Kelvin E. Smith

A survey of the high-field 1H NMR spectra of the steroid hormones, their hydroxylated derivatives, and related compounds

1 H NMR chemical shifts are presented for virtually all the protons in 166 steroids. These comprise mainly the hormones testosterone, androst-4-ene-3,17-dione, progesterone, and a wide range of their hydroxylated derivatives, some corticosteroids including aldosterone and a series of its derivatives, together with miscellaneous steroids comprising a variety of androstane and pregnane derivatives, bile acids, and sterols, to provide the first extensive collection of data for use in correlating 1H chemical shifts with structure.Most of the spectra were assigned with the aid of two-dimensional 1H homonuclear correlated spectroscopy (COSY), or in a few cases by use of ω1,-decoupled COSY (COSYDEC) spectroscopy. Limited use was made of other techniques, especially selective nuclear Overhauser effect difference spectroscopy (NOEDS), to complete the more difficult assignments. Procedures for the rapid analysis of high-field 1H NMR spectra of steroids are suggested: they include the application of templates for the recognition of signals due to particular protons from their characteristic profiles, which generally vary only slightly between different steroids unless simplified by adjacent substitution. Substituent increments for all protons are reasonably additive, except where drastic conformational change occurs or adjacent substituents interact strongly. The conformational dependences of hydroxy-substitution increments are analysed empirically, and some regularities are identified.

Thermus oshimai sp. nov., isolated from hot springs in Portugal, Iceland, and the Azores, and comment on the concept of a limited geographical distribution of Thermus species

We examined aerobic, thermophilic, gram-negative bacteria that were isolated from hot springs in Portugal and were identified as Thermus strains and placed in phenetic groups on the basis of their phenotypic characteristics. We determined the composition of the peptidoglycan, identified the respiratory quinones, and determined the mean base composition of the DNA, and the levels of DNA-DNA homology were determined by both the filter hybridization and reassociation rate methods. Thermus aquaticus, Thermus brockianus, and Thermus filiformis were not detected in this collection of organisms, although three Thermus thermophilus strains were identified. We propose that the isolates that belonged to phenetic clusters E and F are members of a new species, Thermus oshimai; the type strain of T. oshimai is strain SPS17.

Microbial transformations of steroids—VIII. Transformation of progesterone by whole cells and microsomes of Aspergillus fumigatus

The filamentous fungus, Aspergillus fumigatus, efficiently hydroxylated exogenous progesterone producing, after 3 h of incubation, 11 alpha- and 15 beta-hydroxyprogesterone as major products, 7 beta-hydroxyprogesterone as a minor product and trace amounts of 7 beta, 15 beta- and 11 alpha, 15 beta-dihydroxyprogesterone. After 72 h the dihydroxyprogesterones were the sole metabolites in the culture medium. Microsomes, prepared by Ca2+ precipitation, catalysed only monohydroxylation of progesterone at the same sites as whole cells. Hydroxylation was dependent on NADPH (but not NADH) which was replaceable by NaIO4. Hydroxylation was inhibited by carbon monoxide and by the azole fungicide, ketoconazole. Microsomes gave a dithionite-reduced, carbon monoxide difference absorbance spectrum with a peak at 448 nm and a Type-I progesterone-binding spectrum typical of cytochrome P450 interaction with substrate. Ketoconazole inhibition studies suggest the presence of two non-inducible cytochrome P450 progesterone hydroxylases, one possessing 7 beta site-selectivity, the other 11 alpha/15 beta site-selectivity.

Microbial transformations of steroids—X. Cytochromes P-450 11α-hydroxylase and C17–C20 lyase and a 1-ene dehydrogenase transform steroids in Nectria haematococca

Nectria haematococca contains four enzymes that metabolise exogenous steroids. Two of these are microsomal cytochromes P-450 which act sequentially on progesterone producing firstly, by side-chain cleavage, the C19 steroid androstenedione (C17-C20 lyase), and then, in a subsequent set of transformations, 11 alpha-hydroxylated derivatives (11 alpha-hydroxylase). Two other conversions occur after side-chain cleavage. Unsaturation, in the form of a double bond at C1-C2, is introduced into the A ring by a catalytically self-sufficient microsomal 1-ene dehydrogenase. This enzyme is specific for C19 substrates. A C17-specific oxidoreductase is also involved in the production of androstenedione and testosterone from progesterone. The lyase, 11 alpha-hydroxylase and 1-ene dehydrogenase were purified to homogeneity.

Progesterone metabolism by the filamentous fungus Cochliobolus lunatus

Studies of Cochliobolus lunatus m118 steroid metabolism by thin-layer chromatography, mass spectrometry and NMR spectroscopy revealed that the fungus hydroxylates progesterone at positions 7 alpha, 11 beta and 14 alpha, and oxidizes the 11 beta-hydroxy group to the ketone. The 1H NMR spectra of two of the steroid metabolites, 11 beta,14 alpha-dihydroxyprogesterone and 11-oxo-14 alpha-hydroxyprogesterone, are reported for the first time. It is still not known if all the hydroxylation reactions are performed in C. lunatus by a single, non-specific, steroid hydroxylase, structurally different from the 11 beta-hydroxylase found in higher eucaryotes, or if different forms of the enzyme are involved.

Progesterone 6-hydroxylation is catalysed by cytochrome P-450 in the moderate thermophile Bacillus thermoglucosidasius strain 12060

The moderate thermophile, Bacillus thermoglucosidasius, transforms progesterone into four metabolites. These are 6alpha- and 6beta-hydroxyprogesterone, androstenedione and testosterone. This is the first report of bacterial 6alpha-hydroxylation of steroids. The identity of the progesterone metabolites shows that there are three major types of transforming activity in this organism; C-17-C-20 lyase that cleaves the pregnane side chain of the substrate, C-17 oxidoreductase that interconverts the metabolites androstenedione and testosterone, and 6-hydroxylation. 6-hydroxylation activity was purified virtually to homogeneity and was shown to be catalysed by a cytochrome P-450 monooxygenase enzyme. This is the first report of a thermostable cytochrome P-450.

Microbial transformations of steroids—VI. transformation of testosterone and androstenedione by Botryosphaerica obtusa

The 7 beta progesterone-hydroxylating microorganism Botryosphaerica obtusa was tested for its ability to hydroxylate at this site the C-19 androstene-based compounds, androstenedione (androst-4-ene-3,17-dione) and testosterone (17 beta-hydroxyandrost-4-en-3-one). Only very limited 7 beta hydroxylation of both substrates was observed. The products included traces of 7 beta-monohydroxytestosterone and 6 beta,7 beta-dihydroxyandrostenedione from testosterone, and of 6 beta,7 beta-dihydroxyandrostenedione from androstenedione. 6 beta,7 beta-Dihydroxyandrostenedione does not appear to have been reported previously as a microbial transformation product. Both substrates were monohydroxylated in significant amounts at the isomeric 7 alpha site and at the 6 beta site. Testosterone was also significantly monohydroxylated at the 15 alpha site and in minor amounts at the 11 alpha and 12 beta sites. Some monohydroxytestosterones had also been oxidised at their 17-OH group, converting them into the corresponding monohydroxy androstenediones. The 7 alpha-hydroxy metabolites and 15 alpha-hydroxytestosterone being chemically demanding to synthesis are valuable microbial transformation products.

Production of malodorous steroids from androsta-5,16-dienes and androsta-4,16-dienes by Corynebacteria and other human axillary bacteria.

The biotransformations of a number of steroids, chiefly 5,6,16,17-tetradehydro-androstanes, are reported. The strains investigated were Corynebacteria sp. G38, G40, G41, B, Brevis sp. CW5 and Micrococcus sp. M-DH2. Corynebacterium sp. G41 proved remarkably efficient in effecting oxidative isomerisation of 5-ene-3-sterols into the corresponding 4-en-3-ones. The main biochemical reactions involved were oxidation at C-3; no reduction processes were observed. Conversions of 3beta-sterols into the C-3 oxo-steroids were high, but were correspondingly low for the 3alpha-sterol epimers. Androsta-4,16-dien-3-one and 5beta-androsta-16-en-3-one are crucial to the formation of malodour. The rate of formation of these compounds was measured over 72 h incubation periods using three substrates: androsta-5,16-dien-3beta-ol, androsta-4,16-dien-3beta-ol and androsta-5,16-dien-3-one. Induction studies of the transformation of the androsta-5,16-dien-3beta-ol into the very odorous compound androsta-4,16-dien-3-one showed that cells incubated with a mixture of antibiotics displayed the same extent of biotransformation as normal cells if the concentration of antibiotic was low (1, 3, 5 and 7 microg/ml), although at concentrations higher than 10 microg/ml, biotransformation yields were reduced. Pre-incubation with a 3beta-fluoro-steroid inhibited the formation of the odorous androsta-4,16-dien-3-one.

Microbial transformations of steroids. III: Transformation of progesterone by Sepedonium ampullosporum

The 16 alpha-steroid hydroxylating fungus Sepedonium ampullosporum (CMI strain 203 033) transformed progesterone into 16 alpha-hydroxyprogesterone and four other major metabolites which have not been reported previously for this organism, 6 beta-hydroxyprogesterone, 17 alpha-hydroxyprogesterone, 16 alpha-hydroxyandrostenedione and 16-oxotestosterone (16-ketotestosterone). Among the minor metabolites we have been able to identify 15 alpha-hydroxyprogesterone. This compound has not been reported for S. ampullosporum. The conditions used for transformation had comparatively little effect on the relative proportions of products formed, 16 alpha-hydroxyprogesterone always being the predominant metabolite, but had a major effect on the total yields of metabolites isolatable. These findings suggest that one or more constitutive enzyme systems were responsible for the transformations.

Microbial transformations of steroids--V. Transformation of progesterone by whole cells and extracts of Botryosphaerica obtusa.

Members of the genus Botryosphaerica are reported 7 alpha steroid hydroxylators [1]. We found that the species B. obtusa efficiently hydroxylated progesterone in a 1-day transformation but it gave 7 beta-hydroxyprogesterone as the main product rather than the expected 7 alpha-hydroxy isomer, which was produced in only trace amounts. Also formed in minor amounts were 6 beta-, possibly 9 alpha- (see main text), 14 alpha- and 15 beta-monohydroxyprogesterones. The transformation mixtures included appreciable amounts of dihydroxylated progesterones which were mainly based on 7 beta-hydroxyprogesterone. The second hydroxyl group was at one of the minor monohydroxylation sites. The relative concentrations of the progesterone diols increased and those of the mono-alcohols concomitantly decreased when transformation was extended beyond 1 day. Monohydroxylated 6-dehydroprogesterones began to accumulate after about 3 days and these compounds seemed to have been formed by 6,7-dehydration of the dihydroxyprogesterones. We prepared mycelial cell-free extracts which were capable of transforming progesterone and retained the site-specificity of whole cells. These extracts converted 7 beta-hydroxyprogesterone to its 6-dehydro derivative, confirming that ring B desaturation occurs in this organism by dehydration. The dehydratase activity necessary for the conversion was separable from the hydroxylase activity by ultra-centrifugation. All hydroxylase activity co-sedimented with the membrane fraction, implying that steroid hydroxylation is effected by a membrane-bound enzyme(s). Dehydratase activity was present in both the pellet and the supernatant fractions, which suggests that it may involve a loosely bound, and easily removed, membrane-associated enzyme.