Chapter 2: Synthesis of steroid glucuronides
2.1 Introduction & previous work
2.2.2 Screening the steroid library
While a few steroid substrates were tested previously by Wilkinson, the broader scope of the synthase protocol remained unexamined. Therefore a screen of a suitably diverse steroid library was performed. For example, different stereochemistries of the alcohols, different structural features etc. would be desirable to demonstrate the broad applicability of this protocol.
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The steroids chosen for the primary screen are shown in figure 10 and 11 with the expected sites of glucuronylation highlighted in blue. There are a range of stereochemistries about the alcohol acceptor site (compare epiandrosterone 35, androsterone 43, etiocholanolone 46 or testosterone 13 and epitestosterone 41) and different structural features (compare epiandrosterone 35 with methandriol 44, mesterolone 45, nandrolone 37 and estrone 22).
Figure 10: the initial steroid library for the glucuronylsynthase protocol screen. Highlighted in
blue are the expected sites of glucuronylation and numbering at the sites are provided for ease of reference (e.g. “the C3 position is an expected site of glucuronylation for epiandrosterone”)
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In addition there are a couple of tertiary alcohols present that could potentially also react productively (methandriol 44 and methyltestosterone 48). However, these sites are very sterically hindered and it was expected that if glucuronylation were to occur at all (an interesting prospect by itself) then the conversion to the glucuronide would be low. Some representative steroids from the cholesterol family were also chosen (compare lithocholic acid
49 with cholestene-3β,25-diol 52, for example).
A qualitative assessment was performed for the steroid library to determine how well the enzyme could process each steroid. The 1H NMR spectra and mass spectra of the purified
compounds were examined and the relative intensities of the key peaks were assessed, such as those for the anomeric proton, the steroid backbone etc.
The results of this screen and the predicted products are presented in table 2 with the end assessment of each reaction given as tick marks, with three ticks indicating excellent reactivity (e.g. a high quality 1H NMR spectrum), two ticks indicating moderate reactivity (e.g. an average 1H NMR spectrum where some key peak are either indistinguishable or undetectable) and one
tick indicating poor reactivity (e.g. poor signal-to-noise ratio in the 1H NMR spectrum or only
detectable by mass spectrometry).
Table 2: the results of the qualitative screen of the steroid library
Entry Glucuronylsynthase reactiona Assessment
1
2
39 4 5 6 7 8 9 10
40 11 12 13 14 b 15 16 17 NRc
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aReaction conditions: steroid (~1 mg, 1.0 equiv., 0.69 mM), α-D-glucuronyl fluoride (5.0 equiv.),
tert-butanol (10% v/v), NaPi (50 mM, pH 7.5), E504G glucuronylsynthase (final concentration =
0.2 mg/mL), 37 °C, 3 d. bContained some parent steroid in the final product. cNo reaction.
Pleasingly, several important steroid glucuronides such as those of testosterone 13, epitestosterone 41, nandrolone 37 and methandriol 44 afforded sufficient quantities of the corresponding glucuronides for 1H NMR spectra with excellent signal-to-noise ratio. In addition,
glucuronides of etiocholanolone 46 and androsterone 43 were part of a successful WADA research project on longitudinal monitoring of steroid abuse and their successful syntheses here directly demonstrates the potential utility of this approach for anti-doping efforts157.
Androsterone 43 exhibited the lowest activity when compared to etiocholanolone 46 and epiandrosterone 35, which could most likely be attributed to the axial orientation of the C3 hydroxyl in androsterone 43 as opposed to the equatorial orientation of the same hydroxyl group for etiocholanolone 46 and epiandrosterone 35. However, etiocholanolone 46 is also somewhat more hindered than epiandrosterone 35 as the cis-fused A & B rings contribute to steric bulk around the alcohol (figure 12).
Figure 12: the three-dimensional representations of epiandrosterone 35, etiocholanolone 46
and androsterone 43 showing the arrangement of the C3 hydroxyl groups
The successful syntheses of glucuronides of estrone 22 and estradiol 51 were also achieved, with mono- and bis-glucuronylation observed for estradiol 51, suggesting that phenol-based glucuronides are readily accessed by the glucuronylsynthase, even when another reactive alcohol is present.
For estradiol 51, by 1H NMR it appeared that approximately two-thirds of the mixture contained
a C17 glucuronide, and that approximately one-third of the mixture was bis-glucuronide, assuming that the C17 hydroxyl is more reactive due to the delocalisation of the lone pairs on the phenolic oxygen into the aromatic ring. However, this remained conjecture until further quantitative analyses could be performed.
The poorer reactivity of the cholesterol derivatives may be explained by their higher relative hydrophobicity conferred by the long hydrophobic alkyl chain attached to the C17 carbon. This
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translated into difficult reaction preparations as dissolving them in aqueous media even with 10% v/v tert-butanol proved to be impossible and all resisted complete dissolution with the final reactions performed with a saturated solution of the steroids. It appears that further hydroxylation on the backbone of the steroid does not improve yield (a glucuronide of cholestane-3β,5α,6β-triol 53 was not observed even by mass spectrometry), but the presence of a polar group on the pendant alkyl chain, as found in lithocholic acid 49 (shorter chain with a carboxylic acid) and cholestene-3β,25-diol 52 (a hydroxyl group at C25) seems to greatly improve solubility, although again saturated solutions resulted.
The sample of lithocholic acid glucuronide 64 was unfortunately contaminated with some parent steroid though this was not unexpected as the presence of a carboxylate would aid retention of the parent steroid on the cartridge sorbent during the methanol wash. However, this would prevent isolation and consequent characterisation of the pure glucuronide. At the time of screening alternative methods of purification were not available within the group but with recent developments in C18 SPE methodology it is envisaged that future experiments with steroids such as lithocholic acid 49 using C18 SPE purification would be able to separate the more polar glucuronide from the parent steroid.
Methyltestosterone 48 was expected to be a difficult prospect due to the greater steric hindrance as a tertiary alcohol compared to the other secondary alcohols in the screen. While trace amounts were detected in mass spectrometry for methyltestosterone glucuronide 61, no bis-glucuronide was observed for methandriol 44 and cholestene-3β,25-diol 52, with glucuronylation observed to take place solely at the C3 hydroxyl group. This suggested that in the presence of alternative reactive centres the glucuronyl moiety will react preferentially at the less sterically hindered sites, as expected.
Curiously, boldenone 47 also afforded only trace amounts of glucuronide, despite its similarities to the other successful C17 hydroxylated steroids. Whether the different shape of the A ring of the steroid is the cause for such a significant reduction in yield of glucuronide remained unknown.
From the initial screen a general trend began to emerge from the data. It appeared that the C3 β- hydroxylated steroids have the highest reactivity, followed by their C17 β-hydroxylated counterparts. The α-hydroxylated steroids make up the next tier of reactivity, with the least reactive steroids those that are either poorly soluble under the conditions (e.g. coprostanol 50) or particularly sterically hindered, such as methyltestosterone 48. To put this another way the reactivity trend appears to be C3 β-OH > C17 β-OH > C3 α-OH ≈ C17 α-OH.
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Figure 13: the low-resolution negative-mode electron-spray ionisation (LR-ESI) mass spectrum
for methyltestosterone 17-glucuronide 61 showing the small peak observed for the glucuronide It was also pleasing to see that a broad range of steroid substrates could be processed by the glucuronylsynthase albeit with varying degrees of success. This broad substrate scope reflects the comment in section 1.5.2.2 that E. coli must compete with other gut microflora for a limited supply of nutrients and therefore the β-glucuronidase enzyme with which it uses to harvest the glucuronic acid as a carbohydrate source from glucuronides must then be fairly non-specific for the aglycone portion of the molecule.