Lipid peroxidation of a human hepatoma cell line (HepG2) after incorporation of linoleic acid, arachidonic acid, and docosahexaenoic acid

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography-mass spectrometry (GC-MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H(2)O(2) (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H(2)O(2). However, the enhancement of lipid peroxidation by H(2)O(2) was not observed in DHA-supplemented cells. The same result was obtained in the GC-MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA-MHP and a small amount of DHA-MHP were observed in AA- and DHA-supplemented cells respectively. GC-MS analysis also indicated the specific positional distribution of DHA-MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.     

Two γ-Methyl Biosides from Dregea volubilis (L.) Benth

From the hydrolysate of the crude glycosides from the roots, of Dregea volubilis(L.) Benth in Dehong, Yunnan, two α-methyl biosides Ⅰ and Ⅱ (yields: 0.016%and 0.0097%, respectively) were isolated by silica gel column chromatography. Theirchemical structures were established by interpretation of MS, IR,1H,13C-NMR, andgas chromatographic analysis of their degradation products, and comparison of thephysical properties of Ⅰ, Ⅱ and their acetates which were reported in literatures asfollows: α-methyl-pachybioside for Ⅰ, and α-methyl-[3-O-methyl-6-deoxy-D-allose(1→4)-D-olivoside] for Ⅱ. Ⅱ named α-methyl-dredehongbioside, is reported for the first time. Ⅱ, α-methyl-dredehongbioside, colorless needles (from MeOH), bitter, mp. 184–186℃,[α]D22 +74.5˚~(c= 0.52, MeOH). Anal. Cald(%) for C14H26O8:C52.17, H8.07;Found; C52.23,H8.22. Irvmaxkbr: 3370, 1443, 1419, 1375, 1268, 1218, 1168,1127,1060cm-1. MS(m/e,%): 322(M+,3),291(M+-OCH3,15), 273(M+-OCH3-H2O,12), 258,246,232, 222, 159, 145, 141, 128, 95, 87, 85, 74 (base peak, 100), 59. 1H NMRδ(CDCl3): 4.73(1H, dd, J= 4.0 Hz, J= 1.5Hz, C-1-H), 4.55(1H, d, J= 8.0Hz,C-1′-H), 3.79(1H, dd, J=3.0Hz, J= 3.0Hz, C-3′-H), 3.00(1H, dd, J= 9.0Hz,J= 9.0Hz, C-4-H), 2.22(1H, m, C-2-Ha), 1.60(1H, m, C-2-He), 1.33(3H, d,J= 6.0Hz,C-5-CH,), 1.31(3H,d,,J= 6.5Hz, C-5′-CH), 3.68(3H,s,,C-3′-OCH),3.31(3H,s,C-1-OCH). 13C NMR data were seen in Table 1. Ⅳ, tri-acetyl-α-methyl-dredehongbioside, colorless granular (from MeOH),mp. 135--137℃, [a]D22+ 88.2˚(c= 0.50, MeOH). MS(m/e, %): 488 (M+, 2), 388(M+-HOAc,2), 357(M+-OCH3-HOAc,33), 288, 187, 127, 116, 85, 74, 59, 43(basepeak, 100). 1H NMR,δ(CDCl3): 5.25(1H, ddd,.J= 11.0Hz, J=9.0Hz,.J= 5.5Hz,C-3-H), 4.86(1H, d, J= 8.0Hz, C-1′-H), 4.69 (1H, dd, J= 4.0Hz, J= 1.5Hz,C-1-H), 4.58(1H,m,C-2′-H),3.94(1H, dd, J= 3.0Hz,J= 3.0Hz,C-3′-H),3.64(1H,m,C-5-H), 3.22(1H,dd, J= 9.5Hz, J= 8.5Hz, C-4-H), 2.30(1H, m, C-2-Ha),1.67(1H,m,C-2-He), 1.31(3H, d, .J= 6.5Hz, C-5-CH3), 1.17(3H, d, J= 6.0Hz,C-5-CH3), 3.47(3H, s, C-3′-OCH3), 3.30(3H,s, C-1-OCH3), 2.10(6H, s, C-2′, C- 4′ -OCH3), 2.03(3H,s,C-3-OCH3).     

The stereochemistry of beta-lactam formation in penicillin biosynthesis.

1. (2R,3S)-[U-14C,3-3H1]- and (2R,3R)-[U-14C,2,3-3H2] Cysteine hydrochlorides have been separately synthesised. The latter compound has been shown to have uniform distributions of tritium between C-2 and C-3. 2. The abvoe cysteines and (2R)-[U-14C,3,3,3',3'-3H4]cystine have been converted to samples of penicillin G by Penicillium chrysogenum. 3. Incorporation results indicate that all but 14% of the tritium is lost from the (2R,3S)-[3-3H1]isomer; that 42% of tritium is retained by the non-stereospecifically C-3 tritiated cystine; and that 58% of tritium is retained by the (2R,3R)-[2,3-3H2]isomer on conversion to penicillin G. 4. Degradation of the penicillin G derived from (2R,3R)-[U-14C,2,3-3H2]cysteine hydrochloride has indicated that in fact about 87% of the original C-3 tritium of cysteine is retained at C-5 of penicillin G. 5. The results indicate stereospecificity in the cyclisation giving rise to the beta-lactam ring in penicillin G in nature with loss of the 3-pro-S-hydrogen and rentention of the 3-pro-R-hydrogen of cysteine. Thus there is net retention of stereochemistry in the cyclisation.     

Selective abstraction of 2H from C-1' of the C residue in AGC.ICT by the radical center at C-2 of activated neocarzinostatin chromophore: structure of the drug/DNA complex responsible for bistranded lesion formation.

Glutathione-activated neocarzinostatin chromophore (NCS-Chrom) generates bistranded lesions at AGC.GCT sequences in DNA, consisting of an abasic site at the C residue and a strand break at the T residue on the complementary strand, due to hydrogen atom abstraction from C-1' and C-5', respectively. Earlier work showed that 2H from C-5' of T was selectively abstracted by the radical center at C-6 of activated NCS-Chrom, supporting a proposed model of the active-drug/DNA complex. However, since under the conditions used breaks at the T exceeded their inclusion in bistranded lesions, it was not clear what fraction of the hydrogen transfer represented bistranded lesions. Since virtually all abasic sites at the C are part of a bistranded lesions, hydrogen transfer from C-1' of C into the drug should reflect only the bistranded reaction. Accordingly, a self-complementary oligodeoxynucleotide 5'-GCAGCICTGC-3' was synthesized in which the C contained 2H at the C-1' position. In order to eliminate an 2H isotope effect on the transfer and to increase the extent of the bistranded reaction, an I residue was substituted for the G opposite the C residue. Sequencing gel electrophoretic analysis revealed that under one-hit kinetics, 37% of the damage reaction was associated with abasic site (alkali-labile break) formation at the C residue and 48% with direct strand breaks at the T residue. Thus, 74% of the damage involved a bistranded lesion. 1H NMR spectroscopic analysis of the reacted chromophore showed that 2H had been selectively transferred into the C-2 position to the extent of approximately 22%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereochemistry of sodium borohydride reduction of tryptophan synthase of Escherichia coli and its amino acid Schiff's bases

Tryptophan synthase alpha 2 beta 2 complex containing [4'-3H]pyridoxal phosphate was reduced with sodium borohydride in the presence of various substrates and analogs in an attempt to trap reaction intermediates. Reduction in the presence of L-serine gave noncovalently bound radioactive material which was identified as phosphopyridoxylalanine, presumably resulting from reduction of the intermediate Schiff's base formed between pyridoxal phosphate and alpha-aminoacrylate. The tritium in this compound was located in the pro-R position at C-4', indicating that reduction of the Schiff's base double bond had occurred on the Si face at C-4'. On the other hand, analysis of phosphopyridoxyllysine obtained by hydrolysis of the reduced [3H]pyridoxal-P-alpha 2 beta 2 protein showed that the internal Schiff's base had been reduced on the C-4' Re face, suggesting a cofactor reorientation upon substrate binding. Analysis of phosphopyridoxylalanine from a reduction of unlabeled alpha 2 beta 2 complex in the presence of (2S,3R)-[2,3-2H2]serine with tritiated sodium borohydride demonstrated the presence of tritium at C-4' (50%), C-2 (20%), and C-3 (30%). According to the configuration at C-3, reduction of the phosphopyridoxal-alpha-aminoacrylate Schiff's base has occurred from the same side of the molecule at C-4' and C-3.     

Structural features of prostanoid analogues involved in hepatocytes protection against CCl4-induced injury

The ability of natural prostaglandins (PGs) and synthetic prostanoids of B, H and E(1)-types to prevent CCl(4)-induced liver injury in vitro was examined. Seven analogs of PGB, 3 derivatives of PGH and two 11-deoxy-analogs of PGE(1) decreased cytotoxic index of CCl(4) by 2-fold and were more effective then such well-known hepatoprotectors as silymarin, curcumin and PGI(2). These prostanoids reduced trien conjugates formation by 25-95% and strongly prevented LDH leakage through plasma membrane. The results from the structure-activity relationship (SAR) analysis can be summarized as follows: (1) the presence of 2-pyridyl at the 15 position of ω-chain plays the main role in cytoprotection for synthetic analogs of PGH; (2) among 11-deoxy-analogs of PGE(1) the most active substances possess the phenyl residue at C-15 connected with C-13 isoxazolin or C-13 isoxazol; (3) C-13 cyclohexylamine or C-15 phenyl groups in ω-chain and metoxy-group in α-chain are important for protective activity of PGB analogs; (4) synthetic analogs of PGB demonstrate more pronounced cytoprotective properties than prostanoids of H and E-types. This information suggests paths for further exploration in the area of hepatoprotection and the design of novel therapeutic agents.     

Preparation of multiply deuterium-labeled cortisol.

Cortisol labeled with four deuterium atoms at chemically stable sites ([9,11,12,12-(2)H4]cortisol, cortisol-d4) was prepared by hydrogen-deuterium exchange and reductive deuteration reactions. After protecting the C-17 dihydroxyacetone side chain of cortisone (cortisone-BMD), hydrogen-deuterium exchange was carried out with 6.5% NaOD in MeOD, which was followed by protection of the C-3 carbonyl as the semicarbazone. Subsequent reductive deuteration at C-11 with NaBD4 followed by removal of exchangeable deuterium under the same exchange-reaction conditions in a medium of 6.5% NaOH in MeOH and deprotection afforded the desired cortisol-d4 with high isotopic content (d3, 21.2%; d4, 78.1%; d5, 0.74%). The method was applied to the synthesis of cortisol labeled with nine deuterium atoms [( 1,1,9,11,12,12,19,19,19-(2)H9]cortisol, cortisol-d9) starting from [1,1,19,19,19-(2)H5]cortisone (cortisone-d5).     

Biosynthesis of lasalocid A: biochemical mechanism for assembly of the carbon framework

Labeling experiments on the biosynthesis of the polyether antibiotic lasalocid A (1) using carboxylic acid precursors bearing 13C, 2H, and 3H labels at various positions established the following: (1) 2H or 3H at C-2 of propionate or 2H at C-2 of butyrate was partially retained at C-12 and C-14 of 1, respectively. (2) 2H at C-2 of propionate or at C-2 and C-3 of succinate did not label C-10. These and earlier data [Hutchinson, C. R., Sherman, M. M., Vederas, J. C., & Nakashima, T. T. (1981) J. Am. Chem. Soc. 103, 5953; Hutchinson, C. R., Sherman, M. M., McInnes, A. G., Walter, J. A., & Vederas, J. C. (1981) J. Am. Chem. Soc. 103, 5956] are consistent with a hypothesis for the stereochemical control of lasalocid A biosynthesis, whose main tenets are that the configuration of C-12 and C-14 is determined by the stereoselectivity of the carbon chain forming condensation between acyl thio ester and 2-carboxyacyl thio ester intermediates and that the configuration of C-11 and C-15 results from the reduction of 2-keto thio ester intermediates with opposing stereospecificities.     

Association between CFH Y402H Polymorphism and Age Related Macular Degeneration in North Indian Cohort

The purpose of the study was to determine serum complement factor H (CFH) levels in patients of age related macular degeneration (AMD) and examine its association with CFH Y402H polymorphism. 115 AMD patients and 61 normal controls were recruited in this study. The single nucleotide polymorphism was assayed by real time PCR and serum CFH levels were measured by ELISA and standardized to total serum protein. Chi-square test was applied to polymorphism analysis while Mann Whitney U-statistic for CFH-levels. Mendelian randomization approach was used for determining causal relationship. The genotype frequency differed between the AMD patients (TT- 18.3%, TC-41.3% and CC-40.4%) and controls (TT-76.3%, TC-13.6%, and CC-10.1%) (p = 0001). The frequency of alleles was also significantly different when AMD (T-39% and C-61%) was compared to controls (T-83% and C-17%) (p = 0.0001). Level of serum CFH was significantly lower in AMD patients as compared to normal controls (p = 0.001). Our data showed that the CFH Y402H polymorphism is a risk factor for AMD in the North Indian population. Mendelian randomization approach revealed that CFH Y402H polymorphism affects AMD risk through the modification of CFH serum levels.     

The mechanism of glucose 6-phosphate–d-myo-inositol 1-phosphate cyclase of rat testis. The involvement of hydrogen atoms

Comparison of the initial (3)H/(14)C ratios in specifically labelled d-glucose 6-phosphates with the final ratios in myo-inositol produced by glucose 6-phosphate-d-myo-inositol 1-phosphate cyclase from rat testis showed that, during the conversion, the hydrogen atoms at C-1 and C-3 were fully retained, one hydrogen atom was lost from C-6, and that at C-5 was apparently retained to the extent of 80-90%. The loss of (3)H could not be stimulated by addition of unlabelled NADH, and when unlabelled substrate was used (3)H from [(3)H]NADH and [(3)H]water was not incorporated. Treatment of the enzyme with charcoal abolished the activity, and this was restored to 25-50% of the original activity by NAD(+). The charcoal-treated enzyme again apparently gave 85% retention of hydrogen with [5-(3)H]glucose 6-phosphate as substrate in the presence of NAD(+) alone, but the retention was decreased to 65% with excess of NADH. The results are interpreted as indicating that the cyclization proceeds by an aldol condensation in which C-5 is oxidized by NAD(+) in a tightly-bound ternary complex, and that the apparent loss of (3)H when untreated enzyme is used is due to an isotope effect. It is suggested that after treatment with charcoal some exchange of NADH with an external pool may take place.     

Isotope-edited 1D and 2D n.m.r. spectroscopy of 13C-substituted carbohydrates.

Three isotope-edited n.m.r. methods have been applied to selectively 13C-substituted monosaccharides and nucleosides to simplify their spectra and/or measure 1H-1H, 13C-1H, or 13H-13C spin-couplings detected via the labeled site. 1D INADEQUATE spectra allowed the selective detection of the natural-abundance carbons that are spin-coupled to the labeled carbon, and adjustment of the mixing time permitted further discrimination between one-bond and longer-range 13C-13C coupling pathways. Geminal and vicinal 13C-1H coupling constants were determined from the analysis of 1H-1H COSY cross-peaks for those protons coupled to the labeled carbon. Long-range 13C-(HETCOR) and 1H-detected (HMBC) 13C-1H chemical-shift correlation spectra permitted the selective observation of those protons coupled to the labeled site, and JH,H values were measured from data projections. The implications of these methods for structural studies of more complex systems is briefly discussed.     

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.     

5,6,7,8-Tetrahydrofolic acid. Conformation of the tetrahydropyrazine ring

It is suggested from analysis of proton spin-spin coupling constants that the tetrahydropyrazine ring of tetrahydrofolate is a roughly equal mixture of two half-chair conformations, one with the C-6 proton axial and the other with the C-6 proton equatorial. The chemical shifts and spin-spin coupling constants for the carbon-bound protons of (+/-)-L-, (-)-L-, and (-)-L-[6-2H] 5,6,7,8-tetrahydrofolate were measured at 25 degrees and at 300 MHZ. The resonances corresponding to the two C-7 protons in the deuterated compound constituted an AB quartet with JAB of 12 Hz and chemical shift difference of 92 Hz or 0.307 ppm; the C-7 protons are proposed to be a geminally coupled axial-equatorial pair whose rapid equilibration does not result in equivalence due to the adjacent chiral center at C-6. The spin-spin splitting in the C-7 resonances were 3.0 and 6.6 Hz for the low field and high field resonances, respectively, reflecting coupling to the C-6 proton. These coupling constants reflect the conformational equilibrium. The resonances assignable to C-9 protons are nearly equivalent in the 6-2H compound, but exhibit the resonances corresponding to a complex spin system in the 6-H compound.     

Biosynthesis of the pyrimidine moiety of thiamin in Escherichia coli: incorporation of stable isotope-labeled glycines.

Methods are described for the cleavage, extraction, and subsequent gas chromatographic-mass spectrometric analysis of the pyrimidine moiety of thiamin as 2-methyl-4-amino-5-[(ethylthio)methyl]pyrimidine. The methods are of a general nature and can be applied to any system. Using these methods to evaluate the incorporation of 13C-, 15N-, and 2H-labeled glycines into the pyrimidine moiety of thiamin by Escherichia coli, we established that the nitrogen and carbon atoms of glycine are incorporated as a unit into the pyrimidine. 13C- and 15N-labeled glycines are incorporated at greater than 60% but deuterium from [2-(2)H2]glycine was incorporated at only 18%. A detailed analysis of the mass fragmentation pattern of the pyrimidine derivative has established that the glycine nitrogen atom supplies the N-1 of the pyrimidine and that the C-1 and C-2 of the glycine supplies the C-4 and C-6 of the pyrimidine, respectively. This evidence is consistent with the substitution of a C2 unit between the C-5 and C-4 of the 4-aminoimidazole ribonucleotide precursor during the biosynthesis of the pyrimidine moiety of thiamin in E. coli.     

The occurrence of a reductase of delta2-carboxylic acids in Clostridium kluyveri with a stereospecificity different from that of butyryl-CoA dehydrogenase (author's transl)]

A Clostridia strain (R-strain) which hydrogenates tiglinate (1b) and alpha-methylcinnamate (1c) in the presence of hydrogenase gas in 2H2O to (2R, 3S)2-methyl-[2,3-2H]butyrate (5b, H = 2H) and (alphaR, betaR)alpha-methyl[alpha,beta-2H]dihydrocinnamate (5c, H = 2H), respectively, was isolated. The configuration at C-3 was determined by 1H-NMR spectroscopy in the presence of Eu(fod)3. The stereochemistry of this hydrogenation is the mirror image of that which has been determined with intact cells of another strain of Clostridium kluyveri (S-strain). In the presence of hydrogen gas, the R-strain hydrogenates crotonate in 2H2O to butyrate with the following deuterium distribution: C-2, 1.85; C-3, 1.35; and C-4, 0.63 deuterium atoms. Crotonate seems to be the substrate of two reductases with sterically different actions. Tiglinate (1b) and alpha-methylcinnamate, however, are hydrogenated only by that reductase which is different from the butyryl-CoA dehydrogenase.     

An isocratic separation of underivatized gentamicin components, 1H NMR assignment and protonation pattern

A simple method for the separation of the major components of commercial gentamicin sulfate (C-1, C-1a, C-2, C-2a) by high-performance liquid chromatography (HPLC) on an analytical and a semipreparative scale was developed. The method utilized ion-pair reversed-phase chromatography, isocratic elution with an aqueous solution containing 9% trifluoroacetic acid and 2.5% acetonitrile as the mobile phase at a flow rate of 2 and 9 mL/min for analytical and semipreparative columns, respectively. Detection was carried out at 213 nm without derivatization. The protonation pattern of the separated gentamicins was determined by potentiometry and 15N and 1H NMR. The full proton NMR assignment for gentamicin C-1 was obtained through the use of 1H 1D and 2D 1H-1H COSY measurements.     

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.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Lactones 27 [1]: Transformations of γ-lactones fused to a dimethylcyclohexane ring in Absidia cylindrospora cultures

Two saturated (4a,b) and one unsaturated (5) bicyclic γ-lactones containing a dimethylcyclohexane ring were subjected to biotransformation using the fungal strain Absidia cylindrospora. Six new compounds (6–11) and one known (12) [K.W. Rosenmund, H. Herzberg, H. Schutt, Chem. Ber. 87 (1954) 1258] [2] were isolated. All substrates were stereoselectively hydroxylated by the microorganism at either the C-4 (in the case of 4a and 5) or C-2 position (in case of 4a and 4b) giving the corresponding hydroxylactones with tertiary (6 and 9) or secondary (8 and 10) hydroxy groups, respectively.

The hydroxy group was also introduced into C-3 (in the case of 4a) and C-6 (in the case of 4b) positions. The structures of all obtained products were established on the basis of their spectral data. In the case of lactones 8–10 these structures were undoubtedly confirmed by their X-ray analysis.