of resveratrol and piceid on lipid peroxidation in micelles and monolamellar liposomes

The antioxidant activities of trans-resveratrol (trans-3,5,4′-trihydroxystilbene) and trans-piceid (trans-5,4′-dihydroxystilbene-3-O-β-d-glucopyranoside), its more widespread glycosilate derivative, have been compared measuring their inhibitory action on peroxidation of linoleic acid (LA) and the radical scavenging ability towards different free radicals (such as DPPH) and radical initiators. It has been found that the two stilbenes have similar antioxidant capacity, while the comparison with BHT (2,6-di-tert-butyl-4-methylphenol) and -tocopherol (vitamin E, vit. E), taken as reference, points out a slower but prolonged protective action against lipid peroxidation. Furthermore, piceid appears more efficacious than resveratrol as a consequence of the reaction of the latter with its radical form.

The DSC profiles of phosphatidylcholine liposomes of various chain lengths, and EPR measurements of spin labelled liposomes demonstrated that the susceptible hydroxyl group of these compounds are located in the lipid region of the bilayer close to the double bonds of polyunsatured fatty acids, making these stilbenes particularly suitable for the prevention and control of the lipid peroxidation of the membranes.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.     

Phloretin - an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation

The effect of phloretin on respiration by isolated mitochondria and submitochondrial particles was studied. In submitochondrial particles, both NADH- and succinate-dependent respiration was inhibited by phloretin. 50% maximum inhibition was reached at phloretin concentrations of 0.1 mM (NADH oxidation) and 0.7 mM (succinate oxidation). In isolated mitochondria, phloretin inhibited glutamate oxidation in both State 3 and State 4; 50% maximum inhibition occurred at about 30 microM. Succinate oxidation is inhibited in State 3 by phloretin, inhibition being half its maximum value at 0.5 mM, but in State 4 it is stimulated about 2-fold by phloretin at a concentration of 0.6 mM. Ascorbate oxidation is stimulated in both State 3 and State 4, maximum stimulation being equal to that obtained with an uncoupler of oxidative phosphorylation. Under all circumstances, phloretin lowered the transmembrane electrical potential difference in isolated mitochondria. These results are discussed in terms of mosaic non-equilibrium thermodynamics. We conclude that phloretin is both an uncoupler and an inhibitor of oxidative phosphorylation.     

Analysis of light-induced proton uptake in isolated chloroplasts

1. 1. A simple kinetic analysis of light-induced proton uptake into chloroplasts is presented. It is derived from a model of the reaction in which the incoming proton is obligatorily bound by an intra-chloroplast component, and allows quantitative analysis of the effect into parameters of light and dark rate constants and the availability of the chloroplast component.

2. 2. The effect of the following agents on the derived parameters has been measured: electron and energy transfer inhibitors, uncouplers, NaCl concentration, light intensity and pH.

3. 3. A maximal ratio of 4 protons taken up per electron transported has been observed, using ferricyanide as an electron acceptor.

4. 4. A stimulation of light-induced proton uptake by phosphate or arsenate, ADP and Mg has been observed. It was not sensitive to concentrations of Dio-9, which eliminated ATP synthesis.

5. 5. The results are seen as inconsistent with the chemiosmotic theory of energy coupling as presently presented. It is suggested that they may be interpreted in terms of a model in which the function of the proton pump is to enable co-transport into the chloroplasts of the negatively charged complex of phosphate, ADP and Mg.

The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria

1. The effects of phosphate and electron transport on the ATPase induced in ratliver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin.

2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase.

3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (> 0.2 μM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin.

4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations.

5. It is proposed that the steady-state concentration of endogenous P_i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P_i.     

Methylenedioxy- and methoxyflavones from Melicope coodeana syn. Euodia simplex

Three new natural products, 3,8-dimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone, 3,6,8-trimethoxy-5,7-dihydroxy-3′,4′-methylenedioxyflavone and 3,6,8,3′,4′-pentamethoxy-5,7-dihydroxyflavone were isolated from Melicope coodeana syn. Euodia simplex (Rutaceae) along with 3,6,3′-trimethoxy-5,7,4′-trihydroxyflavone and 3,3′-dimethoxy-5,7,4′-trihydroxyflavone. The structural assignments are based on ¹H and ¹³C NMR data, including discussion of the chemical shifts of C-2 in 3,5-dihydroxy- and 3-methoxy-5-hydroxyflavones. The presence of highly methoxylated and methylenedioxyflavones is characteristic of the genus Melicope, and the present findings support the recent transfer of Euodia simplex to Melicope.     

Structure—activity studies leading to potent chloride channel blockers: 5e-tert-butyl-2-[4-(substituted-ethynyl)phenyl]-1,3-dithianes

5e-tert-Butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes with selected functional groups (R) on the ethynyl moiety are potent blockers of the GABA-gated chloride channel measured as inhibitor concentration (IC₅₀) for 4-n-[³H]propyl-1-(4-ethynylphenyl)-2, 6,7-trioxabicyclo[2.2.2]octanebinding to bovine brain membranes. The terminal R substituents were introduced by coupling 5e-tert-butyl-2e-(4-iodophenyl)-1,3-dithiane with HC ≡ CR or 5e-tert-butyl-2e-(4-ethynylphenyl)-1,3-dithiane with XR. The potency of the parent compound (R=H) with an IC₅₀ of 21 μM is equaled or exceeded by up to 7-fold (i.e. IC₅₀ = 3–21 μM) by several carboxylic acids [R = (CH₂)_nCO₂H (n = 0–3), (CH_2nOCH₂CO₂H (n = 1–3) and CH₂SCH₂ CO₂H] and their esters and two phosphonic acids (CH₂CH₂PO₃H₂ and CH₂OCH₂PO₃H₂) but not their esters. These carboxyl and phosphonic acids (and their salts) include the most potent water-soluble chloride channel blockers known. Conversion to the monosulfones increases activity of the R = H and CH₂OH analogs by 1.2- to 3-fold but decreases that of the R = CH₂CH₂CO₂R′ (R′ = H or CH₃) derivatives by 3- to 13-fold. Quantitative structure-activity analyses for 44 2-[4-(substituted-ethynyl)phenyl]-dithianes suggests that the principal feature of the R substituent for high activity is its polarizable volume modeled as molecular refractivity, i.e. this substituent is not a well-defined pharmacophore and undergoes a structurally non-specific interaction with the receptor. These observations lay the background for preparing candidate affinity probes.     

Characterization of a Chinese hamster ovary cell line resistant to uncouplers

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.     

Azide inhibition of mitochondrial electron transport I. The aerobic steady state of succinate oxidation

The azide inhibition of the succinate oxidase activity of rat-liver mitochondria is specific for active (State 3) respiration with no observable inhibition of resting (State 4) respiration. In the range of azide concentrations which inhibit State 3 to rates less than those of State 4, a negative control of respiration by ADP and inorganic phosphate is observed. The inhibition is specific for a site between cytochromes a and a₃, causing a crossover between these two cytochromes with cytochrome a becoming reduced and cytochrome a₃ remaining highly oxidized. Trapped steady-state difference spectra at liquid nitrogen temperatures show that the reduced cytochrome a in the azide-inhibited system has an band at 596 mμ, 6 m μ displaced from its usual position at 602 mμ.

The azide inhibition is released by uncouplers of oxidative phosphorylation such that the uncoupled respiration requires up to ten times as much azide as does coupled (State 3) respiration for comparable inhibition. The release of inhibition by uncouplers occurs with no change in the steady-state concentration of reduced cytochrome a₅₉₆ and the increased respiration is attributed to an increased rate of oxidation of the cytochrome a₅₉₆. This cytochrome is postulated to be either an intermediate in electron transport and energy conservation reactions or an azide compound of such an intermediate.     

Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

Multinucleating ligands from multiple palladium(0)-catalysed cross-coupling reactions — synthesis and characterisation of a trinuclear cyclometallated ruthenium(II) complex and a hexanuclear EPR-active molybdenum complex

Two multinucleating ligands have been prepared from 1,3,5-tris(3,5-dibromophenyl)benzene by multiple Pd(0)-catalysed cross-coupling reactions. 1,3,5-Tris[3,5-bis(4-pyridylethenyl)phenyl]benzene (L¹) has six remote pyridyl moieties, each of which can coordinate a 17 valence-electron Mo(tp^*)(NO)Cl fragment (tp^* = hydrotris(3,5-dimethylpyrazolyl)borate), affording the hexanuclear complex [Cl(NO)(tp^*)Mo₆(L¹) (1). 1,3,5-Tris[3,5-bis(2-pyridyl)phenyl]benzene (L²) incorporates three potentially terdentate, cyclometallating N,C,N-donor sets, and can coordinate three Ru(tpy)²⁺ fragments (tpy = 2,2′:6′,2″-terpyridine) giving the trinuclear complex [(tpy)Ru₃(L²)][PF₆]₃ (2). Complex 1 is EPR active, with nearest-neighbour pairs of molybdenum centres displaying magnetic exchange interactions. Electrochemical studies of the two complexes suggest that there is little ground-state interaction between the metal centres in either case.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates.

1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.

2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM⁻¹ · cm⁻¹) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.

3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min⁻¹ · mg⁻¹ of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.

4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.

Competition for energy between phosphoenolpyruvate and citrulline synthesis in guinea-pig liver mitochondria

1. The interrelationship between citrulline synthesis and phosphoenolpyruvate formation has been studied in guinea-pig liver mitochondria incubated with glutamate in State 3 and in the presence of uncoupler and oligomycin.

2. In coupled mitochondria the rate of phosphoenolpyruvate production was limited by a higher capacity of aspartate aminotransferase than that of phosphopyruvate carboxylase for the intramitochondrial oxalacetate. Citrulline formation was low due to the small production of NH₃ since glutamate oxidation in State 3 proceeds via the transamination pathway.

3. Inhibition of aspartate aminotransferase by aminooxyacetate in State 3 resulted in increases in both phosphoenolpyruvate and citrulline synthesis. Under uncoupled conditions, however, an increase of phosphoenolpyruvate formation was accompanied by a decrease of both citrulline production and the ATP content of the incubation medium. Restoration of the citrulline production was observed on the addition of exogenous ATP.

4. The results indicate that when energy is generated via substrate-level phosphorylation, the inhibition of citrulline production is probably due to a higher availability of GTP to the phosphopyruvate carboxylase than to the nucleoside diphosphate kinase.     

Three Ni/Co(II) complexes with 4,4′-bipyridine and benzoic acid derivatives as mixed coligands: Hydrothermal syntheses, structures, and magnetisms

The hydrothermal reactions of 3,5-dinitrobenzoic acid, or p-toluic acid, 4,4′-bipyridine, with transition metal chlorides in basified solvent gave rise to three coordination polymers, [Ni₂(3,5-DNBC)₄(4,4′-bpy)₂(H₂O)] (1), [Co(3,5-DNBC)₂(4,4′-bpy)₂] (2), [Co₃(4,4′-bpy)(p-ToC)₆] (3), and characterized by elemental analysis, IR, single-crystal X-ray diffraction and variable-temperature magnetic measurements. Compound 1 crystallizes in the monoclinic system, P2₁/c space group, the structure determination reveals that 1 has a 2D network based on lozenge grids, where each Ni^II is in 4O + 2N coordination mode. Compound 2 is also monoclinic system, P2/c space group, and the X-ray structural analysis shows that 2 also has a 2D network but based on rectangular grids with the Co^II atom in a CoN₂O₄ environment. Compound 3 crystallizes in the triclinic system, space group, and polymerizes through nitrogen and oxygen atoms giving 1D chains with the Co^II(1) atom in a CoO₆ environment and Co^II(2) is in N + 4O coordination mode. In good agreement with the expected behavior, magnetic susceptibility measurements show weak ferromagnetic interactions for 1 and 3, whereas compound 2 exhibits moderate antiferromagnetic coupling.     

The synthesis of 2,1′-anhydro-,2,1′:3,6-dianhydro-, and 2,1′:3,6: 3′,6′-trianhydro-sucrose

1′-O-Mesyl-6,6′-di-O-tritylsucrose and the corresponding 1′-O-tosyl derivative were prepared from 6,6′-di-O-tritylsucrose by selective sulphonylation. Both sulphonates underwent intramolecular cyclisation reactions, to give 2,1′-anhydrosucrose in high yields rather than the isomeric 1′,4′-anhydride. Sequential benzoylation, detritylation, and mesylation of the 2,1′-anhydride afforded 2,1′-anhydro-6,6′-di-O-mesylsucrose tetrabenzoate which, in the presence of base, gave 2,1′:3,6:3′,6′-trianhydrosucrose that was not identical with the product previously claimed to have this structure. Several derivatives of 2,1′-anhydrosucrose were prepared possessing different functional groups at either the 6,6′- or 4,6′-positions. Dimolar mesitylene-sulphonylation of 3,3′,4′6′-tetra-O-acetylsucrose gave the 6,1′-disulphonate, which, in the presence of alkali, gave 2,1′:3,6-dianhydrosucrose, which was transformed into the 2,1′:3,6:3′,6′-trianhydride by sequential bromination at C-6′ (carbon tetrabromide-triphenylphosphine) and base-catalysed cyclisation. Treatment of 3,3′,4′,6′-tetra-O-benzoylsucrose with sulphuryl chloride furnished the 4,6,1′-trichloro derivative, which, on alkaline hydrolysis, was converted into 2,1′:3,6-dianhydro-4-chloro-4-deoxy-galacto-sucrose.     

Synthesis of 4′-methoxyadenosine and related compounds

Addition of iodine and methanol to N⁶,N⁶-dibenzoyl-9(2,3-O-carbonyl-5-deoxy-β-d-erythro-pent-4-enofuranosyl)adenine (4) selectively gives N⁶,N⁶-dibenzoyl-2′,3′-O-carbonyl-5′-deoxy-5′-iodo-4′-methoxyadenosine (5). Compound 5 can be converted into 4′-methoxyadenosine via hydrolysis of the carbonate followed by benzoylation, displacement of the 5′-iodo function by benzoate ion, and hydrolysis with ammonia. Configurational assignments are based upon comparisons of ¹H- and ¹³C-n.m.r. spectra with those of previously characterised analogues in the uracil series and by borate electrophoresis. Intermediates in the above scheme have also been converted into 5′-amino-5′-deoxy-4′-methoxyadenosine, 4′-methoxy-5′-O-sulfamoyladenosine, and ethyl 4′-methoxyadenosine-5′-carboxylate, each of which is a 4′-methoxy analogue of biologically active derivatives of adenosine.