Guillonein,an epoxyguaianolide from Guillonea scabra, X-ray structure determination

From the roots of Guillonea scabra a new epoxyguaianolide, guillonein, has been isolated and its structure established by X-ray diffraction analysis. A ¹H NMR spectroscopic study of this new sesquiterpene reveals a conformational difference in its seven-membered ring between the crystal and CDCl₃ solution states. The implications of this conformational change with respect to the C-8 configurations previously assigned to shairidin and desangeloylshairidin are briefly discussed.     

13C-NMR assignments and biosynthetic data for the ether lipids of Caldariella

Fully assigned ¹³C-NMR spectra confirm the C₄₀ (16,16 -biphytanyl) structures of the alkyl chains in the ether lipids of extreme thermoacidophile bacteria of the Caldariella group. The incorporations of ¹³C- and ₁₄C- labelled acetate and mevalonate provide further structural confirmation and define their biosynthetic origin.     

Conformational analysis of an anti-androgenic, (E,E)-8-hydroxygermacrene B,using NOESY and dynamic NMR spectroscopy

(E,E)-8-Hydroxygermacrene B was prepared by ketone reduction of germacrone, a naturally occurring compound from Curcuma aeruginosa Roxb. with NaBH₄ at low temperature (4 °C). This compound showed remarkable in vitro anti-androgenic activity (IC₅₀ 0.15 ± 0.022 mM) applicable to male baldness treatments. NMR analysis at −50 °C indicated that there were four conformational isomers of (E,E)-8-hydroxygermacrene B in a ratio of 48:40:8:4. The major conformers were assigned by ¹H NMR and 2D-NOESY NMR spectroscopy as having methyl groups at C-10 and C-4 in up–down (UD) orientations (48% predominance) and UU (40%). ¹H NMR spectra implied another two minor conformers with these methyls having DU (8%) and DD (4%) orientations.     

Structure of fomitellan A, a mannofucogalactan from the fruiting bodies of Fomitella fraxinea

Chemical structure of fomitellan A, a polysaccharide with a mitogenic effect isolated from the fruiting bodies of Fomitella fraxinea, has been assigned as a mannofucogalactan with a repeating unit of penta-saccharide, which was composed of a (1→6)-linked d-galactopyranosyl backbone having a C-2 position substituted with disaccharide units of 3-O-d-mannopyranosyl-l-fucopyranosyl residue. The ¹H and ¹³C NMR signals of fomitellan A have been completely assigned by extensive NMR experiments.     

X-Ray crystal and molecular structure of 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose,and conformations of aldopentofuranose analogs having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the ³T₂ conformation with a tendency towards the E₂ form, the substituents on C-1, C-4, and P-5 are linked bisectionally, and the acetoxyl groups on C-2 and C-3 are respectively attached axially and equatorially. Based on the X-ray crystallographic and ¹H-n.m.r.-spectral data, favored conformations of P-in-ring analogs of aldopentofuranose peracetates in solution are discussed.     

13C NMR spectra of 1, 3, 6 -trihydroxy-7-methoxy-8-(3, 7 -dimethyl-2,6-octadienyl) xanthone and its dimethyl derivative

The ¹³C NMR spectra of 1, 3, 6-trihydroxy-7-methoxy-8-(3, 7-dimethyl-2, 6-octadienyl)xanthone and its dimethyl derivative are discussed. The data obtained confirmed the assigned structures. The geometrical configuration of the C₁₀ dienyl side-chain has been deduced as trans.     

The incorporation of [5,6-13C2]Nicotinic acid into the tobacco alkaloids examined by the use of 13C nuclear magnetic resonance

[5,6-¹⁴C,¹³C₂]Nicotinic acid was prepared from [¹⁴C,¹³C]methyl iodide via nitromethane, 2-nitroacetaldehyde oxime, 3-nitroquinoline, 3-aminoquinoline, and quinoline in 20% overall yield. Administration of this material to Nicotiana tabacum and N. glauca afforded labeled anabasine, anatabine, nicotine, and nornicotine. Qualitative and quantitative incorporation (0.07–4.5% specific incorporation) was determined by radioactive assay and by examination of the ¹³C NMR spectra of these alkaloids. Satellites due to spin-spin coupling of the incorporated contiguous ¹³C atoms were observed at the resonances due to C-5 and C-6 in anabasine, nicotine, and nornicotine. In anatabine, satellites were found at C-5, C-6, C-5′, and C-6′.     

13C- and 1H-N.M.R. spectroscopy of permethylated gluco-, galacto-, and manno-pyranoses and their 6-deoxy analogues

The ¹³C- (25.16 MHz) and ¹H-n.m.r (220, 300 MHz) spectra of permethylated mannopyranoses, their 6-deoxy analogues, and permethylated 6-deoxy-gluco- and -galacto-pyranoses have been analysed with the aid of specific trideuteriomethylation, heteronuclear spin-decoupling, and spectrum simulation. Comparison of spectral data for the aldohexose derivatives and their 6-deoxy analogues shows that the ring conformation is not significantly affected by the presence or absence of MeO-6; all compounds are present in the ⁴C₁ (D) or ¹C₄ (L) conformation. Changes in orientation of the MeO groups have distinct effects on the chemical shifts of carbons and protons of the pyranoid rings and of the MeO groups. The possible origins of these effects are discussed.     

Conformational studies on pertrimethylsilyl derivatives of some 6-deoxyaldohexopyranoses and of four less-common aldohexopyranoses by 220-MHz P.M.R. spectroscopy. Substituent and configurational effects on the chemical shifts of the ring protons

The complete interpretation of 220-MHz p.m.r. spectra and the accurate chemical shifts and coupling constants, obtained after computer simulation of the spectra, of the per-O-trimethylsilyl (Me₃Si) derivatives of a number of 6-deoxy-aldohexopyranoses and of β-D-altro-, β-D-allo-, and α- and β-D-talo-pyranose are given. By means of an adapted Karplus equation, the structure of the derivatives has been studied in detail. All of the pyranoid rings occur in the ⁴C₁(D) or ¹C₄(L) chair conformation. The preferred conformation of the C-5—CH₂OSiMe₃ group in the four aldohexopyranoses was found to be dependent on the configuration at C-4. By comparison of Me₃Si-aldohexopyranoses with the corresponding 6-deoxy analogues, it was found that the 6-OSiMe₃ group has no marked effect on the conformation of thering. The influence of this group on the chemical shifts of the ring protons is discussed in terms of electric field and inductive effects. Rules are presented for the estimation of the chemical shifts of the ring protons of Me₃Si-aldohexopyranoses and Me₃Si-6-deoxyaldohexopyranoses.     

X-ray crystal,and molecular,structure of 1,2,3-tri-O-acetyl-4,5-dideoxy-4-C-[(R)-phenylphosphinyl]-α-l-lyxofuranose: The first x-ray analysis of a pentofuranose having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure of 1,2,3-tri-O-acetyl-4,5-dideoxy-4-C-[(R)-phenylphosphinyl]-α-l-lyxofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the E₃ conformation, with a tendency towards the ³T₂ form, the substituents at P-5 and C-5 are linked bisectionally, the acetoxyl group at C-2 and the methyl group at C-4 are linked quasiequatorially, and the acetoxyl group at C-3 is linked axially.     

The crystal structure of 6-epi-desacetyllaurenobiolide,a germacra-1(10),4-diene-12,8α-olide from Montanoa grandiflora

A chloroform extract of Montanoa grandiflora afforded a novel 6β-hydroxy-germacradien-8,12-olide. Its structure was shown to be 6-epi-desacetyllaurenobiolide by spectral studies, chemical transformations and single crystal X-ray diffraction. The X-ray data demonstrate that the ten-membered ring exists in the crystal in the highly unusual [₁₅D⁵,₁D¹⁴] conformation, in which the methyl group at C-4 is α-oriented and the methyl group at C-10 is β-oriented. The two double bonds are approximately parallel rather than crossed.     

Properties of metal-ion coordinated imidazoles: nmr and C-2 H Exchange in Co(III) Complexes

The ¹H and ¹³C nmr spectra of Co(NH₃)₅ImH³⁺ and the ¹H nmr spectra of αCotrien(ImH)₂³⁺ and βCotrien(ImH)₂³⁺ are reported. The pK_a values determined from the dependence of the chemical shift on pH are 10.0, 9.6, and 10.1, respectively. The range of the chemical shift between the acid and base forms is unusually small in the ¹H nmr, 0.5–0.7 ppm for the C-2 H and about 0.25 ppm for the C-4 H and C-5 H. In the ¹³C nmr, C-2 and C-4 have large shifts to low field and C-5 a small shift to high field on deprotonation. The C-2 proton is not exchanged with solvent ²H under acidic or basic conditions, in marked contrast to the corresponding proton in both imidazole and 1-methylimidazole. These spectroscopic and chemical properties should be useful for the direct identification of metal-ion coordinated histidines in proteins.     

Dihydrofolate reductase from a methotrexate-resistant Escherichia coli: Proton magnetic resonance studies of complexes with folate and methotrexate

Proton magnetic resonance studies of 1:1 complexes of E. coli dihydrofolate reductase with folate and methotrexate were performed. A resonance at 1850 Hz in 1:1 enzyme-folate was assigned as the C-7 proton of bound folate by comparison with the spectra of enzyme complexed with folate specifically deuterated at C-7. The first order rate constant for folate dissociation was calculated to be less than 110 sec^?1. Four of the five histidine residues exhibited the same pK's and chemical shifts in the two complexes with pK values of 8.0, 7.3, 6.5 and ～5. However, one histidine increased its pK by 0.7 units (6.25→6.95) and its C-2 proton resonance shifted upfield 50 Hz when folate was substituted for methotrexate. Comparison of these results with those of chemical modification and ultraviolet difference spectroscopy experiments suggests that this histidine may be in the folate binding site — possibly near the pteridine portion of that site.     

A 13C-N.M.R.-Spectral study of a gel-forming,branched (1→3)-β-d-glucan, (lentinan) from lentinus edodes, and its acid-degraded fractions. Structure,and dependence of conformation on the molecular weight

The structure and conformation of lentinan, an anti-tumor, branched (1→3)-β-d-glucan from Lentinus edodes, and its acid-degraded, lower molecular-weight fractions have been investigated by ¹³C-n.m.r. spectroscopy. It is found that their ¹³C-n.m.r. spectra are considerably changed, depending on the molecular weight. The conformational behavior as studied by ¹³C-n.m.r. spectroscopy is consistent with that revealed by a study of the shift in the absorption maximum of Congo Red complexed with lentinan and its acid-degraded fractions. It is found that the water-soluble fraction II (mol. wt. 3,640) gives rise to well-resolved ¹³C-n.m.r. spectra; the ¹³C-signals are assigned to (1→3)-β-d-glucan and branch points at C-6. The branched structure is also confirmed by examination of the ¹³C-n.m.r. spectra of the compounds in dimethyl sulfoxide. For the gel state of the fractions of higher molecular-weight, lentinan (mol. wt. 1,000,000) and fraction IV (mol. wt. 16,200), however, ¹³C-n.m.r. spectra of considerably attenuated signal-amplitude are observed. The fact that the ¹³C-signals of the β-d-(1→3)-linked main chain and side chains are completely suppressed is explained as a result of immobilization caused by their taking an ordered conformation. The ¹³C-resonances observed in the gel state, which are assigned to β-d-(1→6)-linkages, are unequivocally assigned to the side chains (of disordered conformation). Finally, the ordered conformation of both the β-d-(1→3)-linked main chain and side chains is identified as the single-helix conformation, which tends to form multiple helixes as junction zones for gel structure.     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.     

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.     

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.     

Mechanism of C4 photosynthesis in Chloris gayana: pool sizes and kinetics of 14CO2 incorporation into 4-carbon and 3-carbon intermediates.

In one group of C₄ species, including Chloris gayana, C₄ acids are decarboxylated via phosphoenolpyruvate carboxykinase to give phosphoenolpyruvate as the initial C₃ product. This paper presents an analysis of the kinetics of labeling of various photosynthetic intermediates in Chloris gayana leaves exposed to ¹⁴CO₂, and the pool sizes of these intermediates, primarily to provide information about the subsequent metabolism of phosphoenolpyruvate. Saturation labeling of the C-4 of aspartate and malate, and the C-1 of 3-phosphoglycerate, indicated photosynthetically active pools of 0.45, 0.22, and 0.95 μol/mg chlorophyll, respectively. For aspartate and 3-phosphoglycerate, the total leaf pools and the photosynthetic pools were of similar size, but the total pool of malate was about 100 times larger than the photosynthetically active pool. From the relative rates of labeling of phosphoenolpyruvate, pyruvate, alanine, and C-1, C-2 plus C-3 of aspartate, during steady-state ¹⁴CO₂ assimilation, relative pool sizes were calculated to be about 10:11:78:100, respectively. Pulse/chase labeling of leaves provided estimates of relative photosynthetic pool sizes in the ratio of about 6:15:90:100, respectively, where aspartate is arbitrarily assigned a value of 100 in both cases. Notably, labeling of alanine was consistent with its derivation from the C-1, C-2 plus C-3 carbons of aspartate, and the alanine pool was at least eight times larger than the phosphoenolpyruvate pool that showed similar labeling kinetics. Results were consistent with the view that at least most of the phosphoenolpyruvate produced by C₄ acid decarboxylation is metabolized via alanine.     

Effects of carbohydrate-structure changes on induced shifts in differential isotope-shift 13C-N.M.R.

Deuterium-induced, ¹³C-isotope shifts are shown to vary considerably from the initially predicted values calculated for ordinary pyranose and furanose sugars, when minor structural changes are introduced into the carbohydrate ring. Both substitution of C-OH groups or reduction of C-OH to CH₂ permitted the evaluation of γ effects of OD without the contribution of β-OD-induced shifting. The observed γ-shift values for these modified structures were twice as large as those previously noted. This difference is most probably due to favored salvation. Substitution of OH at C-6 led to the predicted loss of differential isotope-shift (d.i.s.) at C-6 because of its isolation from all β and γ OD groups. The ³¹P resonances of d-glucose 6-phosphate show downfield deuterium shifts. Based on d.i.s. values, new ¹³C-shift assignments are proposed for isomaltose and 2-amino-2-deoxy-α-d-glucose. A study of acidic carbohydrates has demonstrated that isotope shifts are somewhat larger for sp²-hybridized carbon atoms whose OH groups are acidic. Relaxation times for sp² carbon atoms isolated from dipolar interaction with protons were very long in D₂O relative to their relaxation time in the H₂O environment.