Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

1,2 Hydrogen-shifts in the biosynthesis of the thujane skeleton

Degradation of (+)-isothujone (trans-thujan-3-one) biosynthesized in Tanacetum vulgare from (3RS)-mevalonic acid (MVA)-[2-¹⁴C, 2-³H₂] showed that one hydrogen from C-2 of the precursor was specifically incorporated at C-4 of product whereas the other was lost. Feeding of α-terpineol-[9-¹⁴C, 4-³H₁, 10-³H₃] (p-menth-1-en-8-ol) yielded isothujone with the same isotope ratios as in precursor. These results indicate 1,2 hydrogen-shifts at two locations in the construction of the the thujane skeleton from α-terpineol or its biogenetic equivalent, and are consistent with a mechanism involving direct cyclization of the latter to a product that by-passes the formation of the biogenetic equivalent of terpinen-4-ol (p-menth-1-en-4-ol) as an intermediate. (3R)-MVA-[¹⁴C, ³H] was more effectively incorporated (up to 1.5 %) into (+)-isothujone in vivo during autumn or winter than in summer (up to 0.02%).     

Synthesis of esters of saturated and unsaturated sixteen-carbon acids deuterated at the terminal or penultimate carbons

General syntheses of saturated and unsaturated fatty acids, specifically trideuterated at the terminal carbon or dideuterated at the penultimate carbon, from ω-hydroxy esters, have been developed. Methyl [16-²H₃]hexadecanoate was synthesized from methyl 16-hydroxyhexadecanoate. The hydroxyl group was protected as the tetrahydropyranyl ether and the ester group reduced with lithium aluminum deuteride, first to an alcohol and then, by way of the derived mesylate, to a trideuteromethyl group. The new ester group was formed by oxidation of the hydroxyl group. Methyl 16-hydroxy[2-²H₂]hexadecanoate was prepared, from 16-hydroxy-hexadecanoate, by exchange of the α protons and, by the reductive route above, with lithium aluminum hydride, gave methyl [15-²H₂]hexadecanoate. Methyl 16-hydroxy-7-hexadecynoate was synthesized from 6-chlorohexanol and was converted, by means of the above reactions, to methyl [16-²H₃]- and [15-²H₂]-9-hexadecynoates. Lindlar reduction gave methyl [16-²H₃]- and [15-²H₂]cis-9-hexadecenoates. Overall yields ranged from 30% to 38%.     

The effect of headgroup class on the conformation of membrane lipids in Acholeplasma Laidlawii: A 2H-NMR study

[2-²H₂]Oleic, [2-²H₂]palmitic, [2-²H₂]dihydrosterculic and [3-²H₂]oleic acids were biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B. ²H-NMR spectroscopy and spectral ‘de-Parking” (M. Bloom, J.H. Davis and M.I. Valic, Can. J. Phys., 58 (1980) 1510) were used to study the effect of lipid headgroup class on the conformational order in the vicinity of the C-2 position of the acyl chains of lipids in the liquid crystalline phase. The results indicate that although the orientation and conformations of the membrane lipids in the region of the C-2 position of the chains are qualitatively very similar among the various lipid classes, quantitatively there are some differences, particularly between the glycolipids and the phospholipids. These differences do not exted to the C-3 position. Unlike the headgroup class, the membrane proteins appear to have little if any effect on the molecular ordering of the lipids.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

Mechanism of alkylation at C-24 during ergosterol biosynthesis in Phycomyces blakesleeanus

Ergosterol isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃] contained two ²H atoms showing that one ²H atom is lost during transmethylation. Ergosterol isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁] had a ¹⁴C:³H atomic ratio of 5:3. Chemical degradation of 2,3-dimethylbutanal obtained by ozonolysis of the doubly-labelled ergosterol showed that the ³H atom originally at C-24 of lanosterol is transferred to C-25 of ergosterol during transmethylation. The mechanism of formation of the ergosterol side chain in P. blakesleeanus is presented.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Trichodiene biosynthesis and the stereochemistry of the enzymatic cyclization of farnesyl pyrophosphate

Cyclization of trans,trans-[1-³H₂,12,13-¹⁴C]farnesyl pyrophosphate (2a) by a preparation of trichodiene synthetase isolated from the fungus, Trichothecium roseum, gave trichodiene (5a), which was shown by chemical degradation to retain both tritium atoms of the precursor at C-11. Incubation of 1S-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2b) and 1R-[1-³H,12,13-¹⁴C]farnesyl pyrophosphate (2c) with trichodiene synthetase and degradation of the resulting labeled trichodienes, 5b and 5c, established that the displacement of the pyrophosphate moiety from C-1 of the precursor and formation of the new C-C bond in the formation of trichodiene takes place with net retention of configuration. These results are accounted for by an isomerization-cyclization mechanism involving the intermediacy of nerolidyl pyrophosphate (4).     

Synthesis of 2,6-difluoro-N-(3-[11C]methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide as a new potential PET agent for imaging of B-RafV600E in cancers

The authentic standard 2,6-difluoro-N-(3-methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide was synthesized from 2,6-difluorobenzoic acid and 3-amino-5-hydroxypyrazole in 9 steps with 1% overall chemical yield. Direct desmethylation of the reference standard with TMSCl/NaI gave the precursor 2,6-difluoro-N-(3-hydroxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide for radiolabeling in 70% yield. The target tracer 2,6-difluoro-N-(3-[¹¹C]methoxy-1H-pyrazolo[3,4-b]pyridine-5-yl)-3-(propylsulfonamidio)benzamide was prepared from the precursor with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–50% decay corrected radiochemical yields with 370–740 GBq/μmol specific activity at end of bombardment (EOB).     

Biosynthesis of vitamin E and of the plastoquinones in chloroplasts: Steric course of the decarboxylation

Samples of (3R)- and (3S)-4′hydroxyphenyl[3-²H₁, 3-³H]pyruvate were prepared by taking advantage of the known stereospecificity of phenylpyruvate keto-enol isomerase (tautomerase). 4′-Hydroxyphenyl[3-¹⁴C]pyruvate was obtained by the action of l-amino acid oxidase on dl-[3-¹⁴C]tyrosine, whereas a simple base-catalyzed exchange procedure yielded samples of 4′-hydroxyphenyl[3-³H]- and 4′-hydroxyphenyl[3-²H₂]pyruvate. All labeled samples were converted in situ into the corresponding homogentisic acids on 4′-hydroxyphenyl-pyruvate dioxygenase that is known to catalyze the migration of the acetate side chain with retention of configuration. The isolated doubly labeled homogentisic acids were incubated with chloroplasts from Raphanus sativus cv. saxa Treib, and from the lipophilic products a fraction containing inter alia tocopherol, tocoquinone, and plastoquinone was obtained by chromatographic procedures. The incorporation of radioactivity was between 0.5 and 11% based on homogentisate. Reductive acetylation of the quinones yielded crystalline diacetylhydroquinones, which were submitted to Kuhn-Roth degradation. The radioactive acetate samples thus obtained were analyzed for chirality by an enzymatic procedure previously published. (2R)-[2-²H₁, 2-³H]Homogentisate gave mainly (S)-acetate, whereas (2S)-[2-²H₁, 2-³H]homogentisate was converted mainly into (R)-acetate. It is concluded that the decarboxylation of the side chain occurred with stereochemical retention during the biosynthetic process.     

Synthesis of methyl [18-2H3], [17-2H2], [16-2H2], [14-2H2] and [12-2H2] cis-9-octadecenoates

Methyl [17-²H₂]oleate was prepared by stepwise reduction from 17-oxooleate in 24% yield. Methyl [18-²H₃], [16-²H₂], [14-²H₂] and [12-²H₂] oleates were synthesized from appropriately deuterated octylbromides by conversion to deuterated 7-hexadecyn-1-ols and chain extention to deuterated stearolates followed by semihydrogenation; overall yields were about 17%.     

Hydroxyl Radical-Induced Reactions in Polyadenylic Acid as Studied by Pulse Radiolysis: Part I. Transformation Reactions of Two Isomeric OH-Adducts

The absorption spectra of polyadenylic acid (polyA) radicals in N₂0 saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4μS electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken.¹

The reaction of OH^· radicals with the adenine moiety in poly A results in the formation of two hvdroxvl adducts at the positions C-4 [polyA40H^·] and C-8 [polyA80H^·]. Each OH-adduct undergoes a unimol-ecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and _pH dependencies. The polyA40H^· adduct undergoes a dehydration reaction to yield a neutral N⁶ centered radical (rate constant K_deh= 1.4 × 10⁴s^-1 at ^pH7.3). This reaction is strongly inhibited by H⁺. In comparison with the analogous reactions in adenosine phosphates, the kinetic _pK value for its inhibition is two ^pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA80H^· adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (k^r.o. = 3.5 × 10⁴ s ^-1 at pH7.3). This reaction in contrast is strongly catalysed by H⁺and OH^-, similar as for adenosine but different compared to the nucleotides.     

Ergosterol biosynthesis in Mucor pusillus

Ergosterol, 22-dihydroergosterol, obtusifoliol and 24-methylene-24,25-dihydrolanosterol, isolated from Mucor pusillus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. Ergosterol and 22-dihydroergosterol, isolated from M. pusillus grown in the presence of mevalonic acid-[2-¹⁴C, (4R)-4-³H₁] had ¹⁴C:³H atomic ratios of 5:3. The significance of these results in terms of sterol biosynthesis in this organism in general and alkylation at C-24 in particular is discussed.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

Synthesis of carbon-11-labeled CK1 inhibitors as new potential PET radiotracers for imaging of Alzheimer’s disease

The reference standards methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate (5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-methoxybenzamide (5c), and their corresponding desmethylated precursors 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoic acid (6a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-hydroxybenzamide (6b), were synthesized from 5-amino-2,2-difluoro-1,3-benzodioxole and 3-substituted benzoic acids in 5 and 6 steps with 33% and 11%, 30% and 7% overall chemical yield, respectively. Carbon-11-labeled casein kinase 1 (CK1) inhibitors, [¹¹C]methyl 3-((2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)carbamoyl)benzoate ([¹¹C]5a) and N-(2,2-difluoro-5H-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-d]imidazol-6-yl)-3-[¹¹C]methoxybenzamide ([¹¹C]5c), were prepared from their O-desmethylated precursor 6a or 6b with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 40–45% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (MA) at EOB was 370–740?GBq/μmol with a total synthesis time of ～40-min from EOB.     

The stereochemistry of hydroxylation of the carotenoid lutein in Calendula officinalis

Excised, opening inflorescences of Calendula officinalis incorporated (3RS, 5R)- and (3RS, 5S)-[2-¹⁴C,5-³H₁]mevalonates into the carotenoid fraction. The ¹⁴C:³H ratios of lutein isolated from these tissues showed the hydrogen atom at C-3 of the β-ring is derived from the 5-pro-S position of mevalonate, while that at C-3 of the ε-ring is derived from the 5-pro-R position of mevalonate. Oxidation of lutein to monoketolutein showed that both hydrogen atoms at the C-15,15′ central double bond are derived from the 5-pro-R position of mevalonate.     

The stereochemistry of hexahydroprenol, ubiquinone and ergosterol biosynthesis in the mycelium of Aspergillus fumigatus Fresenius

1. The mycelium of Aspergillus fumigatus has been shown to incorporate mevalonate into squalene, ubiquinone, ergosterol and hexahydroprenol. 2. The ³H/¹⁴C ratio in ubiquinone, biosynthesized from [2-¹⁴C-(4R)-4-³H₁]mevalonate, is the same as in the squalene; essentially no ³H was incorporated from [2-¹⁴C-(4S)-4-³H₁]mevalonate, indicating the biosynthesis of biogenetically trans-isoprene units. 3. The ³H/¹⁴C ratio for ergosterol (from `4R-mevalonate') was 3:5, showing that the proton at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25. 4. As ³H from both mevalonates was incorporated into the hexahydroprenols the biosynthesis of both cis- and trans-isoprene units must occur. 5. The saturated ω- and ψ-isoprene units are shown to be biogenetically trans, as are two of the unsaturated residues. 6. The saturated α- and unsaturated β-isoprene residues are both biogenetically cis. 7. An inexplicable loss of approximately half of the olefinic protons from the cis-portion of hexahydroprenol occurs; possible reasons for this loss are discussed. 8. Increase in chain length of the hexahydroprenols is by a cis addition. 9. A biosynthesis of hexahydroprenols by addition of cis-isoprene units to all-trans-geranylgeranyl pyrophosphate, or a dihydro or tetrahydro derivative thereof, is suggested.     

Biosynthesis of methyl chloride in the fungusPhellinus pomaceus

The biosynthetic origin of themethyl group in the methyl chloride produced by cultures ofPhellinus pomaceus (Pers.) Maire has been investigated using stable isotope labeled substrates. Feeding ofd-[6,6-²H₂] glucose,Dl-[3,3-²H₂] serine andl-[methyl-²H₃] methionine led to the production of deuterated methyl chloride in which the major labeled species contained 2, 2, and 3 deuterium atoms, respectively. The data are consistent with the methyl chloride produced by this organism being derived solely from methionine with retention of all of the methyl protons.Abbreviation SAM S-adenosylmethionine - FH₄ tetrahydrofolate - N⁵-CH₃FH₄ N⁵-methyltetrahydrofolate - B₁₂ cobalamin     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.