Zur lokalisierung funktioneller gruppen mit hilfe der massenspektrometrie—VIII : 6-hydroxy-androstan-3,17-dione und 6,17β-dihydroxy-androstan-3-one

5β-androstan-3-ones carrying a 6α-OH group show in their mass spectra a key-ion indicating the loss of water and C-1 to C-4 as C₄H₅O? particle. 6β-OH isomers lose instead C-1 to C-4 in form of C₄H₇O?.In 6α-hydroxy-androstan-3-ones differentiation between the connection of the A/B-ring system is possible, because in 5α-isomers the loss of C-3 to C-7 occurs as a C₅H₆O₂ particle, while the 5β-isomers lose the same C atoms as a C₅H₇O? unit.Compounds with a 6β-OH group in an A/B trans connected ring system show a tendency for thermal water elimination. After rearrangement of the double bond in 4,5 position the typical fragments for 3-keto-Δ⁴-steroids are obtained.Occasionally a strong influence of a 6-OH group on fragmentation reactions in the D-ring system is observed: The presence of a 6α-OH group in an androstan-3,17-dione enhances the loss of C-16 and C-17 in the form of acetaldehydenol. Also the connection of the A/B-ring system may have a considerable influence on this type of reaction: In 6,17β-dihydroxy-androstan-3-ones only by trans connection of the A/B-ring system, C-16 and C-17 are lost with high probability after water elimination.     

Optical rotary dispersion and configuration in cardenolides

In accordance with the results of Weiss & ZIFFER , the ORD spectra of cardenolides with few exceptions show a distinct Cotton effect, caused by the butenolide ring. The two extrema lie at about 260 and 228 nm; the extremum at shorter wave length is very difficult to measure, and that at longer wavelength shows fine structure in dioxane. 14α,17β-cardenolides having either a hydrogen atom or a hydroxyl group at C-14 and no double bond in rings C and D show a negative Cotton effect, whereas compounds of the other three stereoisomeric types (14α,17α; 14β,17α and 14β,17β) show a positive Cotton effect. In the case of cardenolides having an oxo group, the extremum of the oxo group at shorter wavelength overlaps that of the butenolide ring at longer wavelength. In spite of this the latter extremum could still be seen distinctly in all cases investigated to date.     

Steroids and steroidases—XIX : Comparison of diazomethane and tiffeneau-demjanov homologations of 5α-3-oxosteroids. Evidence for predominant equatorial approach of the C-3 carbonyl group by diazomethane

Quantitative comparisons of the product ratios of the mechanistically similar diazomethane and Tiffeneau-Demjanov homologations of 17β-hydroxy-5α-androstan-3-one and 5α-cholestan-3-one have shown that equatorial approach of diazomethane to the C-3 CO group predominates to the extent of 70–79%. The data for both the C-17β-OH and -C₈H₁₇ substituted steroids are in close agreement thereby confirming that the C-17 substituents do not exert any significant long range effect on the reactions studied.     

Zur lokalisierung funktioneller gruppen mit hilfe der massenspektrometrie—XI : 3,12,17β-trihydroxy-androstane, 12,17β-dihydroxy-androstan-3-one,3,12-dihydroxy-androstan-17-one und 12-hydroxy-androstan-3,17-dione

Mass spectrometric degradation reactions of steroids with hydroxy groups in positions 12 and 17β depend on the configuration of the C-12 hydroxy group. In compounds with a 12α-hydroxy group, this group and the hydrogen in position 17α is eliminated as H₂O. This reaction is followed by loss of a methyl radical. In the isomers with a 12β-hydroxy group this reaction is not possible. Here the loss of carbon 15–17 dominates the production of an ion by loss of two molecules of water. Key ions of mass 97 as well as M-44 and M-74 ions are produced by 17 keto steroids with a hydroxy group in position 12. If the rings A and B are cis-connected less specific degradation reactions are observed.     

Mass spectrometry in structural and stereochemical problems—CCXLIV : The influence of substituents and stereochemistry on the mass spectral fragmentation of progesterone

The complete high resolution mass spectra of progesterone (Δ⁴-pregnene-3,20-dione) and twenty-nine stereoisomers and alkyl substituted analogs have been analyzed with the aid of the recently developed computer program INTSUM. Progesterone analogs with “normal” configuration at the six chiral skeletal carbon atoms give rise to abundant ions corresponding to cleavage of the 1–2 and 3–4 bonds (ketene elimination), to cleavage of the 6–7 and 9–10 bonds (ring B cleavage), and to cleavage of the 13–17 and 15–16 bonds (partial ring D cleavage); these reactions are frequently followed by elimination of alkyl radicals. Alkyl groups at C-6 and C-10 exert a pronounced influence on the formation and fragmentation of the [M-ketene] ions. Reversal of configuration at C-10 increases the importance of ring B cleavage, whereas reversal at C-17 favors the partial cleavage of ring D. The fragmentation of 17-alkylprogesterones differs significantly from the general pattern, with acetyl loss (cleavage of the 17–20 bond) and partial ring D cleavage as the predominating reactions. Loss of ring D by cleavage of the 13–17 and 14–15 bonds is not an important reaction of progesterones. Direct interaction of the two ketonic functions was not observed.     

The incubation of 13α,17-dihydroxystemodane with Cephalosporium aphidicola

The biotransformation of 13α,17-dihydroxystemodane (3) with the fungus Cephalosporium aphidicola afforded 13α,17,18-trihydroxystemodane (4), 3β,13α,17-tri-hydroxystemodane (5), 13α,17-dihydroxy-stemodan-18-oic acid (6), 3β,11β,13α,17-tetra-hydroxystemodane (7), 11β,13α,17,18-tetrahydroxystemodane (8) and 3β,13α,17,18-tetra-hydroxystemodane (9). The hydroxylation at C-18 of the substrate points to a biosynthetically-directed transformation, because aphidicolin (2) is hydroxylated at this carbon. However, the C-3(β) and C-11(β) hydroxylations seem to indicate a xenobiotic biotransformation.     

Triple quadrupole tandem mass spectrometry of sesquiterpene lactones: a study of goyazensolide and its congeners

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the fragmentation pattern of ten sesquiterpene lactones of the goyazensolide type under low-energy collision-induced dissociation (CID) using a triple quadrupole mass spectrometer. The analysis revealed that loss of CO(2)[M + H - 44](+) is the predominant process for compounds that exhibit a hydroxyl at C-8. In contrast, compounds with different acyloxy groups at C-8 fragment by means of elimination of the corresponding carboxylic acids [M + H - (R(2)CO(2)H)](+) and consecutive losses of CO and H(2)O. Our results also demonstrate the influence of both the stereochemistry of the acyloxy group at C-8 on the relative abundances of product ions and the hydroxyl at C-15, which creates an additional pathway, resulting in highly diagnostic product ions. This work clearly demonstrates the utility of tandem quadrupole low-resolution mass spectrometry for studies on the rationalization of the fragmentation of a series of compounds with a highly conserved core structure, but differing in substituent groups.     

Zur lokalisierung funktioneller gruppen in steroiden mit hilfe der massenspektrometrie X : 5-Hydroxy-cholestane und 3,5-dihydroxy-cholestane sowie deren acetylderivate

Mass spectra of steroids with hydroxygroups in position 3 and 5 are characterized by ions produced by elimination of C-1 to C-4. These degradation reactions are especially important for steroids with a 5β-configurated hydroxygroup.In addition 3α,5β-dihydroxysteroids loose the carbon atoms of ring A together with C-6 in a structure specific cleavage process, enabling an unambigiuous determination of the configuration. Apart from these degradation reactions caused by the presence of functional groups derivatives of cholestanes show the well known cleavage processes of ring D. 3α,5-Dihydroxycholestanes may be differentiated from their 3β-isomeres by the preferential production of an ion of mass 247.     

A novel fragmentation of trimethylsilyl ethers of 3β-hydroxy-Δ5-steroids

An ion formed by loss of 56 mass units from the molecular ion is often seen in mass spectra of trimethylsilyl ethers of C₁₉ and C₂₁ steroids having a 3β-hydroxy-Δ⁵ structure and an oxo group at C-17 or C-20. The nature of this fragment was investigated by the use of perdeuteriotrimethylsilyl ether derivatives and of [4-¹⁴C], [3-¹⁸O], [4,4-²H₂] and [2,2,4,4-²H] labelled derivatives of 3β-hydroxy-5-androsten-17-one and 3β-hydroxy-5-pregnen-20-one. Evidence is presented to show that the neutral fragment of mass 56 is composed of carbon atoms 1, 2 and 3, the oxygen at C-3 and four hydrogen atoms. During the fragmentation process, the trimethylsilyl group and one of the hydrogens at C-2 are transferred to the fragment that carries the charge.     

ZUM MECHANISMUS MASSENSPEKTROMETRISCHER FRAGMENTIERUNGSREAKTIONEN BEI A/B-CIS- VERKNüPFTEN 3α-HYDROXYSTEROIDEN MIT UND OHNE 11-KETOGRUPPE

Mass spectra of steroids containing a carbonyl group in position 11 and a 3α-hydroxy group in a cis connected A/B ring system are characterised by very strong [M – 72]⁺· key ions and may therefore be clearly differentiated from the spectra of their isomers. The mechanism of this fragmentation reaction was investigated by deuterium labelling and the DADI technique. The 3α-hydroxy group is eliminated together with the 9α-H atom. Next a hydrogen atom is transferred from the A ring to the B/C/D ring system. This causes the cleavage of the C-3? C-4 bond and expulsion of C atoms 1 to 4 as butadiene. In 3α-hydroxy-5α-androstanes possessing no 11-keto group an analogous [M – 18]⁺. fragment is fromed, followed by the elimination of ethylene originating mostly from C-1 and C-2.     

Mass spectral fragmentation of functionalized lanostanes-I

Mass spectra of various functionalized lanostanes and their deuterated analogs are compared. It is proposed that the transfer of a hydrogen from the 18-methyl group to the 11-oxo group via a McLafferty rearrangement is made geometrically possible by prior ionization of the 13,14 bond which allows an optimum interatomic distance between the oxygen and hydrogen to be acquired; the 8,9 double bond is a requisite for electronic induction of this specific process. Transfer of the hydrogen from the 7β-hydroxy group to position 14 is implicated in the mass spectrum of 3β-acetoxy-7β-hydroxy-5α-lanostan-11-one.     

Mechanisms of water elimination and other degradations of 2-hydroxy-5-ketobornane cations in the gas phase

The mechanisms of water elimination in 2-hydroxy-5-ketobornanes have been investigated and compared with those in borneol and isoborneol in order to ascertain whether and how such processes are affected by incorporation of a remote second functional group of lower ionization energy. Analysis of metastable ion peak shapes and specific labelling with ²H and ¹⁸O are employed as methods for differentiating between individual reaction paths. In addition, those degradation reactions which give rise to the most prominent fragments and pattern differences in the mass spectra of the epimeric keto alcohols were scrutinized. The elimination of water is shown to exhibit entirely different features due to the presence of the carbonyl group. The other most important degradation reactions all proceed by hydrogen transfer from the hydroxyl group onto the charged product and concomitant loss of the hydroxyl oxygen in a neutral entity. The results are interpreted in terms of initial charge radical localization on the carbonyl group initiating the relevant chemical reactions. Peak shape analysis proves a very valuable, and at times the only useful way to distinguish individual reactions.     

Mass spectrometry of stanozolol and its analogues using electrospray ionization and collision-induced dissociation with quadrupole-linear ion trap and linear ion trap-orbitrap hybrid mass analyzers

Mass spectrometric identification and characterization of growth-promoting anabolic-androgenic steroids in biological matrices has been a major task for doping control as well as food safety laboratories. The fragmentation behavior of stanozolol, its metabolites 17-epistanozolol, 3'-OH-stanozolol, 4alpha-OH-stanozolol, 4beta-OH-stanozolol, 17-epi-16alpha-OH-stanozolol, 16alpha-OH-stanozolol, 16beta-OH-stanozolol, as well as the synthetic analogues 4-dehydrostanozolol, 17-ketostanozolol, and N-methyl-3'-OH-stanozolol, was investigated after positive electrospray ionization and subsequent collision-induced dissociation utilizing a quadrupole-linear ion trap and a novel linear ion trap-orbitrap hybrid mass spectrometer. Stable isotope labeling, H/D-exchange experiments, MS3 analyses and high-resolution/high mass accuracy measurements of fragment ions were employed to allow proposals for charge-driven as well as charge-remote fragmentation pathways generating characteristic product ions of stanozolol at m/z 81, 91, 95, 105, 119, 135 and 297 and 4-hydroxylated stanozolol at m/z 145. Fragment ions were generated by dissociation of the steroidal A- and B-ring retaining the introduced charge within the pyrazole function of stanozolol and by elimination of A- and B-ring fractions including the pyrazole residue. In addition, a charge-remote fragmentation causing the neutral loss of methanol was observed, which was suggested to be composed by the methyl residue at C-18 and the hydroxyl function located at C-17.     

Biotransformation of ursolic acid by an endophytic fungus from medicinal plant Huperzia serrata

Endophytic fungi were used not only for their producing bioactive products but also for their ability to transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was identified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina afforded three products, 3β-hydroxy-urs-11-en-28,13-lactone (2), 3β,7β-dihydroxy-urs-11-en-28,13-lactone (3), 1β,3β-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can hydroxyate the C12-C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively. In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.     

Characteristic fragmentation behavior of some glucuronide-type triterpenoid saponins using electrospray ionization tandem mass spectrometry

The fragmentation behavior of some glucuronide-type triterpenoid saponins from Symplocos chinensis, and their analogues escin Ia and Ib, were investigated by positive ion electrospray ionization tandem mass spectrometry using a quadrupole linear ion trap mass spectrometer. The fragmentation patterns of these saponins significantly changed in accordance with structural variations in the glucuronyl residue of the oligosaccharide chain. It was found that the carboxyl group and hydroxyl group at the C-3' position of the glucuronyl residue were the key sites for determining the fragmentation behavior of these compounds. When the carboxyl group was esterified, only the C(2alpha) ion, and no B(2alpha) ion, and cationized aglycone were observed. When the hydroxyl group at C-3' was acylated, the inherent cross-ring cleavage was hindered. However, glycosidic cleavages always occurred, regardless of the crucial structural variations. The results of the present studies can benefit the determination of trace triterpenoid saponins of this type in crude plant extracts, and also provide background information to aid the structural investigations of similar glycoconjugates.     

Identification of diastereomeric chlorophyll allomers by atmospheric pressure chemical ionisation liquid chromatography/tandem mass spectrometry

Atmospheric pressure chemical ionisation liquid chromatography/mass spectrometry (APCI-LC/MS) has been used for identification of the epimers of hydroxy, methoxy and methoxylactone allomers of chlorophyll a (13(2)-HO-chl a, 13(2)-MeO-chl a and 15(1)-MeO-lact-chl a), the hydroxy allomer of bacteriochlorophyll a (13(2)-HO-bchl a) and the hydroxy and methoxylactone allomers of bacterioviridin a (13(2)-HO-bvir a and 15(1)-MeO-lact-bvir a). The APCI mass spectra show that facile fragmentations involve the methoxyl or hydroxyl groups at the C-13(2) or C-15(1) chiral centres. Losses involving the C-13(2) or C-15(1) hydroxyl or methoxyl groups occur more easily from the S-epimer than from the R-epimer due to the greater relief of the steric strain associated with interaction with the bulky C-17 substituent. The differences in mass spectrometric fragmentation can be used as a diagnostic tool for the assignment of the stereochemical configuration at the C-13(2) or C-15(1) chiral centres.     

Reaktionen mit phosphororganischen Verbindungen. XLII. Nucleophile Substitutionen an Hydroxysteroiden mit Hilfe von Triphenylphosphan/Azodicarbonsäureester

Nucleophilic Substitution Reactions of Hydroxysteroids using Triphenylphosphane/diethylazodicarboxylate Nucleophilic substitution reactions by means of the title reagent on various more or less hindered steroid alcohols with suitable nucleophils in benzene is described. It was not possible to run this substitution process in the hitherto used solvent THF. Cholestan-3α-ol ( 1 ) was transformed to the 3β-substituted products 3β-benzoyloxy-cholestane ( 1a ) and 3β-azido-cholestane ( 1b ). Testosterone ( 2 ) affords with the corresponding nucleophils after short heating in benzene the inverted 17α-substituted products 3a, 3b and 3c . Analogously the 17α-azido-derivative 5a arises from 17β-hydroxy-androst-3-on ( 4 ). In the presence of a ketogroup in the substrate a competitive reaction can occur as it is shown in the case of cholestan-3-on ( 6 ): the products are the en-hydrazo-dicarboxylate-steroids 7a and 7b . The sterically very hindered 11α-position in 11α-hydroxy-4-pregnen-3,20-dion ( 8 ) can be transformed also to the 11β-azide 9a . The substitution of a 6 β-hydroxy group in androstane-3β, 6β, 17β-triol-3,17-diacetate ( 10 ) to the 6α-azide 11a affords the elimination product 12 as main component. Trans-diaxial vicinal diols such as cholestane-2β,3α-diol ( 13 ) give a mixture of the α- and β-oxiranes 14a and 14b .     

Biotransformation of two stemodane diterpenes by Mucor plumbeus

The microbiological transformation of 13α,17-dihydroxy-stemodane (2) by the fungus Mucor plumbeus afforded 13α,17,19-trihydroxy-stemodane (3), 3β,13α,17-trihydroxy-stemodane (5), 3-oxo-13α,17-dihydroxy-stemodane (7), 7α,13α,17,19-tetrahydroxy-stemodane (8), 3β,11α,13α,17-tetrahydroxy-stemodane (10), 3β,7α,13α,17-tetrahydroxy-stemodane (12), 3β,8β,13α,17-tetrahydroxy-stemodane (14), 2α,13α,17-trihydroxy-stemodane (16), 2α,13α,17,19-tetrahydroxy-stemodane (17), 2α,3β,13α,17-tetrahydroxy-stemodane (20) and 3β,11β,13α,17-tetrahydroxy-stemodane (22), whilst the incubation of 13α,14-dihydroxy-stemodane (25) gave 3β,13α,14-trihydroxy-stemodane (28), 2α,13α,14-trihydroxy-stemodane (29) and 13α,14,19-trihydroxy-stemodane (30). Preference for hydroxylations of ring A at C-2(α), C-3(β) and C-19 were observed in both incubations. An interesting rearrangement of 13α,14α-dihydroxy-stemodanes to 14-oxo derivatives with an unusual carbon framework has been observed under acetylation conditions. We have named this skeleton prestemodane, which, as a hydrocarbon ion, had been postulated as a biogenetic precursor of stemodane.     

Steroide—XL: 16,17-azido- und 16,17-aminoalkohole des östra-1,3,5(10)-trien-3-methyläthers

The four epimeric azido alcohols of estra-1,3,5(10)-trien-3-methyl ether with nitrogen at C-16 and oxygen at C-17 were prepared by the following reactions: cleavage of the 16α,17α-epoxide 1 with sodium azide affords the 16β,17α-azido alcohol 2a. The analogous reaction of the 16β,17β-epoxide 4 gives the 17α,16β-azido alcohol 5a and the desired 16α,17β-azido alcohol 6a in low yield. 6a is obtained in a smooth reaction by substitution of the 16β,17β-bromohydrine 8 with sodium azide. Sodium borohydride reduction of the 16β-azido-17-ketone 9 yields the 16β,17β-azido alcohol 10a, reduction of 16α-azido-17-ketone 13 with lithium borohydride gives the 16α,17α-azido alcohol 14a. From the azido alcohols the corresponding amino alcohols 3a, 7a, 11a and 15a are prepared with hydrazine hydrate/Raney nickel. The amino alcohols give the acetic anhydride the corresponding acetylamino alcohols. The cis-amino alcohols 11a and 15a react with acetone to the corresponding oxazolidines 12 and 16.     

Synthesis of the aglycone of 26-O-deacetyl pavoninin-5

The aglycone of 26-O-deacetyl pavoninin-5, (25R)-cholest-5-en-3β,15α,26-triol, 5a, was synthesized in 10 steps in 17% overall yield from diosgenin, 3. Removing mercury from the Clemmensen reduction of diosgenin 3, gave a higher yield of (25R)-cholest-5-en-3β,16β,26-triol, 4, by a method, that is also more environmentally friendly. Attempted methods for the transposition of the C-16β hydroxyl to the 15α position are described. A successful method for this transposition via the 15α-hydroxy-16-ketone, 13, using the Barton deoxygenation reaction on the 16-alcohol, 15, is reported.