Steroid hormone modulation of 3H-prostaglanding E1 binding to bovine corpus leteum cell membranes.

The specific binding of 3H-prostaglandin E1 (3H-PGE1) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0x10-minus-6M. At concentrations above 2.5 x 10-minus-6M; estrone, 17beta-estradiol (but not 17alpha-estradiol or 17beta-estradiol glucuronide), estroil, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of 3H-PGE1. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced 3H-PGE1 binding. These findings permitted the following correlations between steroid structure and modulation of 3H-PGE1 binding: steroids with a free phenolic ring and a 17beta-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of 3H-PGE1. PGE receptors are heterogeneous with respect to affinity for 3H-PGE1. The steroids that decreased 3H-PGE1 binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased 3H-PGE1 binding caused appearance of new low affinity PGE receptors. Association rate constants for 3H-PGE1 binding were decreased by 17beta-estradiol (61%) and increased by DHT (59%).     

The formation of covalent adducts between benzo[a]pyrenediol epoxide and RNA: structural analysis by mass spectrometry

Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention times with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribonucleotides were poly(G) greater than yeast RNA greater than poly(A) greater than poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Several adducts exhibited molecular ions by both LSIMS and EIMS. Large fragments observed by EIMS usually resulted from the loss of CH3OH, CH3O., CH2O, CH3., and H. from the molecular ion. Major fragmentation pathways also resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions. Each of these major ions in turn resulted in further characteristic fragmentation patterns.     

Synthesis of deuterium-labeled tetrahydrocortisol and tetrahydrocortisone for study of cortisol metabolism in humans

A method is described for the preparation of multi-labeled tetrahydrocortisol (3alpha,11beta,17alpha,21-tetrahydroxy-5beta-[1, 2,3,4,5-2H5]pregnan-20-one, THF-d5), allo-tetrahydrocortisol (3alpha,11beta,17alpha,21-tetrahydroxy-5alpha-[1 ,2,3,4,5-2H5]pregnan-20-one, allo-THF-d5), and tetrahydrocortisone (3alpha,17alpha,21-trihydroxy-5beta-[1,2,3,4,5-2H5]pre gnane-11,20-dione, THE-d5) containing five non-exchangeable deuterium atoms in the steroid ring A. Reductive deuteration at C-1, C-2, C-3, C-4, and C-5 of prednisolone or prednisone was performed in CH3COOD with rhodium (5%) on alumina under the deuterium atmosphere. The isotopic purities of the labeled compounds as [2H5]-form were estimated to be 86.17 atom%D for THF-d5, 74.46 atom%D for allo-THF-d5 and 81.90 atom%D for THE-d5, based on the ion intensities in the region of the molecular ion of methoxime-trimethylsilyl (MO-TMS) derivatives measured by GC-MS.     

NAD(P)+ analogues: tools for the investigation of the active site of oestradiol 17beta-dehydrogenase from human placenta.

Oestradiol-17beta:NAD+ 17-oxidoreductase from human placenta can accept coenzyme analogues of NAD+ and NADP+ where the amide group is replaced by methyl ketone, nitrile or thioamide. The inhibition with analogues of NAD+ has been studied. The presence of a substituent at C-3 of the pyridinium ring is necessary for the binding. The inhibition by C-4 methylated analogues is very poor, and the effect of a methyl group at C-5 depends on the substituent at C-3. The 1,4,5,6-tetrahydronicotinamide adenine dinucleotide is a competitive inhibitor. Nicotinamide 8-bromoadenine dinucleotide and nicotinamide 8-thioadenine dinucleotide are efficient hydrogen acceptors.     

The influence of protonation or alkylation of the phosphate group on the e.s.r. spectra and on the rate of phosphate elimination from 2-methoxyethyl phosphate 2-yl radicals.

The e.s.r. spectra of 1-yl, 2-yl and 3'-yl methoxyethyl phosphate radicals derived from CH3OCH2CH2-OPO3H2 by hydrogen abstraction have been measured in aqueous solutions and the hyperfine constants determined. The coupling constants vary strongly with protonation or alkylation of the phosphate group. The 2-yl radicals eliminate phosphate. The rate-constants for the elimination (ke) have been estimated by e.s.r. measurements and by product studies as a function of pH using 60Co gamma-radiolysis. The ke values vary from approximately 0.3 s(-1) for the CH3OCHCH2OPO3--radical and approximately 10(3) s-1 for CH3OCHCH2OPO3H-, to approximately 3 X 10(6) S-1 for CH3OCHCH2OPO3H2. Alkylation of the phosphate group increases the elimination rate-constant to a similar extent as protonation. The results support a recent mechanism which described the OH-radical-induced single-strand breaks of DNA in aqueous solution starting from the C-4' radical of the sugar moiety. It is further concluded the C-4' radical of DNA eliminates the 3'-phosphate group faster than the 5'-phosphate group.     

Microbial metabolism of pyridinium compounds. Radioisotope studies of the metabolic fate of 4-carboxy-1-methylpyridinium chloride

Extracts of Achromobacter D formed CO(2), methylamine, succinate and formate as metabolic end-products from N-methylisonicotinic acid (4-carboxy-1-methylpyridinium chloride). The origin of the CO(2) in the 4-carboxyl group and of the methylamine in the N-methyl group of N-methylisonicotinate was demonstrated with carboxyl-(14)C- and N-Me-(14)C-labelled substrates respectively. The carbon skeletons of formate and succinate were shown to arise from the C-2 and the C-3-C-6 atoms of the heterocyclic ring respectively by using N-methyl[2,3-(14)C(2)]isonicotinate. This result is consistent with ring cleavage by the organism between C-2 and C-3.     

Production of 16beta-(acetoxy)acetoxy derivatives by reaction of 17-keto steroid enol acetates with lead (IV) acetate

Treatment of enol acetates of 3beta-acetoxyandrost-5-en-17-one and its 5alpha-reduced analog, 5alpha-androstan-17-one, and estrone acetate, 1-4, with Pb(OCOCH(3))(4) in acetic acid and acetic anhydride gave the previously unreported products, 16beta-(acetoxy)acetoxy-17-ketones 8-10 and 12, in 9-15% yields along with the known major products, 16beta-acetoxy-17-ketones 5-7 and 11. Similar treatment of the 16beta-acetoxy-17-ketones with the lead reagent did not yield the corresponding (acetoxy)acetates. Reaction of the enol acetate 3 with Pb(OCOCD(3))(4) in CD(3)COOD yielded principally the labeled (acetoxy)acetate 10-d(3), which had a CD(3)COOCH(2)COO moiety at C-16beta. In contrast, when the deuterated enol acetate 3-d(3), which was obtained by treatment of the 17-ketone 14 with (CD(3)CO)(2)O in the presence of LDA and which had a CD(3)COO moiety at C-17, was reacted with Pb(OCOCH(3))(4), the resulting product was the labeled compound 10-d(2). This product had a CH(3)COOCD(2)COO function at C-16beta. Based on these results, along with further isotope-labeling experiments, it seems likely that the (acetoxy)acetate is produced through a lead (IV) acetate-catalyzed migration of the 17-acetyl function of the enol acetate to the C-16beta-position followed by attack of an acetoxy anion of the lead reagent.     

Stereochemistry and deuterium isotope effects associated with the cyclization-rearrangements catalyzed by tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases, and the chimeric CH4 hybrid cyclase

Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS and HPS) catalyze the cyclizations and rearrangements of (E,E)-farnesyl diphosphate (FPP) to the corresponding bicyclic sesquiterpene hydrocarbons. The complex mechanism proceeds through a tightly bound (R)-germacrene A intermediate and involves partitioning of a common eudesm-5-yl carbocation either by angular methyl migration, or by C-9 methylene rearrangement, to form the respective eremophilane and spirovetivane structures. In this work, the stereochemistry and timing of the proton addition and elimination steps in the mechanism were investigated by synthesis of substrates bearing deuterium labels in one or both terminal methyl groups, and in the pro-S and pro-R methylene hydrogens at C-8. Incubations of the labeled FPPs with recombinant TEAS and HPS, and with the chimeric CH4 hybrid cyclase having catalytic activities of both TEAS and HPS, and of unlabeled FPP in D2O, together with gas chromatography-mass spectrometry (GC-MS) and/or NMR analyses of the labeled products gave the following results: (1) stereospecific CH3-->CH2 eliminations at the cis-terminal methyl in all cases; (2) similar primary kinetic isotope effects (KIE) of 4.25-4.64 for the CH3-->CH2 eliminations; (3) a significant intermolecular KIE (1.33+/-0.03) in competitive cyclizations of unlabeled FPP and FPP-d6 to premnaspirodiene by HPS; (4) stereoselective incorporation of label from D2O into the 1beta position of epiaristolochene; (5) stereoselective eliminations of the 1beta and 9beta protons in formation of epiaristolochene and its delta(1(10)) isomer epieremophilene by TEAS and CH4; and (6) predominant loss of the 1alpha proton in forming the cyclohexene double bond of premnaspirodiene by HPS and CH4. The results are explained by consideration of the conformations of individual intermediates, and by imposing the requirement of stereoelectronically favorable proton additions and eliminations.     

The C-4 hydroxyl group of galactopyranosides is the major determinant for ligand recognition by the lactose permease of Escherichia coli.

Binding specificity in lactose permease toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by nonspecific hydrophobic interactions with the non-galactosyl moiety [Sahin-Tóth, M., Akhoon, K. M., Runner, J., and Kaback, H. R. (2000) Biochemistry 39, 5097-5103]. To characterize the contribution of individual hydroxyls, binding of structural analogues of p-nitrophenyl alpha-D-galactopyranoside (NPG) was examined by site-directed N-[(14)C]ethylmaleimide (NEM) labeling of the substrate-protectable Cys148 in the binding site. NPG blocks NEM alkylation of Cys148 with an apparent affinity of approximately 14 microM. A deoxy derivative at position C-2 binds with 25-fold lower affinity (K(D) 0.35 mM), and the deoxy analogue at C-3 exhibits ca. 70-fold decreased binding (K(D) 1 mM), while binding of 6-deoxy-NPG is at least 130-fold diminished (K(D) 1.9 mM). Remarkably, the C-4 deoxy derivative of NPG binds with almost 1500-fold reduced affinity (K(D) approximately 20 mM). No significant substrate protection is afforded by NPG analogues with methoxy (CH(3)-O-) substitutions at positions C-3, C-4, and C-6. In contrast, the C-2 methoxy analogue binds almost normally (K(D) 26 microM). The results confirm and extend the observations that the C-2, C-3, C-4, and C-6 OH groups of galactopyranosides participate in important H-bonding interactions. Moreover, the C-4 hydroxyl is identified as the major determinant of ligand binding, suggesting that sugar recognition in lactose permease may have evolved to discriminate primarily between gluco- and galactopyranosides.     

Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.

Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.     

Establishing the structure of anthracyclinones produced by Streptomyces griseoruber

Streptomyces grisoruber strain 1618-306 produces three types of anthracycline antibiotics, derivatives of epsilon-pyrromycinone (methyl (7S, 9R, 10R)-9-ethyl-5,7,8,9,10,12-hexahydro-1,4,6,7,9-pentahydroxy-5,12-di oxo-10- naphthacenecarboxylate), epsilon-1-hydroxyauramycinone and epsilon-1-hydroxysulfurmycinone, differing in C-9 substituent in D ring of anthracyclines (Et, Met or CH2COCH3, respectively). Besides 7-O-glycosides of these aglycones, complex of antibiotics contains corresponding 7-deoxy- and 7,8,9,10-bisanhydroanthracyclinones.     

Crystal structure of steroidal monoenes obtained by reduction of the aromatic A ring of 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol

The crystal structures of 13-ethyl-gona-1(10)-ene-11alpha,17beta-diacetate (3b) and 13-ethyl-10alpha-gona-4-ene-11alpha,17beta-diacetate (5b), two steroidal monoenes obtained as minor products from the reduction, then acetylation, of the aromatic A ring of 13-ethyl-3-ethoxy-gona-1,3,5(10)-triene-11alpha,17beta-diol (1), were determined by X-ray diffraction. The conformations of the rings A, B, C, and D and the unusual stereochemistry at C-10 of the 10alpha-gona-4-ene (5b) are discussed.     

A tandem mass spectrometric study of bile acids: interpretation of fragmentation pathways and differentiation of steroid isomers

Bile acids are steroids with a pentanoic acid substituent at C-17. They are the terminal products of cholesterol excretion, and play critical physiological roles in human and animals. Bile acids are easy to detect but difficult to identify by using mass spectrometry due to their poly-ring structure and various hydroxylation patterns. In this study, fragmentation pathways of 18 free and conjugated bile acids were interpreted by using tandem mass spectrometry. The analyses were conducted on ion trap and triple quadrupole mass spectrometers. Upon collision-induced dissociation, the conjugated bile acids could cleave into glycine or taurine related fragments, together with the steroid skeleton. Fragmentations of free bile acids were further elucidated, especially by atmospheric pressure chemical ionization mass spectrometry in positive ion mode. Aside from universally observed neutral losses, eliminations occurred on bile acid carbon rings were proposed for the first time. Moreover, four isomeric 5β-cholanic acid hydroxyl derivatives (3α,6α-, 3α,7β-, 3α,7α-, and 3α,12α-) were differentiated using electrospray ionization in negative ion mode: 3α,7β-OH substituent inclined to eliminate H(2)O and CH(2)O(2) groups; 3α,6α-OH substituent preferred neutral loss of two H(2)O molecules; 3α,12α-OH substituent apt to lose the carboxyl in the form of CO(2) molecule; and 3α,7α-OH substituent exhibited no further fragmentation after dehydration. This study provided specific interpretation for mass spectra of bile acids. The results could contribute to bile acid analyses, especially in clinical assays and metabonomic studies.     

The synthesis of functionalized 13,14-seco-steroids via Grob fragmentation

A synthetic methodology for the synthesis of 13,14-seco-steroids with substituents at C-14 and C-17 is described. The approach involves Grob fragmentation of 14beta-hydroxy-17beta-tosylates, hydroboration-oxidation of the intermediate delta13(17)-olefin, and hydride reduction of the 14-ketone. An unambiguous structural assignment of (13R,14S,17S)-14,17-diacetoxy-3-methoxy-7alpha-methyl-13,14-secoestra-1,3,5(10)-triene was determined by X-ray analysis.     

Mass spectrometry of sterols. Electron ionization induced fragmentation of C-4-alkylated cholesterols

The electron impact ionization of C-4-alkylated cholest-5-en-3β-hydroxysterols has been investigated. The mass spectra of the C-4-alkylated cholesterols contain a number of ions in the high mass region for which analogous ions are not found in the spectrum of cholesterol. Detailed studies of the composition and origin of these ions have been made by high resolution mass spectrometry and analysis of metastable ions. In addition, a large number of isotopically (deuterium and ¹⁸O) substituted C-4-alkylated analogues have been prepared to assist in the interpretation of the spectra. The combined results indicate the occurrence of a number of very complex and unusual electron ionization induced fragmentations. Most notable of the findings reported herein concerns the demonstration of the formation of an ion involving loss of the elements of ring A with an intramolecular shift of the oxygen and hydrogen atoms of the hydroxyl function to the charge-retaining species.     

Fragments formed by the side chain cleavage of a 20-aryl analog of 20alpha-hydroxycholesterol by adrenal mitochondria.

An analog of 20alpha-hydroxycholesterol, (20R)-20-phenyl-5-pregnene-3beta,20-diol, which is completely substituted at C-22 was prepared with radioisotopes at various positions. The analog labeled with 3H at C-M and 14C at C-4 and C-IU was converted into radioactive pregnenolone by an enzyme preparation derived from adrenal mitochondria. Cleavage of the phenyl analog labeled with 3H in the aromatic ring by the same enzyme preparation led to the formation of [3H]phenol. Using the substrate doubly labeled with 14C at C-4 and 3H in the aromatic ring, it appeared that the products of the reactions, pregnenolone and phenol, were formed in equal amounts. During incubation of the side chain labeled substrate, another labeled fragment was formed. It was identified as acetophenone, a product resulting from cleavage of the C17,20 bond. The steroidal fragment corresponding to this C8 ketone was traced using nuclear label analog. From its nonpolar chromatographic properties it appears to be a C-17-deoxy-C19 steroid.     

The functional importance of structural features of ergosterol in yeast.

As an approach to the study of the relationship between the structure of sterols and their capacity to function in the lipid leaflet of membranes, various sterols were examined for their ability to support the growth of anaerobic Saccharomyces cerevisiae. A marked dependence on precise structural features was observed in growth-response and morphology. Of the chemical groups which distinguish ergosterol, the main sterol of S. cerevisiae, the hydroxyl group at C-3 was obligatory, and the other groups were found to be of the following relative importance: 24beta-methyl-delta22-grouping greater than 24beta-methyl group greater than delta5,7-diene system = delta5-bond approximately or equal to no double bond. Methyl groups at C-4 and C-14 were inconsistent with activity. Consequently, the data strongly suggest that the normal biosynthetic processes removal of methyl groups from the nucleus and introduction of one in the side chain are of functional significance. A double bond between C-17 and C-20 joining the steroidal side chain to the nucleus had no deleterious effect on the growth process but only if C-22 was trans-oriented to C-13. In the cis-case no growth at all proceeded. This means the natural sterol probably acts functionally in the form of its preferred conformer in which C-22 is to the right ("right-handed") in the usual view. Since the placing of a substituent (OH or CH3) in the molecule at C-20 in such a way that it appears on the front side in the right-handed conformer completely destroyed activity, the sterol apparently presents its front face to protein or phospholipid when complexing occurs.     

Structure-activity relationships of progesterone derivatives that bind to the digitalis receptor: modifications in A and B rings

A number of progesterone derivatives, having a 17 alpha-acetoxy group and various functions at C-3 and C-6, interact at the cardiac glycoside (CG) binding site, using [3H]ouabain in a radioligand binding assay (RBA) with membranes from dog myocardium. We now report on results of structure-activity studies concerned with modification of the A and B rings as they influence potency in the RBA. Some progesterone derivatives with 5 alpha- or 5 beta-stereochemistry show weak receptor competing activity. Among the congeners highest potency is associated with the presence of C-4 or C-4,6 unsaturation and a C-6 substituent (CH3, Cl, Br) whose importance appears to reside in its steric rather than electronic character. The C-3 function may be carbonyl, 3 beta-hydroxy or 3 beta-acetoxy when associated with C-4 or C-4,6 unsaturation. In compounds with other substituents that promote activity, C-6 alpha substitution with -CH3, -Cl, or -Br strongly enhances activity; -F, -OCH3, carbonyl, or the unsubstituted compound promotes weak binding; and -OC2H5, -OAc, -OCOOCH3, or -OH eliminates binding activity. Receptor interaction with the double bond at C-4, but not C-5, appears to be particularly important for binding. The most potent analog identified thus far is chlormadinone acetate (17 alpha-acetoxy-6-chloropregna-4,6-diene-3,20-dione), which has 1/20 the potency of ouabain in the RBA. Studies to determine optimal structural requirements for CG-receptor binding by these hormonal steroid congeners, in conjunction with appropriate biological assays, may provide insight into the nature of a putative endogenous counterpart, lead to a better understanding of the mode of action of the CG and yield CG-like compounds with superior therapeutic properties.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

Advanced NMR approaches for a detailed structure analysis of natural products

Some new nuclear magnetic resonance (NMR) approaches to elucidate chemical structures, which have not been determined by routine NMR methods, are presented. Selective detection of methine (CH), methylene (CH(2)), or methyl (CH(3)) signals in each subspectrum by editing NMR methods was utilized to reduce the complexity in crowded spectra. It also increased the peak separation and enhanced the sensitivity by limiting the measuring area of the 2D spectra. Several 2D methods to measure (2,3)J(CH) values, which are useful for stereochemical assignment are then introduced. To determine the structure of a highly hydrogen-deficient molecule, efficient correlation methods for long-range (13)C-(13)C coupling and (1)H-(15)N HMBC are also described.