Synthesis of steroid bisglucuronide and sulfate glucuronide reference materials: Unearthing neglected treasures of steroid metabolism

Article abstract of DOI:10.1016/j.steroids.2018.11.017

Doubly or bisconjugated steroid metabolites have long been known as minor components of the steroid profile that have traditionally been studied by laborious and indirect fractionation, hydrolysis and gas chromatography-mass spectrometry (GC–MS) analysis. Recently, the synthesis and characterisation of steroid bis(sulfate) (aka disulfate or bis-sulfate) reference materials enabled the liquid chromatography-tandem mass spectrometry (LC–MS/MS) study of this metabolite class and the development of a constant ion loss (CIL) scan method for the direct and untargeted detection of steroid bis(sulfate) metabolites. Methods for the direct LC–MS/MS detection of other bisconjugated steroids, such as steroid bisglucuronide and mixed steroid sulfate glucuronide metabolites, have great potential to reveal a more complete picture of the steroid profile. However, access to steroid bisglucuronide or sulfate glucuronide reference materials necessary for LC–MS/MS method development, metabolite identification or quantification is severely limited. In this work, ten steroid bisglucuronide and ten steroid sulfate glucuronide reference materials were synthesised through an ordered combination of chemical sulfation and/or enzymatic glucuronylation reactions. All compounds were purified and characterised using NMR and MS methods. Chemistry for the preparation of stable isotope labelled steroid {¹³C₆}-glucuronide internal standards has also been developed and applied to the preparation of two selectively mono-labelled steroid bisglucuronide reference materials used to characterise more completely MS fragmentation pathways. The electrospray ionisation and fragmentation of the bisconjugated steroid reference materials has been studied. Preliminary targeted ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis of the reference materials prepared revealed the presence of three steroid sulfate glucuronides as endogenous human urinary metabolites.

Full text of DOI:10.1016/j.steroids.2018.11.017

Accepted Manuscript
Synthesis of Steroid Bisglucuronide and Sulfate Glucuronide Reference Mate-
rials: Unearthing Neglected Treasures of Steroid Metabolism
Andy Pranata, Christopher C. Fitzgerald, Olha Khymenets, Erin Westley,
Natasha J. Anderson, Paul Ma, Oscar J. Pozo, Malcolm D. McLeod
PII:
S0039-128X(18)30221-6
https://doi.org/10.1016/j.steroids.2018.11.017
STE 8350
DOI:
Reference:
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Steroids
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Revised Date:
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13 August 2018
24 October 2018
26 November 2018
Please cite this article as: Pranata, A., Fitzgerald, C.C., Khymenets, O., Westley, E., Anderson, N.J., Ma, P., Pozo,
O.J., McLeod, M.D., Synthesis of Steroid Bisglucuronide and Sulfate Glucuronide Reference Materials: Unearthing
Neglected Treasures of Steroid Metabolism, Steroids (2018), doi: https://doi.org/10.1016/j.steroids.2018.11.017
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Synthesis of Steroid Bisglucuronide and Sulfate Glucuronide
Reference Materials: Unearthing Neglected Treasures of
Steroid Metabolism
a
a
b
a
Andy Pranata , Christopher C. Fitzgerald , Olha Khymenets , Erin Westley , Natasha J.
a
a
b
a,
Anderson , Paul Ma , Oscar J. Pozo , Malcolm D. McLeod *
a
Research School of Chemistry, Australian National University, Canberra, ACT 2601,
Australia.
b
Integrative Pharmacology and Systems Neuroscience Group, IMIM, Hospital del Mar,
Doctor Aiguader 88, Barcelona, Spain.
*
Corresponding author.
E-mail address: malcolm.mcleod@anu.edu.au (M. D. McLeod)
1

Abstract
Doubly or bisconjugated steroid metabolites have long been known as minor components
of the steroid profile that have traditionally been studied by laborious and indirect
fractionation, hydrolysis and gas chromatography-mass spectrometry (GC-MS) analysis.
Recently, the synthesis and characterisation of steroid bis(sulfate) (aka disulfate or bis-
sulfate) reference materials enabled the liquid chromatography-tandem mass spectrometry
(
LC-MS/MS) study of this metabolite class and the development of a constant ion loss
CIL) scan method for the direct and untargeted detection of steroid bis(sulfate)
(
metabolites. Methods for direct LC-MS/MS detection of other bisconjugated steroids, such
as steroid bisglucuronide and mixed steroid sulfate glucuronide metabolites, have great
potential to reveal a more complete picture of the steroid profile. However, access to
steroid bisglucuronide or sulfate glucuronide reference materials necessary for LC-MS/MS
method development, metabolite identification or quantification is severely limited. In this
work, ten steroid bisglucuronide and ten steroid sulfate glucuronide reference materials
were synthesised through an ordered combination of chemical sulfation and/or enzymatic
glucuronylation reactions. All compounds were purified and characterised using NMR and
13
MS methods. Chemistry for the preparation of stable isotope labelled steroid { C }-
6
glucuronide internal standards has also been developed and applied to the preparation of
two selectively mono-labelled steroid bisglucuronide reference materials used to
characterise more completely MS fragmentation pathways. The electrospray ionisation
and fragmentation of the bisconjugated steroid reference materials has been studied.
Preliminary targeted ultra-high-performance liquid chromatography-tandem mass
spectrometry (UHPLC-MS/MS) analysis of the reference materials prepared revealed the
presence of three steroid sulfate glucuronides as endogenous human urinary metabolites.
2

Highlights





Ten steroid bisglucuronide reference materials synthesised and characterised
Ten steroid sulfate glucuronide reference materials synthesised and characterised
18
13
Stable isotope labelled internal standards using O and C prepared
Electrospray ionisation and fragmentation of reference materials studied
Three steroid sulfate glucuronide metabolites confirmed in human urine
Keywords:
Steroid bisglucuronide; steroid sulfate glucuronide; steroid conjugate; phase II metabolism;
stable isotope labelled internal standard; mass spectrometry.
Abbreviations
CID = collision induced dissociation, CIL = constant ion loss, DHEA =
dehydroepiandrosterone, EA = epiandrosterone, E. coli = Escherichia coli, GC-MS = gas
chromatography-mass spectrometry, LC-MS = liquid chromatography-mass spectrometry,
LC-MS/MS = liquid chromatography-tandem mass spectrometry, NL = neutral loss, PORD
=
cytochrome P450 Oxido-Reductase Deficiency, SIM = single ion monitoring, SLOS =
Smith-Lemli-Opitz Syndrome, SPE = solid phase extraction, SRM = selected reaction
monitoring, STSD = Steroid Sulfatase Deficiency, UHPLC-MS/MS = ultra-high-
performance liquid chromatography–tandem mass spectrometry, WAX = weak anion
exchange.
3

1. Introduction
Steroids are a large family of compounds with diverse roles as lipids, hormones and
secondary metabolites, and as a result, numerous functions in biology and medicine [1].
Many current therapeutic interventions target steroid biosynthesis or signalling pathways
[
2],[3],[4] and this knowledge is also exploited in steroid abuse that remains a major
problem for world sport and wider society [5]. In mammalian systems, steroids undergo
two phases of metabolism [6]. Phase I metabolism involves changes to the steroidal
carbon skeleton including the oxidation and reduction of functional groups. This metabolic
change intersects and interacts with phase II metabolism involving the conjugation of
steroids with highly polar, charged groups, commonly sulfate [7],[8] and glucuronic acid
[
9],[10]. Phase II metabolism is the major step that increases steroid hydrophilicity,
allowing them to be rapidly and efficiently excreted from the body in biological fluids. At
least 97% of steroids excreted in urine are present as some form of phase II conjugate
[
11]. However, phase II conjugates also serve other important roles in steroid transport
and regulation, with steroids such as dehydroepiandrosterone (DHEA) sulfate and estrone
sulfate serving as an endogenous depot in steroid hormone metabolism [12],[13].
Traditionally, steroid analysis has been conducted using gas chromatography-mass
spectrometry (GC-MS) [14]. However, phase II conjugates such as steroid sulfates and
steroid glucuronides are not volatile or thermally stable enough for direct GC-MS analysis.
For this reason, chemical or enzymatic deconjugation of these metabolites to liberate the
phase I metabolites, prior to derivatisation and GC-MS analysis, is typically employed [11].
The routine deconjugation of phase II metabolites has several drawbacks. Although acid
solvolysis provides a general method of deconjugation, it cannot discriminate between
sulfate and glucuronide conjugates, and is also known to degrade sensitive analytes
[
2],[15]. Milder enzymatic hydrolysis with Escherichia coli (E. coli) β-glucuronidase
neglects the contribution of steroid sulfate metabolites and can result in incomplete
hydrolysis [16], while the use of crude enzyme preparations containing glucuronidase and
sulfatase enzymes can also lead to undesired steroid conversions [11] and is unable to
hydrolyse sulfates at several positions such as steroid 20-sulfates [17]. More recently,
purified bacterial arylsulfatase enzymes have been developed [18] to selectively hydrolyse
steroid sulfates under conditions compatible with those employed for E. coli β-
glucuronidase hydrolysis, but further work is required to establish the scope of these
methods for analytical applications [19]. More generally, the routine use of deconjugation
4

is undesirable as it destroys any information available from the study of conjugation
patterns or levels.
Rapid advances in liquid chromatography-mass spectrometry (LC-MS) technology provide
an improved method for the direct detection of the intact phase II conjugates, as they
ionise well by electrospray ionisation (ESI), and time-consuming hydrolysis and
derivatisation steps are not required [20]. Typically, mono-conjugated steroid sulfate and
glucuronide conjugates have been studied using this approach [16],[21]. On the other
hand, doubly conjugated steroids that are also present as a minor component of the
steroid profile, have generally only been studied through a laborious process of
chromatographic fractionation and solvolysis [22],[23], typically coupled to GC-MS analysis
[
24],[25]. These steroidal conjugates (Figure 1) including bis(sulfates), sulfate
glucuronides, bisglucuronides (single conjugation at two sites), or diglucuronides (double
conjugation at one site) have received little attention over past decades, in large part due
to an absence of suitable reference materials to aid analytical method development.
In 2017, an ultra-high-performance liquid chromatography–tandem mass spectrometry
(UHPLC-MS/MS) constant ion loss (CIL) scan method for the direct and untargeted
detection of steroid bis(sulfate) metabolites was reported [26]. This method revealed a
wide range of endogenous bis(sulfates) including examples from the estrane, androstane,
and pregnane steroid families. The CIL scan method was applied to identify metabolites
associated with sports doping and has also been employed in the analysis of maternal
urine for the prenatal diagnosis of inborn errors of steroid biosynthesis associated with
Smith-Lemli-Opitz Syndrome (SLOS), Steroid Sulfatase Deficiency (STSD), and
cytochrome P450 Oxido-Reductase Deficiency (PORD) [27].
Access to reference materials is central in the development of MS methods to detect or
quantify steroidal metabolites. In the study described above, a collection of 23 synthetically
derived steroid bis(sulfates) was used to investigate the ionisation and fragmentation of
this family, leading to the development of the selective, direct and untargeted CIL scan
method [26]. Currently, well-characterised reference materials for the other bisconjugate
families are not readily available. This limits analytical MS method development and
precludes the unambiguous identification or quantification of these under-explored
compounds. Few examples of the quantitative synthesis of steroid bisglucuronides
[
28],[29],[30], sulfate glucuronides [31],[32],[33] or diglucuronides [34] have been reported.
Examples of small scale, qualitative chemical and biochemical synthesis of steroid
5

bisglucuronides [35],[36],[34],[37],[38] and sulfate glucuronides [39] have also been
reported where the products have generally not been fully purified or characterised
spectroscopically.
In this work, the synthesis and characterisation of ten steroid bisglucuronide and ten
steroid sulfate glucuronide reference materials is reported. The MS ionisation and
fragmentation of these metabolites has been explored and three of these steroid sulfate
glucuronides have been confirmed as endogenous human urinary metabolites by UHPLC-
MS/MS analysis. In addition, the development of stably labelled glucuronide reference
materials is described, including selectively mono-labelled bisglucuronides, suitable for
use as internal standards or as probes to interrogate the site selectivity of fragmentation
processes. This chemistry will facilitate the development of new LC-MS methods for the
direct detection of bisconjugates and open avenues in the study of this fascinating but
neglected family of steroid metabolites in fields such as sports drug testing and medical
science.
Figure 1.
2. Experimental
Materials and instruments associated with the chemical synthesis and characterisation of
bisconjugates are reported in the supplementary material.
2.1. LC-MS/MS methods for steroid bisconjugate analysis
Negative mode LC-MS/MS studies for the ionisation and fragmentation of reference
materials was undertaken using a Waters Acquity triple quadrupole mass spectrometer
equipped with an ESI source and interfaced to a Waters 2695 Alliance Separations
Module. Nitrogen was used as desolvation and cone gas. The desolvation gas flow was
602 L/h, and the cone gas flow was 1 L/h. The nitrogen desolvation temperature was 349
°
C, and the source temperature was 148 °C. Steroid sulfate glucuronides or steroid
-
bisglucuronides were monitored for the mono-anion ([M-H] , cone voltage 70 V) and di-
2-
anion ([M-2H] , cone voltage 26 V) using ESI in negative scan MS (m/z 150-1000) or
targeted MS/MS mode (m/z 50-700, collision energy 10-50 eV) and with 4 kV capillary
voltage. The LC separation of reference materials was performed using a Waters
Symmetry C18 column (150 x 2.1 mm i.d., 5 µm) and a column temperature of 30 °C,
eluting with a gradient consisting of the following mobile phases: methanol and water, both
containing 0.01% formic acid and 10 mM ammonium formate. A gradient elution program
was applied, where the percentage of organic solvent was linearly changed at a flow rate
6

-1
of 0.3 mL min : 0 min, 30%; 9 min, 90%; 10 min, 30%; 15 min, 30%. The total analysis
time was 15 min.
The UHPLC-MS/MS analysis of urine samples was carried out using a Waters XEVO TQ-
S micro triple quadrupole mass spectrometer equipped with an ESI source and interfaced
to a Waters Acquity UPLC system for the chromatographic separation. Nitrogen was used
as desolvation and cone gas. The desolvation gas flow was 1200 L/h, and the cone gas
flow was 50 L/h. The nitrogen desolvation temperature was 600 °C, and the source
temperature was 150 °C. A cone voltage of 26 V and a capillary voltage of 0.4 kV were
used in negative ionization mode. The UHPLC separation for urine samples was
performed using a Waters Acquity UPLC CSH Phenyl-Hexyl column (100 x 2.1 i.d., 1.7
μm) and a column temperature of 30 °C, eluting with a gradient consisting of the following
mobile phases: acetonitrile:water (9:1, v/v), and water, both containing 0.01% formic acid
and 25 mM ammonium formate (VWR Prolabs Chemicals, Leuven, Belgium). A gradient
elution program was applied, where the percentage of organic solvent was linearly
changed at specified flow rates: 0 min (0.4 mL/min), 15%; 0.5 min (0.4 mL/min), 15%; 26
min (0.4 mL/min), 32%; 27 min (0.5 mL/min), 100%; 28 min (0.5 mL/min), 100%; 29 min
(0.5 mL/min), 15%; 29.5 min (0.4 mL/min), 15%, 30 min (0.4 mL/min) 15%. The total
analysis time was 30 min.
2.2. Urine sample preparation
The collection of human urine samples was conducted with approval of the Comité Ètic
d’Investigació Clínica of our institute (CEIC-IMAS no. 94/467) and the Spanish Health
Ministry (DGFPS no. 95/75). Three sub-sets of urine samples were selected for
confirmation of the presence of steroid sulfate glucuronide metabolites: urine samples from
healthy female volunteers (n=11, age 23-40 years), urine samples from healthy male
volunteers (n=11, age 26-46 years), and spot urine samples from healthy pregnant women
(n=8, age 30-39 years) at mid-term (gestation week 18-28). The collection time was not
specified for these samples. All urine samples were stored frozen at -20 °C prior to
analysis.
The procedure was adapted from the literature with minor modifications [40]. An aliquot of
18
urine (2 mL) was fortified with stably labelled 5α-androstan-3α,17β-diol 3,17{ O }-
3
18
-1
bis(sulfate) and 5α-androstan-3β,17β-diol 3,17{ O }-bis(sulfate), (25 ng mL ) internal
3
standard, treated with sodium phosphate buffer (50 mM, pH 7.5, 1 mL) and then
centrifuged (2000 rpm, 5 min). The supernatant was then loaded onto an Oasis weak
7

anion exchange (WAX) solid phase extraction (SPE) cartridge (Waters, 3 cc) that was pre-
conditioned with methanol (1 mL) and water (2 mL), and then washed with aqueous
sodium hydroxide solution (0.1 M, 2 mL), sodium phosphate buffer (50 mM, pH 7.5, 2 mL),
water (2 mL), and methanol (2 mL). The urinary steroid conjugates were then eluted with a
solution of ethyl acetate:methanol:diethylamine (25:25:1 v/v/v, 2 mL). Concentration under
a stream of nitrogen at 60 °C afforded a residue, which was reconstituted in water (150 µL)
and transferred to a sealed vial. The extract was injected (5 µL) into the instrument for
analysis by UHPLC-MS/MS according to Section 2.1 above.
2.3. Glucuronylation inhibition study
To a tube containing sodium phosphate buffer (50 mM, pH 7.5, 60 µL) and DHEA 1
solution in tert-butanol (c = 500 µM, 10 µL, c = 50 µM) was added diluted E. coli E504G
i
f
-1
glucuronylsynthase in sodium phosphate buffer (c = 0.6 mg mL , 10 µL, c = 0.06 mg
i
f
-1
mL ). In another tube, α-D-glucuronyl fluoride 2 solution in sodium phosphate buffer (c =
i
5
00 µM, 10 µL, c = 50 µM) was mixed with 5α-androstane-3β,17α-diol 3-glucuronide 3
f
solution in sodium phosphate buffer (c = 200 µM, 10 µL, c = 20 µM). The α-D-glucuronyl
i
f
fluoride 2 and additive 5α-androstane-3β,17α-diol 3-glucuronide 3 mix was then added to
the DHEA 1 and enzyme mix. Another four reactions were also set up by varying the final
concentration of the additive 3 to 0, 5, 10, and 15 µM. Negative controls were performed
with only DHEA 1, enzyme, and sodium phosphate buffer, while external standards were
prepared with DHEA 1, enzyme, sodium phosphate buffer, and DHEA glucuronide 4 (c =
i
5
0 µM, 10 µL, c = 5 µM). Reactions, negative controls, and external standards were then
f
incubated in water bath at 37 °C for 10 min, and immediately quenched with methanol
100 µL) containing 50 µM etiocholanolone sulfate as internal standard. The quenched
(
reaction mixture was centrifuged for 10 min (20000g), and then transferred to a 96-well
plate ready for mass spectrometry analysis.
The production of DHEA glucuronide 4 was assayed by UHPLC-MS using an Agilent 6120
quadrupole mass spectrometer equipped with and API-ES source and coupled to an
Agilent 1290 Infinity LC injector, HTS sampler and 1260 Infinity UHPLC system. Nitrogen
was used as drying and nebulizing gas. The drying gas flow was 3.0 L/min, and the
nebulizing pressure was 20 psig. The drying gas temperature was 300 °C. The mono-
-
anions ([M-H] ) of DHEA glucuronide 4 (m/z = 463.2), 5α-androstane-3β,17α-diol
bisglucuronide 5 (m/z = 643.3), and etiocholanolone sulfate (m/z = 369.2) were monitored
using ESI in negative single ion monitoring (SIM) MS mode with 200 V fragmentor and 3
kV capillary voltage. The UHPLC separation was performed with an Agilent Poroshell 120
8

C18 column (30 mm x 2.1 i.d. mm, 2.7 µm) and column temperature at 30 °C, eluting with
a gradient consisting of the following mobile phases: 10% v/v methanol:water, and 90% v/v
methanol:water, both containing 10 mM ammonium acetate. A gradient elution program
was applied, where the percentage of organic solvent was linearly changed at a flow rate
-1
of 0.2 mL min : 0 min, 42%; 1 min, 42%; 6 min, 80%; 7 min, 100%; 8 min, 100%; 9 min,
42%; 14 min, 42%. The total analysis time was 14 min.
2.4. Synthesis
2.4.1. General procedure for small scale steroid conjugate purification by SPE
This step was performed to separate a steroid conjugate (such as a steroid sulfate, steroid
glucuronide, or steroid bisglucuronide) from any unreacted starting steroid or steroid diol
after a conjugation reaction (sulfation or glucuronylation). The procedure was adapted
from literature methods [37],[41]. A WAX SPE cartridge (6 cc) was pre-conditioned with
methanol (5 mL) followed by water (15 mL). The reaction mixture was then loaded onto the
cartridge and eluted under a positive pressure of nitrogen at a flow rate of approximately 2
-1
mL min with the following solutions: formic acid in water (2% v/v, 15 mL), water (15 mL),
methanol (15 mL) and saturated aqueous ammonia solution in methanol (5% v/v, 15 mL).
The methanolic ammonia fraction was concentrated in vacuo to yield the desired steroid
conjugate as the corresponding ammonium salt.
1
2
.4.2. General procedure for determining conversion by H NMR analysis
This step was performed to calculate the ratio of steroid conjugate (steroid sulfate, steroid
glucuronide, or steroid bisglucuronide) to steroid or steroid diol remaining after a
conjugation reaction (sulfation or glucuronylation). The procedure employed a modified
WAX SPE protocol (general procedure 2.4.1) eluting with only formic acid in water (2% v/v,
15 mL), water (15 mL) and saturated aqueous ammonia solution in methanol (5% v/v, 15
mL), followed by concentration of the methanolic ammonia fraction to yield a mixture
containing both the starting steroid or steroid diol and the corresponding steroid conjugate
1
as the ammonium salt. A H NMR spectrum was obtained and integration of a suitable
signal (typically C3-H or C17-H) from both starting steroid or steroid diol and steroid
conjugate was used to determine the percent conversion of the sulfation or glucuronylation
reaction.
9

2.4.3. General procedure for C18 SPE purification of a steroid sulfate glucuronide or
steroid bisglucuronide
A C18 SPE cartridge (3 cc) was pre-conditioned with methanol (2 mL) followed by water (6
mL). The solution of steroid sulfate glucuronide or steroid bisglucuronide mixture in water
-1
(
1 mg mL , 1 mL) was then loaded onto the cartridge and eluted under a positive pressure
-1
of nitrogen at a flow rate of approximately 2 mL min with methanol:water (10-50% v/v, 3
mL), and methanol (3 mL). The methanol:water fraction was concentrated in vacuo to yield
the desired steroid sulfate glucuronide or steroid bisglucuronide as the corresponding
ammonium salt.
2.4.4. General procedure for the small scale reduction reaction of a steroid sulfate or
steroid glucuronide containing a saturated ketone, with purification by SPE
The procedure was adapted from the literature [41]. A solution of steroid sulfate or steroid
glucuronide (3.2-19 µmol) in methanol (100 µL) was treated by the addition of NaBH over
4
1
minute (7.0 mg, 0.19 mmol) with cooling on ice. After the vigorous reaction had
subsided, the reaction was capped, allowed to warm to room temperature and stirred for
6 h. The reaction was quenched by the slow addition of water (3 mL), adjusted to pH 7
universal indicator strips) by the addition of aqueous hydrochloric acid (0.1 M, 2 mL) and
1
(
subjected to SPE purification by general procedure 2.4.2 to afford the desired steroid diol
1
monosulfate or monoglucuronide as the corresponding ammonium salt. A H NMR
spectrum was obtained and integration of a suitable signal (typically C3-H, C16-H or C17-
H) from both the steroidal ketone and alcohol was used to determine the percent
conversion of the reduction reaction.
2.4.5. General procedure for the small scale glucuronylation reaction of a steroid, steroid
diol, steroid diol monosulfate, or steroid diol monoglucuronide with purification by SPE
The procedure was adapted from the literature [37]. The steroid, steroid diol, steroid diol
monosulfate, or steroid diol monoglucuronide (2.1-20 µmol, 0.7 mM final concentration) in
a tube was dissolved in tert-butanol (10% v/v), and sodium phosphate buffer (50 mM, pH
7.5, ~80% v/v), followed by E. coli E504G glucuronylsynthase (final concentration of 0.2
-1
mg mL ). Finally, α-D-glucuronyl fluoride 2 (5.0 equivalents) was dissolved in sodium
phosphate buffer (50 mM, pH 7.5, ~10% v/v) and added to the reaction. The reaction was
incubated without agitation at 37 °C for 2 days. The reaction mixture was then subjected to
a series of SPE purification steps as detailed in the experimental method depending on
which starting material was employed.
10

2.4.6. 5α-Androstane-3β,17β-diol bisglucuronide, ammonium salt 6
2.4.6.1 Method A. The reaction was conducted with 5α-androstane-3β,17β-diol 7 [26] (5.0
mg, 17 µmol, see the supplementary material) by general procedure 2.4.5. Purification of
the reaction by general procedure 2.4.2 gave a mixture of the title compound 6 and 5α-
1
androstane-3β,17β-diol 3-glucuronide 8 in a 2:1 ratio as determined by 400 MHz H NMR
integration of the C20-H and C26-H protons (no starting steroid diol 7 observed).
Performing the C18 purification procedure eluting with methanol:water (25% v/v) by
1
general procedure 2.4.3 afforded the title compound 6 in pure form (> 95% by H NMR).
Characterisation data is reported in the supplementary material.
The steroid bisglucuronides 5 and 9-16 (Figure 2), namely 5α-androstane-3β,17α-diol
bisglucuronide 5, estradiol bisglucuronide 9, androst-5-ene-3β,17β-diol bisglucuronide 10,
androst-4-ene-3β,17β-diol bisglucuronide 11, estr-4-ene-3β,17β-diol bisglucuronide 12,
5
β-cholane-3α,24-diol bisglucuronide 13, 5α-pregnane-3β,20S-diol bisglucuronide 14,
pregn-5-ene-3β,20S-diol bisglucuronide 15, and pregn-5-ene-3β,20R-diol bisglucuronide
6, were prepared using similar methods (see the supplementary material).
1
Figure 2.
.4.6.2 Method B. The reaction was conducted with 5α-androstane-3β,17β-diol 17-
2
glucuronide, ammonium salt [18] (derived in 19% conversion from dihydrotestosterone,
assumed 3.2 µmol, a 1:9 ratio of the 3α:3β diastereomers, see the supplementary
material) by general procedure 2.4.5. Purification of the reaction by general procedure
2.4.2 gave the title compound 6 as a colourless solid with a 90% conversion overall (>
98% conversion from 3β-diol monoglucuronide to the bisglucuronide, with the 3α-diol
1
monoglucuronide unreacted) as determined by 400 MHz H NMR integration of the C3-H
protons. Performing the C18 purification procedure eluting with methanol:water (25% v/v)
1
by general procedure 2.4.3 afforded the title compound 6 in pure form (> 95% by H NMR).
13
13
2
.4.7. 5α-Androstane-3β,17β-diol 3{ C },17-bisglucuronide, ammonium salt { C }-6
6
6
The reaction was conducted with 5α-androstane-3β,17β-diol 17-glucuronide, ammonium
salt [18] (1.0 mg, 2.1 µmol, a 1:7 ratio of the 3α:3β diastereomers, see the supplementary
13
13
material) and { C }-α-D-glucuronyl fluoride { C }-2 by general procedure 2.4.5.
6
6
13
Purification of the reaction by general procedure 2.4.2 gave the title compound { C }-6 as
6
a colourless solid with 87% conversion overall (> 98% conversion from 3β-diol
monoglucuronide to the bisglucuronide, with the 3α-diol monoglucuronide unreacted) as
1
determined by 400 MHz H NMR integration of the C3-H protons. Performing the C18
11

purification procedure eluting with methanol:water (25% v/v) by general procedure 2.4.3
13
1
afforded the title compound { C }-6 in pure form (> 95% by H NMR). Characterisation
6
data is reported in the supplementary material.
13
13
13
13
The steroid { C }-bisglucuronide estradiol 3,17{ C }-bisglucuronide { C }-9, and { C }-
6
6
6
6
13
13
13
monoglucuronides DHEA { C }-glucuronide { C }-4, epiandrosterone (EA) { C }-
6
6
6
13
13
13
13
glucuronide { C }-17, etiocholanolone { C }-glucuronide { C }-18, testosterone { C }-
6
6
6
6
13
13
13
glucuronide { C }-19 and epitestosterone { C }-glucuronide { C }-20 (Figure 3), were
6
6
6
prepared using similar methods (see the supplementary material).
Figure 3.
2.4.8. 5α-Androstane-3β,17β-diol 3-sulfate, ammonium salt 21
The reaction was conducted with EA sulfate, ammonium salt [41] (derived from EA 22, 5.5
mg, 19 µmol, see the supplementary material) by general procedure 2.4.4 to yield the title
compound 21 as a colourless solid with > 98% conversion. Characterisation data is
reported in the supplementary material.
2
.4.9. 5α-Androstane-3β,17β-diol 3-sulfate 17-glucuronide, ammonium salt 23
The reaction was conducted with 5α-androstane-3β,17β-diol 3-sulfate, ammonium salt 21
derived from EA 22, 5.5 mg, 19 µmol) by general procedure 2.4.5. Purification of the
(
reaction by general procedure 2.4.2 gave the title compound 23 as a colourless solid with
1
9
3% conversion as determined by 400 MHz H NMR integration of the C18-H protons.
3
Performing the C18 purification procedure eluting with methanol:water (28% v/v) by
1
general procedure 2.4.3 afforded the title compound 23 in pure form (> 95% by H NMR).
Characterisation data is reported in the supplementary material.
The steroid sulfate glucuronides 24-32 (Figure 4), namely androst-5-ene-3β,17β-diol 3-
sulfate 17-glucuronide 24, estradiol 3-sulfate 17-glucuronide 25, 5α-androstane-3β,17β-
diol 3-glucuronide 17-sulfate 26, 5α-androstane-3β,17α-diol 3-glucuronide 17-sulfate 27,
estradiol 3-glucuronide 17-sulfate 28, androst-4-ene-3β,17β-diol 3-glucuronide 17-sulfate
29, androst-4-ene-3β,17α-diol 3-glucuronide 17-sulfate 30, pregn-5-ene-3β,20R-diol 3-
sulfate 20-glucuronide 31, pregn-5-ene-3β,20S-diol 3-sulfate 20-glucuronide 32, were
prepared using similar methods (see the supplementary material)
Figure 4.
12

13
13
2
.4.10. 5α-Androstane-3β,17β-diol 3-sulfate 17{ C }-glucuronide, ammonium salt { C }-
6
6
23
The reaction was conducted with 5α-androstane-3β,17β-diol 3-sulfate, ammonium salt 21
13
13
(
3.0 mg, 7.7 µmol) and { C }-α-D-glucuronyl fluoride { C }-2 by general procedure 2.4.5.
6 6
13
Purification of the reaction by general procedure 2.4.2 gave the title compound { C }-23
6
1
as a colourless solid with 21% conversion as determined by 400 MHz H NMR integration
of the C18-H3 protons. Performing the C18 purification procedure eluting with
13
methanol:water (20% v/v) by general procedure 2.4.3 afforded the title compound { C }-
6
1
2
3 in pure form (> 95% by H NMR). Characterisation data is reported in the
supplementary material.
3. Results
3.1. Synthesis of steroid bisglucuronide reference materials
Glucuronylation was performed enzymatically using α-D-glucuronyl fluoride 2 as the
glucuronide donor and the E. coli glucuronylsynthase as catalyst [37]. In earlier work, this
method was applied to hydroxylated keto-steroids with various structures and
stereochemistries, and successfully produced a library of 14 steroid monoglucuronides
with 5-90% conversion [37]. Also in earlier work, this method was applied to estradiol and
gave a mixture of estradiol bisglucuronide 9, estradiol 3-glucuronide, and estradiol 17-
glucuronide in 1.1:1.0:1.6 ratio. Given the success of this earlier trial, we initially sought to
access a library of steroid bisglucuronides through the direct enzymatic glucuronylation of
steroid diols.
Steroid bisglucuronides in this study were generally synthesised in a single glucuronylation
reaction of steroid diols using an excess α-D-glucuronyl fluoride 2 donor (5 equivalents)
and the glucuronylsynthase enzyme, as shown for 5α-androstane-3β,17β-diol 7 (Scheme
1) [37].
Scheme 1.
After the reaction, SPE using an Oasis WAX cartridge was performed as outlined in
1
general procedure 2.4.2 and the product mixture analysed by H NMR spectroscopy. All
steroid diols gave > 98% conversion to conjugated steroid mixtures, with the exception of
reactions targeting bisglucuronides 11 and 12 (Figure 2). The starting diols for the
synthesis of bisglucuronides 11 and 12 contained mixtures of 3α and 3β alcohols, and the
3α alcohol did not react [37], thus lower conversions of 90% and 85%, respectively, were
13

observed. Where required, a second WAX SPE purification was performed as outlined in
general procedure 2.4.1 to remove the unreacted steroid diols.
1
When the mixtures only contained conjugated steroid diols, H NMR integration could also
provide a ratio of the steroid diol monoglucuronides and bisglucuronide formed. In the
example shown in Scheme 1 above, bisglucuronide 6 and diol monoglucuronide 8 were
1
produced in 2:1 ratio as determined by H NMR integration of the anomeric protons.
Typically, the steroid bisglucuronide was the major product, except for bisglucuronide 11
where a mixture of steroid diol monoglucuronides were the major products formed (Figure
2). When 3β,17β- or 3β,20S-diols were available (bisglucuronides 6, 10, 11, 12, 14, 15),
reactions produced bisglucuronide and diol 3-glucuronide, except for bisglucuronide 11
where diol 17-glucuronide was also observed in the mixture. In contrast, when 3β,20R- or
3α,24-diols were available (bisglucuronides 13 and 16), diol 20- or 24-glucuronides were
present at the end of the reaction. These data suggested relative reactivity for the E. coli
glucuronylsynthase-promoted glucuronylation that paralleled that revealed earlier by Ma
et. al. [37], with 3β(5α), 3β(5-ene), 3(phenolic), 20R and 24 hydroxy groups showing the
highest reactivity, while 3α(5β), 17β, 17α and 20S hydroxy groups showed lower reactivity,
and 3α(5α) hydroxy groups proved unreactive.
To isolate pure steroid bisglucuronide from conjugated steroid diol mixtures, a C18 SPE
method was used since it separated mixtures based on polarity, much like a reverse-
phase chromatography. The more polar compound (steroid bisglucuronide) was eluted by
lower concentrations of methanol in water. While the less polar compound in the mixture
(steroid diol monoglucuronides) were subsequently eluted with 100% methanol. For
steroid bisglucuronides with similar carbon skeletons such as 5α-androstanes (5 and 6),
androst-4/5-enes (10 and 11), estr-4-ene (12), and pregnane (14) types, the
bisglucuronide could be eluted selectively with 15-25% v/v methanol in water. A lower
methanol concentration was required to selectively elute estradiol bisglucuronide 9 (10%
v/v methanol in water), while the less polar compounds based on cholane (13) and
pregnene (15 and 16) skeletons needed 50% and 40% v/v methanol in water, respectively.
At the end, nine pure steroid bisglucuronides 6 and 9-16 were obtained, with the exception
of bisglucuronide 15 and 16, which were isolated as 20S:20R diastereomeric mixtures as
the bisglucuronides were not separable using the C18 method.
Although the enzymatic glucuronylation was successful in many cases, the bisglucuronide
5
could not be prepared by this approach. Direct glucuronylation of 5α-androstane-3β,17α-
14

diol 33 afforded 5α-androstane-3β,17α-diol 3-glucuronide 3 as the sole conjugated product
Scheme 2). This was despite earlier work [37], where several 17α-hydroxy steroids had
(
been successfully subjected to enzymatic monoglucuronylation. A stepwise approach
proved more productive. Glucuronylation of epidihydrotestosterone 34, followed by a
reduction reaction using sodium borohydride, gave a 1:8 mixture of 3α- and 3β- alcohol
diastereomers 35. A second glucuronylation reaction was then performed, and as desired,
the bisglucuronide 5 was obtained completing a library of ten steroid bisglucuronides
(Figure 2).
Scheme 2.
Two hypotheses were advanced to explain the requirement for stepwise synthesis: the
intermediate steroid 3β,17α-diol 3-glucuronide 3 substrate provided a poor fit for the
enzyme active site preventing further glucuronylation, or that the same intermediate 3
bound unproductively in the enzyme active site and so inhibited further reaction. To
explore this, a simple inhibition experiment was conducted using DHEA 1 as a model
substrate and 5α-androstane-3β,17α-diol 3-glucuronide 3 as a potential inhibitory additive
(Scheme 3). The aim was to explore if increasing concentrations of 5α-androstane-3β,17α-
diol 3-glucuronide 3 could decrease the production of DHEA glucuronide 4. The additive 3
was investigated at final concentrations of 0, 5, 10, 15, 20 µM. The LC-MS analysis of
these reactions showed that 15 and 20 µM of the additive 3 significantly reduced the
production of DHEA glucuronide 4 (p < 0.05), and by approximately 50% at 20 µM (Figure
5
). As a steroidal alcohol, the additive 3 could potentially serve as substrate, leading to the
steroid bisglucuronide 5, but as expected based on attempted synthesis from steroidal diol
3, this was not observed by LC-MS. The results show that 5α-androstane-3β,17α-diol 3-
glucuronide 3 inhibits E. coli glucuronylsynthase promoted synthesis of DHEA glucuronide
, and implicates unproductive binding of this intermediate as the reason for the failed
3
4
conversion of 5α-androstane-3β,17α-diol 33 to the bisglucuronide 5 (Scheme 2).
Scheme 3.
Figure 5.
The stepwise synthesis described above (Scheme 2) showed that order of glucuronylation
was important for the E. coli glucuronylsynthase promoted synthesis of bisglucuronides.
The one-step glucuronylation also proved unsuccessful for a number of other steroid diols
including 5β-androstane-3α,17β-diol, androst-4-ene-3β,17α-diol, and 16α-hydroxy-DHEA.
15

The stepwise approach was not investigated for these examples, but could be pursued in
future research. Further investigations (see the supplementary material) revealed that 5α-
androstane-3β,17β-diol bisglucuronide 6 (Figure 2) was accessible from the 5α-
androstane-3β,17β-diol 17-glucuronide (90% conversion, section 2.4.6.2) and not the
corresponding 3-glucuronide 8, estradiol bisglucuronide 9 was accessible from estradiol 3-
glucuronide (> 98% conversion) and not the corresponding 17-glucuronide, and androst-4-
ene-3β,17β-diol bisglucuronide 11 was accessible from both androst-4-ene-3β,17β-diol 3-
glucuronide (42% conversion) and 17-glucuronide (60% conversion). Although longer, the
stepwise method was observed to give clearer conversions in the final step, and in one
case, eliminated the need for C18 purification to remove diol monoglucuronide by-
products. Another advantage of the capacity to make a bisglucuronide from a specific
steroid diol monoglucuronide intermediate was the potential to isotopically label one of the
two glucuronide units of the bisglucuronide selectively to generate MS probes or internal
standards.
3.2. Synthesis of stable isotope labelled steroid monoglucuronides and bisglucuronides
Introduction of stable isotope labels to the glucuronide unit would allow differentiation of
the two conjugated positions of a bisglucuronide and enable the development of internal
standards for both steroidal glucuronides, bisglucuronides and sulfate glucuronides. Stable
labelling of the glucuronide unit would also increase synthetic efficiency. A range of stable
isotope labelled monoglucuronides are available that incorporate deuterium atoms within
the steroid skeleton. New synthetic routes are required for each labelled glucuronide
involving multiple chemical steps. Labelling of the glucuronide unit would provide a more
general method for the introduction of the label in the final step of the synthesis. Given
this, we sought methods to stably label the α-D-glucuronyl fluoride 2 donor employed in the
E. coli glucuronylsynthase-promoted glucuronylation reaction.
Labelling was first attempted using 18O derived from labelled water. The α-D-glucuronyl
fluoride 2 is prepared through an oxidation of α-D-glucosyl fluoride 36 using
bis(acetoxy)iodobenzene (BAIB) and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) in
acetonitrile and sodium bicarbonate buffer. These modified conditions avoided the use of
aqueous bleach as stoichiometric oxidant [42] and permitted a simple substitution with
18
labelled water. Under these conditions, oxidation afforded a mixture of labelled { O }-α-D-
2
18
18
glucuronyl fluoride, { O }-α-D-glucuronyl fluoride { O}-2, and unlabelled α-D-glucuronyl
1
fluoride 2 in approximately 20:12:1 ratio based on the LRMS (-ESI) analysis.
16

Mechanistically, the formation of doubly labelled and unlabelled sugar in the reaction was
18
consistent with the depicted equilibrium between aldehyde 37 and aldehyde hydrate { O}-
8 in the oxidation step (Scheme 4). For example, elimination of unlabelled water from
3
18
aldehyde hydrate { O}-38, followed by addition of labelled water and oxidation would
afford the doubly labelled { O }-α-D-glucuronyl fluoride. The resulting 18O labelled α-D-
18
2
glucuronyl fluoride { O}-2 mixture was reacted with EA 22 to form 18O labelled EA
18
18
18
glucuronide { O}-17. Based on LRMS (-ESI), the ratio between EA { O }-glucuronide, EA
2
{18
O }-glucuronide { O}-17, and EA glucuronide 17 was again 20:12:1.
18
1
Scheme 4.
18
18
The O labelled α-D-glucuronyl fluoride { O}-2 would be suitable for the preparation of a
mass spectrometry probe to distinguish between the glucuronide units in a bisglucuronide,
but could not serve as a stable isotope labelled internal standard due to the presence of
unlabelled material. This prompted a second approach to label the α-D-glucuronyl fluoride
13
13
2
by using the C isotope. A four-step synthesis of α-D-glucuronyl fluoride { C }-2 was
6
13
employed, starting from { C }-D-glucose [42]. This method successfully produced the
6
13
sugar with six C isotopes fully incorporated based on LRMS (-ESI). This was higher than
13
the theoretically expected labelling based on a lower threshold of at least 99 atom % C
13
13
specified by the supplier (94.2% hexa-labelled { C }, 5.7% penta-labelled { C }, 0.1%
6
5
13
13
tetra-labelled { C }, and 0.0% tri-labelled { C }). Two steroid bisglucuronides with one
4
3
13
selectively labelled glucuronide unit were prepared: 5α-androstane-3β,17β-diol 3{ C },17-
6
13
13
13
bisglucuronide { C }-6 and estradiol 3,17{ C }-bisglucuronide { C }-9. These were
6
6
6
synthesised using the stepwise approach described above (Scheme 2) with the final
13
13
glucuronylation step performed using { C }-α-D-glucuronyl fluoride { C }-2. In addition,
6
6
13
13
five C labelled steroid monoglucuronides were also synthesised, including DHEA { C }-
6
13
13
13
13
glucuronide { C }-4, EA { C }-glucuronide { C }-17, etiocholanolone { C }-glucuronide
6
6
6
6
{13
{13
{13
C }-18, testosterone { C }-glucuronide { C }-19 and epitestosterone { C }-glucuronide
13
13
13
6
6
6
6
13
13
13
C }-20. The steroid monoglucuronides { C }-4, { C }-17-{ C }-20, and bisglucuronides
6
6
6
6
13
13
C }-6 and { C }-9 were also shown to have full incorporation of the { C }-glucuronide
6
6
6
13
moiety based on LRMS (-ESI). The presence of the { C }-glucuronide moiety gave
6
1
13
1
distinctive couplings in H and C NMR spectra. In the H NMR spectrum, additional
coupling was observed, when compared to the unlabelled compounds. This was caused
13
1
by both short (one bond) and long-range (two or three bond) C- H coupling, making the
1H NMR spectrum complex. However, characterisation was more straightforward in the
13
13
broadband decoupled C NMR spectrum, due to the characteristic splitting caused by C-
17

13
13
13
C couplings. For example, testosterone { C }-glucuronide { C }-19 had an apparent
6
6
doublet of triplets (δ 176.6, J 58.8, 4.9 Hz) observed for the carbonyl carbon, while a
second doublet of triplets (δ 104.5, J 47.1, 4.9 Hz) was observed for the anomeric carbon.
Another four glucuronide carbons were observed as a multiplet (δ 71.9-79.0). As expected,
13
these labelled glucuronide C NMR signals had significantly greater signal intensity than
the non-enriched carbons of the steroidal backbone. In summary, the 13C labelling
13
13
13
provided fully labelled bisglucuronide ({ C }-6 and { C }-9) and monoglucuronide ({ C }-
6
6
6
13
13
4
, { C }-17-{ C }-20) conjugates suitable for use a stable isotope labelled internal
6 6
standards and mass spectrometry probes.
.3. Synthesis of steroid sulfate glucuronide reference materials
3
A second family of steroid bisconjugates produced in this study was the steroid sulfate
glucuronides. Ten steroid sulfate glucuronides were synthesised on a preparative scale,
generally by a three-step sequence involving sulfation, ketone reduction, and
glucuronylation as shown below for the conversion of EA 22 to 5α-androstane-3β,17β-diol
3-sulfate 17-glucuronide 23 (Scheme 5). This order of synthesis was chosen as initial
glucuronylation would introduce three additional hydroxy groups on the sugar ring, and
subsequent sulfation could then occur unselectively on any of the available hydroxy
groups.
Scheme 5.
The sulfation reaction was performed using sulfur trioxide-pyridine complex according to a
literature method [41]. Ketone reduction was performed using sodium borohydride [41], or
with additional cerium (III) chloride heptahydrate (Luche conditions) for α,β-unsaturated
ketones like testosterone and epitestosterone sulfate [43]. Reduction of the C17 ketones
afforded the 17β-hydroxy steroid as the sole diastereomer. However, reduction of the C3
or C20 ketones gave diastereomeric mixtures. The C3 ketone reduction afforded 1:7-13
mixtures of 3α:3β diastereomers [14]. The C20 ketone reduction proceeded under Felkin-
Anh control to favour the 20R diastereomer (1:6 S:R) as observed in the literature for the
reduction of pregnenolone [44],[45]. Reduction of the toluenesulfonylhydrazone derivative
of pregnenolone sulfate under conditions similar to those reported by Tada et al. [46],
afforded a 1:1 ratio of the 20S:20R diastereomers.
In the final step, steroid diol monosulfates were glucuronylated using the E. coli
glucuronylsynthase and the α-D-glucuronyl fluoride 2 donor [37] to give steroid diol sulfate
18

glucuronides 23-32. Glucuronylation was observed for 3β(5α), 3β(5-ene), 3(phenolic), 17β,
0S and 20R hydroxy groups. The starting diol monosulfates for the synthesis of sulfate
2
glucuronides 26, 27, 29 and 30 contained mixtures of 3α and 3β alcohols, and the 3α
1
alcohol did not react [37], observed by H NMR analysis of the product mixture. The
starting diol monosulfates for the synthesis of sulfate glucuronides 31 and 32 contained
mixtures of 20S and 20R alcohols, and both diastereomers were observed to react. For
glucuronylation at the C3 position to form 17-sulfate 3-glucuronides, higher conversions
(83-86%) were observed for steroid 3β,17β-diols 26 and 29 or estradiol 28, than the
steroid 3β,17α-diols 27 and 30 (42-62%). Glucuronylation at the C17 position to form
steroid 3-sulfate 17-glucuronides 23 and 25, typically proceeded in high conversion (93-
97%). Surprisingly, glucuronylation to afford androst-5-ene-3β,17β-diol 3-sulfate 17-
glucuronide 24 consistently gave low conversion (15%). Purification was achieved by SPE
(WAX and C18) in a manner similar to that described for the bisglucuronides above
1
(
Section 3.1) to afford the products with > 95% purity and these were characterised by H
13
NMR, C NMR, LRMS, and HRMS. In total, ten pure steroid sulfate glucuronides 23-32
were obtained, including diastereomeric mixtures 31 and 32 favouring the 20R- and 20S-
diastereomers respectively, which were not separable using the C18 method.
13
One C labelled steroid sulfate glucuronide was synthesised, 5α-androstane-3β,17β-diol
13
13
3
-sulfate 17{ C }-glucuronide { C }-23 using the same three-step synthesis, but using
6 6
{13
C }-α-D-glucuronyl fluoride { C }-2 in the final glucuronylation step (Figure 4). Full
13
6
6
incorporation of six 13C isotopes from the glucuronide moiety was observed based on
13
1
LRMS (-ESI). The presence of the { C }-glucuronide moiety gave distinctive splitting in H
6
and 13C NMR spectra in a manner similar to that observed for selectively labelled
13
13
bisglucuronides { C }-6 and { C }-9 (Figure 3).
6
6
3.4. NMR analysis of steroid bisglucuronide and sulfate glucuronide reference materials
All reference materials prepared by this study were characterised by H and 13C NMR
spectroscopy. In addition to providing important evidence of compound identity and purity,
the application of NMR chemical shift and multiplicity data within emerging NMR
metabolomics workflows may aid in the rapid assignment of metabolite structure [47].
1
1
Several diagnostic proton signals were observed in the H NMR spectra of the
synthetically derived steroid bisglucuronides 5, 6, 9-16 and sulfate glucuronides 23-32. On
glucuronylation, adjacent C3-H and C17-H protons shifted downfield by 0.20-0.35 ppm.
For example, the C17-H signal in diol monosulfate 21 (δ 3.56, t, J 8.6 Hz) shifted downfield
19

by 0.23 ppm (δ 3.79, t, J 8.6 Hz) on glucuronylation to afford sulfate glucuronide 23
Scheme 5). In contrast, the C20-H protons displayed different behaviour. The 20S
(
diastereomer (more clearly seen with bisglucuronide 14), showed a downfield shift of 0.08
ppm for the C20-H signal. On the other hand, the C20-H signal for the 20R diastereomer
shifted 0.35 ppm in bisglucuronide 16. For the aromatic protons in estradiol bisglucuronide
9, the C1-H proton moved 0.11 ppm (meta-position to the reacting site), and the C2-H and
C4-H protons moved 0.34 ppm (ortho-position to the reacting site). Other than these
1
steroidal proton shifts, new peaks in H NMR that were typical of bisglucuronide and
sulfate glucuronide compounds included the anomeric protons from each glucuronide unit.
For bisglucuronide compounds (5, 6, 9-16), these were typically resolved, with each
appearing as a doublet. For bisglucuronide 6, the 3-glucuronide anomeric proton signal (δ
4.41, d, J 7.7 Hz) was well resolved from that of the 17-glucuronide (δ 4.35, d, J 7.8 Hz).
1
These assignments were readily made by comparisons to previously reported H NMR
data for EA glucuronide 17 and dihydrotestosterone glucuronide [37]. Estradiol
bisglucuronide 9 displayed one anomeric proton signal for the 17-glucuronide (δ 4.40, d, J
7.8 Hz) with the second anomeric proton from the 3-glucuronide obscured by the water
peak from the deuterated methanol solvent (δ 4.85). Eight additional protons from the two
glucuronide units in bisglucuronides and four additional protons in sulfate glucuronides
appeared between δ 3.1-3.7.
Similarly, diagnostic protons for steroid sulfate glucuronides (23, 24, 26-32) were the
oxymethine protons that were shifted downfield after the sulfation reaction. After sulfation,
C3-H or C17-H shifted downfield by 0.65-0.76 ppm as expected from the previously
reported data [41]. For estradiol 3-sulfate 17-glucuronide 25, the aromatic protons were
also shifted, C1-H proton moved 0.18 ppm (meta-position to the reacting site), and the C2-
H and C4-H protons moved 0.54 ppm (ortho-position to the reacting site). After the
glucuronylation reaction, the oxymethine proton shifts were smaller than after the sulfation
reaction as mentioned above and as previously reported for monoglucuronides [37]. In
summary, the protons associated with sulfation and glucuronylation reaction sites were
typically resolved (δ 3.0 to 5.0) from the rest of steroidal backbone protons, and so the
chemical shift and multiplicity of these signals is likely some diagnostic value.
1
Characteristic H NMR signals all steroid bisglucuronide and steroid sulfate glucuronide
reference materials are tabulated in the supplementary material (Table S1).
20

3.5. MS analysis of unlabelled and labelled steroid bisglucuronides
3.5.1. Ionisation
-
In the scan MS with 70 V cone voltage, the mono-anion [M-H] was the major ion observed
-
with some minor [M-H-gluc] in-source fragmentation also found (where “gluc” was the
dehydrated glucuronic acid moiety (C H O ) 176 Da). The highest relative abundance for
6
8
6
-
the [M-H-gluc] ion appeared for estradiol bisglucuronide 9 and 5β-cholane-3α,24-diol
bisglucuronide 13 with 30% and 25% respectively, while only between 5-10% was
observed for the other compounds (5, 6, 10-12, 14-16). In addition, estradiol
-
bisglucuronide 9 also showed another in-source fragment [M-H-2gluc] m/z 271 (15%) in
2-
the scan MS. In contrast, scan MS with 26 V cone voltage formed the di-anion [M-2H] as
-
the major ion, while still forming mono-anion [M-H] with 50-100% relative abundance. In-
source fragmentation was only observed for estradiol bisglucuronide 9, giving 5% [M-H-
-
gluc] . A recent MS study on a library of crude chromatographically resolved steroid
bisglucuronides reported similar findings [38]. The current study highlights potential to
favour the formation of either mono- or di-anion precursors of the bisglucuronides for
subsequent MS/MS studies.
3.5.2. Fragmentation
Collision Induced Dissociation (CID) was then applied to mono- and di-anionic precursor
-
ions to study their MS fragmentation. With the mono-anion [M-H] , the major fragments at
-
5
0 eV collision energy that retained the steroid backbone were [M-H-gluc] and [M-H-
-
C H O ] , corresponding to neutral loss (NL) of 176 Da and 76 Da respectively. The NL of
2
4
3
76 Da was not observed for estradiol bisglucuronide 9 and 5β-cholane-3α,24-diol
-
bisglucuronide 13. Instead, estradiol bisglucuronide 9 showed [M-H-2gluc] (m/z 271) or a
combined NL of 352 Da. Another minor fragment containing the steroid backbone involved
-
the combined NL of the glucuronide unit and water ([M-H-gluc-H O] ), and this was more
2
prominent for the unsaturated steroid bisglucuronides 11 and 12. Fragments from the
glucuronide moiety itself (m/z 175, 157, 129, 113, 85, 75) were also observed as earlier
reported for steroid glucuronides [16],[38]. Fragmentation data for all steroid
bisglucuronide mono-anion precursors is tabulated in the supplementary material (Table
S2).
2-
For the di-anionic precursor ions [M-2H] , fragmentation with 20 eV collision energy, the
-
common fragments formed were derived from ion loss of m/z 175 ([gluc-H] ) and 75
21

-
-
-
(
[C H O ] ), to give [M-2H-(gluc-H)] (equivalent to [M-H-gluc] above) and [M-2H-
2 3 3
-
-
(
C H O )] (equivalent to [M-H-C H O ] above) respectively. These fragments were
2
3
3
2
4
3
noteworthy because of the increase in m/z caused by an ion loss from the precursor ion
during fragmentation. A similar pattern of ion loss from di-anionic precursor ions was
previously identified in steroid bis(sulfates) leading to the development of the CIL scan
method [26]. Estradiol bisglucuronide 9 showed a fragment ion at m/z 271 ([M-2H-(gluc-
-
H)-gluc] ) resulting from the combined ion loss of m/z 175 and NL of 176 Da. Interestingly,
5β-cholane-3α,24-diol bisglucuronide 13 gave an ion loss of m/z 75 giving a fragment ion
at m/z 637 that was not formed by NL 76 from the corresponding mono-anion. The NL of
-
water after ion loss of m/z 175 ([M-2H-(gluc-H)-H O] ) and fragments of the glucuronide
2
moiety (m/z 175, 157, 129, 113, 85, 75) were also typically observed as described earlier
for the mono-anion fragmentation. Fragmentation data for all steroid bisglucuronide di-
anion precursors is tabulated in the supplementary material (Table S3).
The fragmentation behaviour of two selectively mono-labelled steroid bisglucuronide
13
13
compounds, 5α-androstane-3β,17β-diol 3{ C },17-bisglucuronide { C }-6 and estradiol
6
6
13
13
-
3
,17{ C }-bisglucuronide { C }-9 were also studied by CID. When the mono anion ([M-H]
6 6
13
13
)
of 5α-androstane-3β,17β-diol 3{ C },17-bisglucuronide { C }-6 was fragmented, two
6
6
13
-
-
ions m/z 467 ([M-H-({ C }-gluc)] ) and 473 ([M-H-gluc] ) were observed with similar
6
intensity (Table 1). This suggested that similar energies were required for fragmentation of
13
each end of the bisglucuronide. On the other hand, estradiol 3,17{ C }-bisglucuronide
6
{13
C }-9 only showed only one fragment m/z 453 ([M-H-gluc] ) where the unlabelled
-
6
glucuronide at 3-position had been lost, indicating that cleavage of the phenolic
glucuronide was preferred due to the conjugated nature of the linking glycosidic oxygen
atom. Both labelled and unlabelled glucuronide fragments (m/z 175, 157/163, 113/118,
85/89, 75/77) appeared for both selectively labelled bisglucuronides.
2-
Fragmentation of the di-anions ([M-2H] ) showed similar behaviour (Figure 6, Table 2).
13
For the estradiol 3,17{ C }-bisglucuronide, the preferential loss of the unlabelled
6
glucuronide was also observed. In this case, neutral loss of the unlabelled glucuronide unit
2-
to give m/z 226 ([M-2H-gluc] ) was accompanied by reductions in the relative intensity of
the unlabelled glucuronide fragment ions m/z 85 and 75 relative to their labelled
counterparts m/z 89 and 77. Ion loss of the unlabelled glucuronide was also observed to
-
give m/z 453 ([M-2H-(gluc-H)] ). Both labelled and unlabelled glucuronide fragments (m/z
113/118, 85/89, 75/77) appeared for both selectively labelled bisglucuronides. In
conclusion, selectively mono-labelled bisglucuronide reference materials provide a means
22

to study the selectivity of fragmentation associated with both glucuronide units of a
bisglucuronide compound. Collision induced dissociation was also performed on EA
18
13
glucuronide and the stable isotope labelled EA glucuronides { O }-17 and { C }-17, and
2
6
these results are reported in the supplementary material (Table S4).
Figure 6.
Table 1.
Table 2.
3
.6. MS analysis of steroid sulfate glucuronide reference materials
.6.1. Ionisation
3
During the scan MS analysis, 26 V and 70 V cone voltages were used to maximise the
2-
-
response of the di-anion ([M-2H] ) and mono-anion ([M-H] ) respectively. When the 26 V
2-
cone voltage was used, the formation of the di-anion ([M-2H] ) was favoured, with the
-
mono-anion ([M-H] ) observed at lower relative abundance (15-55%). The in-source
-
fragment [M-2H-(gluc-H)] was also observed at relatively low intensity (5-50%). In-source
fragmentation of estradiol 3-glucuronide 17-sulfate 28 also afforded the ion derived from
dehydrated glucuronic acid m/z 175 ([gluc-H]-).
-
When using 70 V cone voltage, mono-anion ([M-H] ) but no di-anion was observed. Similar
to above, an in-source fragment was typically observed corresponding to loss of the
dehydrated glucuronic acid ([M-H-gluc] , 25-100%, corresponding to [M-2H-(gluc-H)]-
-
-
-
above). Other in-source fragments such as [M-H-C H O ] , [M-H-C H O ] and [M-H-gluc-
4
6
5
7
12
7
-
H O] were also commonly observed. The estradiol 3-sulfate 17-glucuronide 25 formed an
2
-
in-source fragment m/z 271, corresponding to ([M-H-gluc-SO ] ).
3
3.6.2. Fragmentation
Collision Induced Dissociation (CID) experiments were performed for all steroid sulfate
-
glucuronides from the mono- and di-anion precursor ions. For the mono-anion ([M-H] ), the
most intense fragment at 50 eV collision energy retaining the steroid backbone was [M-H-
-
gluc] , except for the unsaturated steroid sulfate glucuronides 29 and 30 where [M-H-gluc-
-
-
H O] fragment was more intense. In addition, minor [M-H-gluc-H O] fragment was also
2
2
observed for steroid sulfate glucuronides 26, 27, and 31. Another common ion was found
-
at m/z 97 corresponding to hydrogen sulfate ion ([HSO ] ). The m/z 97 ion was typically the
4
23

most intense fragment throughout the library except for estradiol 3-sulfate 17-glucuronide
2
5, where neutral loss of 80 Da corresponding to sulfur trioxide (SO ) was observed
3
-
instead. The neutral loss SO rather than the ion [HSO ] arose as the A ring was aromatic
3
4
and fragmentation of neutral SO generates a stabilised phenolate anion. Due to this,
3
-
-
fragments such as m/z 447 and m/z 271 corresponding to [M-H-SO ] and [M-H-gluc-SO ] ,
3
3
respectively, were only seen for estradiol 3-sulfate 17-glucuronide 25. The ion at m/z 80
•
-
corresponding to sulfur trioxide radical anion ([ SO ] ) was also formed from this
3
compound. Fragmentation data for all steroid sulfate glucuronide mono-anion precursors is
tabulated in the supplementary material (Table S5).
2-
Fragmentation of the di-anion precursor [M-2H] at 20 eV collision energy gave a greater
-
number of fragments than the mono-anion [M-H] . Similar to the mono-anion, loss of the
-
-
glucuronide derived anion ([M-2H-(gluc-H)] , corresponding to [M-H-gluc] above) or
-
hydrogen sulfate ([HSO ] , except estradiol 3-sulfate 17-glucuronide 25) were typically the
4
two most intense fragments throughout the library. In addition, [M-2H-(gluc-H)-H O]-
2
-
(
corresponding to [M-H-gluc-H O] above) was more commonly observed in the di-anion
2
fragmentation compared to the mono-anion. Another common fragment formed in the di-
-
anion fragmentation was derived from ion loss of m/z 75 ([C H O ] ), to give [M-2H-
2
3
3
-
-
(
C H O )] (corresponding to [M-H-C H O ] above) similar to the steroid bisglucuronide di-
2 3 3 2 4 3
2-
anion fragmentation. Once again, the ion loss fragmentation from [M-2H] to [M-2H-
-
-
-
(
C H O )] , [M-2H-(gluc-H)] , and [M-2H-(gluc-H)-H O] lead to an increase in m/z ratio.
2 3 3 2
-
-
Other fragments were also observed including [M-2H-(C H O )] and [M-2H-(C H O )] . As
3
3
5
3
5
5
described in the mono-anion, the di-anion derived from estradiol 3-sulfate 17-glucuronide
5 underwent neutral loss of 80 Da (SO ), giving rise to fragments m/z 271 and m/z 239
2
3
-
-
that corresponded to [M-2H-(gluc-H)-SO ] and [M-2H-(C H O )-SO ] . On the other hand,
3
7
11
7
3
the aromatic glucuronide estradiol 3-glucuronide 17-sulfate 28 fragmented to give [gluc-H]-
m/z 175), while only glucuronide fragments (m/z 113, 85, 75) were usually observed in the
(
other library members. Fragmentation data for all steroid sulfate glucuronide di-anion
precursors is tabulated in the supplementary material (Table S6).
Figure 7.
3.6.3. UHPLC-MS/MS analysis of real urine samples
The library of ten steroid sulfate glucuronide reference materials was developed according
a range of design criteria including structural diversity and synthetic accessibility, but
without specifically targeting putative metabolites. Despite this, the potential existed for
24

library members to occur as endogenous metabolites. To explore this, a selected reaction
monitoring (SRM) method for the detection of sulfate glucuronide library members as
endogenous metabolites was developed. To increase analytical sensitivity, the di-anion
2-
(
[M-2H] ) was selected as precursor as MS conditions necessary to favour the mono-
-
anion ([M-H] ) typically resulted in lower ion counts. The selected transitions for three of
the analytes (23, 24 and 32) are summarized in Table 3.
This SRM method was applied to a set of 30 real urine samples collected in our laboratory.
Confirmation of the analyte identity was performed using the World Anti-Doping Agency
(WADA) MS criteria for retention time and the relative abundance of three diagnostic
transitions [48]. Three of the synthesized analytes, namely pregn-5-ene-3β,20S-diol 3-
sulfate 20-glucuronide 32, 5α-androstane-3β,17β-diol 3-sulfate 17-glucuronide 23 and
androst-5-ene-3β,17β-diol 3-sulfate 17-glucuronide 24 could be detected in most of the
samples. The occurrence of all of them could be confirmed in several samples by both the
ion ratios of several transitions and the retention time.
The acquisition of the most specific transition (286  397) allowed for the detection of
pregn-5-ene-3β,20S-diol 3-sulfate 20-glucuronide 32 in all the samples analysed (Table 4)
with retention times commonly within 1% of deviation. The identity of that conjugate in
human urine could be confirmed by the relative abundance of the second transition (286

497) in all but a few diluted samples. Additionally, the third acquired transition could be
observed within criteria in more than half of the samples. An example of the confirmation
of a male urine is illustrated in Figure 8a. The 20R isomer 31 was also detected and
confirmed in one of the male samples analysed using the same transitions as those of the
20S isomer 32.
5α-Androstane-3β,17β-diol 3-sulfate 17-glucuronide 23 could be detected in all male and
female samples by using the transition 273  371 (Table 4) with retention time deviations
normally below 1%. In contrast, that analyte could only be detected in one of the urines
collected during pregnancy. The identity of the compound could be confirmed in six out of
the 30 samples analysed by using a second transition. The three selected transitions could
be observed within criteria in three male samples. An example is provided in Figure 8b.
Finally, androst-5-ene-3β,17β-diol 3-sulfate 17-glucuronide 24 could be detected in all
samples except for three urines collected during pregnancy (Table 4). Similar to the other
two analytes, conjugate 24 did not substantially differ its retention time from that of the
reference material (typical deviation below 1%) and could be confirmed in several of the
25

analysed samples from both male and female (14 out of 22 with two transitions, and 11 out
of 22 with three transitions). However, its identity could not be confirmed in any of the
urines collected during pregnancy. An example is provided in Figure 8c.
In summary, our results confirmed the existence of at least three previously unreported
steroid sulfate glucuronide conjugates as endogenous urinary metabolites. Although brief,
this study provides the motivation for more detailed studies on steroid sulfate glucuronides
as endogenous urinary metabolites. Access to a range of steroid sulfate reference
materials provides avenues to develop sensitive and selective UHPLC-MS methods for the
direct and untargeted detection of this metabolite family.
Table 3.
Table 4.
Figure 8.
4. Discussion
Steroidal bisconjugates (Figure 1) have long been known as minor components of the
steroid profile. In the past, these were typically analysed using laborious chromatographic
fractionation and hydrolysis [22],[23], followed by GC-MS detection [24],[25]. Recent
developments in both chemical synthesis and LC-MS technology have created avenues
for the direct detection of these minor metabolites. These advances are exemplified by the
CIL scan method for the direct and untargeted detection of urinary steroid bis(sulfate)
metabolites [26]. The CIL scan method has been employed to study the endogenous
steroid bis(sulfate) profile, including during pregnancy, to identify markers associated with
sports doping [26], and for the analysis of maternal urine to provide discriminating prenatal
diagnosis of inborn errors of steroid biosynthesis associated with SLOS, STSD, and PORD
[
27]. Integral to the development of the CIL scan method was the interplay between
chemical synthesis and MS method development. Synthetic access to a wide range of
steroidal bis(sulfate) reference materials revealed ion loss fragmentation as a common
feature of this compound class and enabled the development of a UPLC-MS CIL scan
method with high selectivity and good levels of detection for the targeted analytes [26].
In this work, the synthesis of other neglected steroidal bisconjugate families, steroid
bisglucuronides and steroid sulfate glucuronides, was achieved using the E. coli
glucuronylsynthase enzyme [37],[42],[49]. The glucuronylsynthase is an engineered
26

glycosynthase variant [50],[51] of the E. coli β-glucuronidase enzyme that is widely
employed in chemical analysis for steroid glucuronide hydrolysis in sample preparation
[
11]. The glucuronylsynthase incorporates an active mutation that disables glucuronide
hydrolysis, but using the enzyme in concert with the synthetically derived α-D-glucuronyl
fluoride 2 substrate promotes the single-step chemical synthesis of glucuronides under
mild conditions. The synthesis of 14 steroid monoglucuronides using the
glucuronylsynthase was the subject of earlier research [37]. This study extends the
glucuronylsynthase approach to the synthesis of ten steroid bisglucuronide and ten steroid
sulfate glucuronide, reference materials. The synthesis was performed on a scale suitable
for purification and characterisation by MS and NMR to confirm compound identity.
As an enzymatic method of glucuronylation, the E. coli glucuronylsynthase is
mechanistically distinct from the UGT-promoted biosynthesis of steroid glucuronides
[
52],[53] but shares several key attributes, including mild and single-step conjugation. One
notable feature of the glucuronylsynthase approach is the ability to adjust reaction scale
using standard laboratory methods [42]. The glucuronylsynthase method is also distinct
from chemical methods of glucuronylation that employ protected and activated glucuronide
donors and may require multiple protection and deprotection steps [54],[55]. Of the
reference materials targeted by this work, estradiol 3-sulfate 17 glucuronide 25 (Figure 4)
has previously been prepared by a five step chemical synthesis from estradiol [31]. Using
the glucuronylsynthase approach, sulfation, reduction and glucuronylation of estrone
afforded the estradiol 3-sulfate 17 glucuronide 25 in three steps. Further, selective
sulfation of estradiol followed by glucuronylation afforded the regioisomeric estradiol 17-
sulfate 3-glucuronide 28 in just two steps. The synthesis described herein significantly
expands the range of steroid bisconjugate reference materials accessible, providing for the
first time access to steroid sulfate glucuronides where the sulfate is conjugated to
saturated rather than phenolic hydroxy groups. The study delineates the scope and some
of the limitations of the glucuronylsynthase promoted synthesis but clearly establishes the
method as a valuable complement to biochemical or chemical synthesis approaches for
the preparation of steroid bisconjugate reference materials.
The E. coli glucuronylsynthase also provides a general approach to prepare stable isotope
labelled steroid bisconjugates through the late stage introduction of a labelled glucuronic
13
13
acid unit (Figure 3). The fully labelled { C }-α-D-glucuronyl fluoride { C }-2 was prepared
6
6
in four steps by an established route [42] from the relatively inexpensive and commercially
13
available { C }-D-glucose. Using a sequential glucuronylation pathway provided for the
6
27

selective labelling of steroid bisglucuronides 6 and 9 suitable for use as internal standards
or mass spectrometry probes. In this work, the MS study of the mono- and di-anions
showed no significant preference for fragmentation of the glucuronide units appended to
13
the saturated A- and D-rings in 5α-androstane-3β,17β-diol 3{ C },17-bisglucuronide
6
{13
C }-6 (Figure 6). In contrast, fragmentation of estradiol 3,17{ C }-bisglucuronide { C }-
6 6 6
13
13
9
revealed fragmentation preferentially occurred at the phenolic A-ring. Similar methods
13
13
provided access to five stable isotope labelled steroid mono-glucuronides { C }-4, { C }-
6
6
13
13
1
7-{ C }-20 and one steroid sulfate glucuronide { C }-23.
6
6
By design, this study has not targeted the preparation of particular steroid bisconjugate
reference materials, instead exploring the scope and limitations of the glucuronylsynthase
method and providing a diverse range of derivatives for MS method development. Despite
this, preliminary investigations have employed the reference materials to study MS
ionisation and fragmentation patterns, so confirming the presence of the steroid
bisconjugates pregn-5-ene-3,20S-diol 3-sulfate 20-glucuronide 32, 5α-androstane-
3β,17β-diol 3-sulfate 17-glucuronide 23 and androst-5-ene-3β,17β-diol 3-sulfate 17-
glucuronide 24 as endogenous human urinary metabolites by UHPLC-MS/MS analysis. In
this respect, the current work is only at an early stage. Future studies using the steroidal
bisconjugates prepared in this work will target the development of selective MS-based
methods for the direct and untargeted detection of these metabolite families using modern
MS instrumentation. Such studies promise to reveal in rich detail the role steroidal
bisconjugates in the steroid profile, unearthing these neglected treasures of steroidal
metabolism.
5. Conclusions
A library of ten steroid bisglucuronides and ten steroid sulfate glucuronides was
synthesised, purified and characterised by MS and NMR methods. The synthesis of stable
isotope labelled internal standards by late-stage introduction of labelled glucuronide units
is also reported, and applied to study the MS fragmentation of selectively labelled steroid
bisglucuronides. Access to steroidal bisconjugate reference materials promised to expand
the MS methods available to detect these minor steroid metabolites in fields such as
sports drug testing or medical research.
Conflict of interest
The authors declare that they have no conflict of interest.
28

Acknowledgements
The authors thank WADA (Science Research Grant 15A29OP) for financial support. The
authors also acknowledge Dr Hideki Onagi (ANU) for MS analysis and Dr Bradley
Stevenson (ANU) for Pseudomonas aeruginosa arylsulfatase (PaS) enzyme preparation.
The Spanish Health National System is acknowledged for the contract of OJP
(CPII16/00027) Strategic Plan for Research and Innovation in Health (PERIS) of the
Catalan government is acknowledged for OK contract (SLT002/16/00007).
Appendix A. Supplementary material
Supplementary material associated with this article can be found in the online version…
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