5α-Androst-16-en-3α-ol β-D-glucuronide, precursor of 5α-androst-16-en-3α-ol in human sweat

Article abstract of DOI:10.1002/cbdv.201300286

5α-Androst-16-en-3α-ol (α-androstenol) is an important contributor to human axilla sweat odor. It is assumed that α-andostenol is excreted from the apocrine glands via a H₂O-soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O-soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α-androstenol, β-androstenol sulfates, 5α-androsta-5,16-dien- 3β-ol (β-androstadienol) sulfate, α-androstenol β-glucuronide, α-androstenol α-glucuronide, β-androstadienol β-glucuronide, and α-androstenol β-glucuronide furanose. The occurrence of α-androstenol β-glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative-ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α-androstenol was observed after incubation of the sterile human sweat or α-androstenol β-glucuronide with a commercial glucuronidase enzyme, the urine-isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β-glucuronidase activities. We demonstrated that if α- and β-androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O-soluble precursor of α-androstenol in apocrine secretion should be a β-glucuronide. Copyright

Full text of DOI:10.1002/cbdv.201300286

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5a-Androst-16-en-3a-ol b-d-Glucuronide, Precursor of 5a-Androst-16-en-3a-
ol in Human Sweat
by Christian Starkenmann*, Fabienne Mayenzet, Robert Brauchli, and Myriam Troccaz
Firmenich SA, Corporate R&D Division, 1 Route des Jeunes, P.O. Box 239, CH-1211 Geneva-8
(phone: þ41-22-7803477; fax: þ41-22-7803334; christian.starkenmann@firmenich.com)
5a-Androst-16-en-3a-ol (a-androstenol) is an important contributor to human axilla sweat odor. It is
assumed that a-andostenol is excreted from the apocrine glands via a H2O-soluble conjugate, and this
precursor was formally characterized in this study for the first time in human sweat. The possible H2O-
soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., a-
androstenol, b-androstenol sulfates, 5a-androsta-5,16-dien-3b-ol (b-androstadienol) sulfate, a-andros-
tenol b-glucuronide, a-androstenol a-glucuronide, b-androstadienol b-glucuronide, and a-androstenol b-
glucuronide furanose. The occurrence of a-androstenol b-glucuronide was established by ultra
performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in neg-
ative-ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25
female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in
male secretions. The release of a-androstenol was observed after incubation of the sterile human sweat or
a-androstenol b-glucuronide with a commercial glucuronidase enzyme, the urine-isolated bacteria
Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus
haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have b-glucuronidase
activities. We demonstrated that if a- and b-androstenols and androstadienol sulfates were present in
human sweat, their concentrations would be too low to be considered as potential precursors of
malodors; therefore, the H2O-soluble precursor of a-androstenol in apocrine secretion should be a b-
glucuronide.
Introduction. – Malodor counteraction is a basic concern in the fragrance industry,
and, therefore, understanding the biological pathways leading to the formation of bad
odors is of high importance. Both eccrine and apocrine glands are present in human
underarms. Apocrine glands were reported to excrete odorless polar compounds, which
are then bio-transformed by axillary microflora to odorous compounds [1]. The present
work focused on understanding the formation of 5a-androst-16-en-3a-ol (a-androste-
nol), known to be involved in human underarm malodors [2][3].
The starting point of this work was a textbook on cosmetic science, edited in 1999
[4], a standard reference in the field of deodorants. The authors stated that apocrine
secretions contain, among others, ꢀsteroid conjugates such as androstenol sulfate and
glycosidesꢁ. The cited publications referred to 3a/b-hydroxy-5a-androstan-17-one
(androsterone and epiandrosterone) sulfates, 3b-hydroxyandrost-5-en-17-one (¼
ꢀdehydroepiandrosteroneꢁ; DHEA) sulfate, and 5-androstene-3b,17b-diol 3-sulfate
[5][6]. The occurrence of odoriferous, volatile a-androstenol in underarms was
confirmed by the analysis of axillary pads [2][7]. The capacity of axillary-isolated
coryneform bacteria to transform synthetic a-androstenol sulfate to odoriferous
ꢂ 2013 Verlag Helvetica Chimica Acta AG, Zꢃrich

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steroids was also clearly demonstrated, but no final evidence of the natural occurrence
of the precursor in underarms was provided [8]. Another biochemical pathway was
proposed, starting with the ꢀnecessary secretionꢁ of androsta-4,16-dien-3b-ol (b-
androstadienol) conjugates to initiate the metabolic sequence toward odoriferous
steroids such as androsta-4,16-dien-3-one, 5a-androst-16-en-3-one (androstenone), and
androstenols [4]. To date, no evidence of the occurrence of b-androstadienol sulfates or
glucuronides in apocrine secretions is available from the abundant literature on this
topic.
Thanks to new liquid chromatography (LC) and mass spectrometry (MS) systems,
the analytical profiling of anabolic androgenic glucuronidated and sulfated steroids in
body fluids has made huge progress [9–11]. We, therefore, decided to elucidate the
natural precursor of a-androstenol in sterile human sweat using these systems.
Results and Discussion. – Preparation of Steroid Sulfates. Androsterone (1) was
reduced via the BamfordꢀStevens reaction [12] to prepare pure a-androstenol (2). b-
Androstenol (4) was also prepared via the BamfordꢀStevens reaction from epian-
drosterone (3). a-Androstadienol (6) was obtained by reduction with LS-Selectride
[12] from androstadienone (5), and b-androstadienol (8) was prepared from DHEA
(7) via the BamfordꢀStevens reaction. The DHEA sulfate is commercially available;
the other sulfates were prepared in pyridine with chlorosulfonic acid (ClSO3H) from
the corresponding alcohols (Scheme 1) [8].
LC/MS Analysis of Steroid Sulfates. The chromatographic conditions and the LC/
MS method to separate and identify steroid sulfates were developed with commercial
DHEA sulfate 13. With HESI in negative-ion mode, the base-ion peak of 13 was at m/z
367, corresponding to [Mꢀ1]ꢀ . In the MS/MS mode, only a unique fragment-ion peak
at m/z 97 could be observed, corresponding to the mass of the sulfate moiety (HSOꢀ4 )
and displaying a small isotopic ion peak (m/z 99) of 4.4% intensity, thereby excluding
the possibility of the presence of a phosphate. The same fragmentation pattern was
observed for all steroid sulfates prepared, i.e., 9–12 and 14. This observation allowed us
to conduct an MS experiment, i.e., parent-ion scanning, which can be performed on a
triple quadrupole mass spectrometer to specifically detect compounds losing HSOꢀ4 .
The consequence of the formation of an ion peak at m/z 97 was that the MS/MS
spectra were not very informative, revealing only the presence of a sulfate moiety.
Therefore, the identification of the steroid could only rely on molecular mass and
retention times.
Analysis of Steroid Sulfates in Axillary Sweat. The sterile sweat was diluted twice in
MeOH prior to injection. The UPLC/MS TSQ Quantum Ultra trace displayed in
Fig. 1,a, represents the total-ion scanning mode of the sterile sweat. The sterile sweat
was then injected in parent-ion mass mode set at m/z 97. We observed that most of the
peaks detected in scan mode correspond to sulfate derivatives (Fig. 1,b). The
androsterone and epiandrosterone sulfates 9 and 11, respectively, and the DHEA
sulfate 13 were well-detected, as was the cholesterol sulfate. None of the peaks
displayed in Fig. 1,b, could correspond to a- or b-androstenol sulfate derivatives 10 and
12 (C19H30O4S; Mr 354) at retention times (tR) of 4.68 and 4.73 min measured for the
synthetic 10 and 12, respectively. The same was observed for b-androstadienol sulfate
(14; C19H28O4S; Mr 352) at 4.59 min, which is the tR for synthetic b-androstadienol

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Scheme 1. Preparation of the Steroid Sulfates Used as Authentic Standards
sulfate (14). This offered us two options: b-androstadienol sulfate (14), and a- and b-
androstenol sulfates 10 and 12, resp., are not present in axillary sweat, or their
concentrations are too low to be detected under our conditions.
Calibration curves were established, and the detection limits were as follows: a-
androstenol sulfate 10 (SIM) m/z 352.8–353.8, (signal-to-noise ratio) S/N 3; 0.05 mg/
ml), b-androstenol sulfate 12 (selected ion monitoring (SIM) m/z 352.8–353.8; S/N, 10;
0.0125 mg/ml) and b-androstadienol sulfate 14 (SIM, m/z 350.8–351.8; S/N 4; 0.025 mg/
ml). From LC/MS in base-peak mode, we easily detected the presence of 9, 11, 13, and
the cholesterol sulfate in sweat (Fig. 1,a) without preconcentration. The concentration
of DHEA sulfate 13 was estimated at 19 mg/ml (ꢁ0.9), which is in agreement with
published data [13].

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Fig. 1. Analysis by UPLC/MS triple quadrupole in negative-ion heated electrospray ionization mode
(HESI; base peak neg.-ion ESI 130.00–500.00). a) Full scan in base peak of male sweat. b) Base peak of
male sweat in parent-ion scan of 97 in negative HESI.
The sweat steroid sulfates were concentrated 27 times by SPE (solid phase
extraction) and reinjected: no a- and b-androstenols 10 or 12, or b-androstadienol
sulfate 14, were detected; therefore, if they were present, their concentrations should
have been below our detection limit.
Preparation of Glucuronide Conjugates. The coupling of methyl acetobromo-a-d-
glucuronidate to a-androstenol 2 with Ag2CO3 under KoenigsꢀKnorr reaction
conditions [14][15] failed to give the desired product in reasonable yield. As described
by Brown et al. [16], the imidate of the methyl 2,3,4-tri-O-acetyl-1-hydroxy-a,b-d-
glucopyranuronate 15 was coupled to a-androstenol 2 to give 16, which was then
deprotected, in 30% yield, to a-androstenol b-glucuronide 17, containing a small
quantity of a-androstenol-a-glucuronide 18 (Scheme 2). The configuration of a-
1
androstenol b-glucuronide (17) was confirmed by H-NMR, based on the coupling
constant (3.75 Hz) between the anomeric H-atom (d(H) 4.90 ppm) and the H-atom at
C(2) of the sugar moiety. a-Androstenol a-glucuronide (18) was a minor product
obtained only in mixture, but this allowed us to compare its 1H-NMR coupling constant

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Scheme 2. Preparation of the Steroid Glucuronides as Authentic Standards
(d(H) 4.33 (J¼7.80 Hz)) to that of 17. b-Androstadienol 8, obtained from DHEA 7,
was also coupled in the same way to prepare b-androstadienol b-glucuronide 21. a-
Androstenol b-glucuronide 17 was assumed to be in a pyranose form, but it could also
be in a furanose form. To exclude this possibility, the a-androstenol b-glucuronide
furanose form 22 was prepared starting from glucuronolactone. The coupling was
performed in BF3 ·Et2O, and the lactone-ring opening was achieved in aq. NaOH
solution; after purification steps, pure a-androstenol b-glucuronide, furanose form, 22
was obtained. A UPLC method was developed to separate ab-, aa-, ba-, and bb-
androstenol glucuronides (Fig. 2,a–c).

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Fig. 2. Analysis by UPLC/MS LXQ in negative-ion mode of male sweat concentrated ten times. Left:
UPLC trace of HESI full MS, monitoring in the range of m/z 448.75–449.75 (exact m/z 449.25). Right:
MS/MS. a) a-Androstenol-a- and -b-glucuronides. b) a-Androstenol-b-glucuronide, furanose form. c) b-
Androstenol-a- and -b-glucuronides. d) Male axillary sweat.
Analysis of Glucuronide Steroids in Axillary Sweat. Sterile sweat was concentrated
ten times and analyzed in full MS scan. The LC/MS trace displayed in Fig. 2,d,
corresponds to the extract ion with a peak at m/z 449.35. At 7.03 min, a peak with a mass
spectrum corresponding to the a-androstenol b-glucuronide (17) was detected. No
peaks with m/z 446.75–447.75, which could correspond to b-androstadienol-b-
glucuronide 21, could be detected. Compound 18, with a detection limit of 0.025 mg/
ml (S/N 8) was not found.
Quantitative Estimation of a-Androstenol b-Glucuronide 17 in Sterile Sweat. The
concentration of 17 was established in an eccrine and apocrine axillary sweat pool
collected from 25 women and 24 men, who used a sauna over a three-year period [17].
A 4-ml aliquot of sterile sweat was concentrated ten times and analyzed by LC/MS
(HESI) in negative-ion mode. From an external calibration curve in SIM at m/z
448.75–449.75, we found 17 at 79 ng/ml (ꢁ2) for the female pool and at 241 ng/ml (ꢁ
70) for the male pool.
Enzymatic Release of a-Androstenol 2 from a-Androstenol b-Glucuronide (17).
Synthetic 17 was incubated with Streptococcus agalactiae, which possesses glucuroni-
dase activity [18], and with an isolated commercially available enzyme, b-glucuroni-

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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dase/arylsulfatase. In both cases, 17 disappeared. The fermentation broth was then
extracted with CH2Cl2 and injected on GC/MS; a-androstenol 2 was detected, but not
in the blank system.
A homology search (a microbial protein blastp search on www.ncbi.nlm.nih.gov)
was carried out between the S. agalactiae ATCC 13813 b-d-glucuronidase amino acid
sequence (621 amino acids, accession No. EFV97073) and the Gram-positive bacteria
genome. Some identities were found with glucuronidases from the underarm bacterial
species Staphylococcus warneri, Staphylococcus haemolyticus, and Propionibacterium
acnes [19–21]. After incubation with a-androstenol b-glucuronide (17), the three
species were able to generate a-androstenol (2). The percentage reductions of 17 after
a one-day incubation at 378 were 99, 29, and 22% with S. warneri DSM20316, S.
haemolyticus DSM20263, and P. acnes ATCC6919, respectively.
In parallel, the male sweat pool was incubated with the commercial b-glucuroni-
dase/arylsulfatase enzyme at 378 for one night. We confirmed by LC/MS the
disappearance of 17; the formation of 2 was confirmed after extraction with CH2Cl2
(containing a-androstanol as internal standard). a-Androstanol was quantified by GC/
MS in SIM mode (m/z 274), and the concentration of 2 (m/z 276) was determined. In
sterile male sweat, 17 was found at a concentration of 241 ng/mlꢁ70 (0.53 mmol/mlꢁ
0.15) and, after incubation, 2 was found at a concentration of 167 ng/mlꢁ12 (0.61 mmol/
mlꢁ0.04).
Discussion. – In human underarm secretions, steroid sulfate derivatives are more
abundant than glucuronides [6][22–24]. This finding could be the reason for the
assumption that sulfate conjugates were the precursors of odorant steroids [4]. In a
previous work, the concentration of DHEA sulfate 13 in Asian and Caucasian axillary
sweat secretions was compared [13], but it is difficult to deduce a correlation between
the concentration of DHEA sulfate 13 and a-androstenol b-glucuronide (17), implying
that the quantification of 13 cannot be directly linked to body odor.
Brooksbank and Haselwood in 1961 claimed [25] that the precursor of a-
androstenol was not a sulfate. They extracted the acid fraction of sweat, and, after
solvolysis in AcOEt, they could not find a-androstenol (2). Sulfate is a good leaving
group, and, under acidic conditions or thermolysis, the formation of odorant androstene
derivatives [8][12] has been observed; therefore, we did not know whether this claim
was valid [2].
The precursor of 2 is its corresponding b-glucuronide, which is not surprising,
because it was already identified in urine [25], and recently we demonstrated its
importance in the odor of stale urine [18]. In the literature, bacterial b-glucuronidase
activities have already been detected in both facultatively and strictly anaerobic strains
belonging to the family Enterobacteriaceae (Escherichia coli), and the genera
Streptococcus, Lactobacillus, Bifidobacterium, Clostridium, anaerobic Corynebacteri-
um, and Bacteroides. Genes encoding B-glucuronidase (GUS) have been characterized
from E. coli, L. gasseri, Staphylococcus spp., Clostridium perfringens, S. aureus,
Thermotoga maritima, and Ruminococcus gnavus [26–29]. In moist areas such as the
human axilla, Corynebacterium spp. and Staphylococcus spp. dominate the resident
flora, which was demonstrated earlier through culture-based approaches [19–21].
Here, we have shown that at least three bacteria present in the underarm, S. warneri

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and S. haemolyticus, and to a minor extent P. acnes, were able to cleave the b-
glucuronide conjugate to generate a-androstenol (2).
Furthermore, we noticed that b-androstenol (4) and b-androstadienol (8) are
enzymatically oxidized to ketones, which is also in agreement with the literature
[23][24]. This could explain the MS fragment ion corresponding to the peak at m/z 271
(Fig. 2,c) for 19, instead of the expected fragment ion with the peak at m/z 273
observed for 17. Even if we cannot clearly confirm this hypothesis now, we strongly
believe that androstenone is bacterially generated from a-androstenol (2), and
probably to a lesser extent from b-androstenol (4), which is in agreement with the
literature [23][24]. Androstenone may, therefore, build up over time to enhance human
sweat odor after the primary liberation of conjugated steroid alcohols.
We are grateful to Dr. Eric Ohleyer to guide us in the synthesis of glucuronide derivatives, Mrs.
Sabine Beccucci for providing the sterile sweat and bacterial solutions, Dr. Eric Frerot for LC/MS
guidance and suggestions, and Dr. Monica Bandera and Laurent Wꢀnsche for reviewing the manuscript.
Experimental Part
Materials. All chemical and reagents were purchased from FlukaꢀSigmaꢀAldrich (CH-Buchs),
Acros (BE-Geel), and TCI Europe N.V. (BE-Zwjindrecht). All solvents, including Et2O p.a. and CH2Cl2
(Atrasol), were purchased from Carlo Erba Reactifs SDS (FR-Val de Reuil Cedex), H2O for ultra-
performance liquid chromatography (UPLC) was purchased from Biosolve (NL-Valkenswaard), and
MeCN for UPLC was purchased from Merck (DE-Darmstadt). HCOONH4 puriss p.a. for the UPLC
solvent was from FlukaꢀSigmaꢀAldrich. Na2SO4 (99%) was obtained from SigmaꢀAldrich (DE-
Steinheim). Silica gel (SiO2, 60A; 32–63 mm) for flash chromatography (FC) was purchased from
Brunschwig (CH-Basel). b-Glucuronidase/arylsulfatase (Helix pomatia) was purchased from Roche
(CH-Rotkreuz). Samples were filtered prior to injection on UPLC with UptiDisc 13-mm syringe filters
(0.45 mm, Acrodisc) and regenerated cellulose membrane from Interchim (FR-MontluÅon). The solid
phase extraction (SPE) cartridges were Supelclean ENVI-18 SPE tubes of 6 or 3 ml from Supelco
(CH-Buchs). Previously urine-isolated Streptococcus agalactiae (100% similarity with Streptococcus
agalactiae ATCC 13813 16S rDNA sequence) were grown in liquid tryptic soya broth (Beckton
Dickinson, FR-Pont de Claix) for 24 h at 378. The culture was then washed two times with 0.9% NaCl
(saline soln.) and concentrated in saline soln. to 1010 cfu/ml. Sterile sweat samples (human apocrine and
eccrine secretions) were collected from the axillae of 49 Caucasian volunteers, who used a sauna over
three winter seasons, and the samples were pooled by sex [17]. Staphylococcus warneri DSM 20316,
Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919 were purchased
from DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, DE-Braunschweig) and
cultivated as described for S. agalactiae, except for P. acnes, which was cultivated on Schaedler agar
´
containing 5% sheep blood (BioMerieux, CH-Geneva) and WilkinsꢀChalgren liquid broth (Oxoid,
CH-Basel) at 378 for 48 h under anaerobic conditions.
1H- and 13C-NMR Spectra. The NMR spectra were recorded in CDCl3 or MeOD on a Bruker
Avance-500 spectrometer (CH-Zurich) at 500.13 and 125.76 MHz (1H and 13C), resp. Chemical shifts d in
ppm were recorded rel. to Me4Si as internal standard. The assignments were completed by 2D
spectroscopy, correlated spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and
heteronuclear multiple-bond coherence (HMBC) experiments with standard Bruker software (Top-
spin).
UPLC/MS. Analyses were performed on a Waters Acquity system (Waters, CH-Baden-Dꢄttwil)
coupled to a mass spectrometer. The separations and quantifications of sulfates were performed on an
Acquity BEH-C18 column (2.1 mm i.d.ꢂ100 mm, 1.7 mm i.d.). One ml was injected; the elution solvents
were H2O containing 5 mm HCOONH4 (solvent A), and a mixture of 90% MeCN and 10% H2O
containing 5 mm HCOONH4 (solvent B). The gradient profile started at 20% B and was then increased

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
2205
to 98% B in 6.0 min. The flow rate was 0.3 ml/min. The separation of glucuronides and their
quantification were performed on an Acquity BEH shield RP-18 column (2.1 mm i.d.ꢂ100 mm, 1.7 mm
i.d.). The gradient started at 35% B and was increased to 45% B in 8.0 min, and then to 98% in 0.1 min
and held for 1 min. The flow rate was 0.3 ml/min.
The mass spectrometer was a Thermo Finnigan LXQ (Thermo Fisher Scientific, CH-Basel) with
heated electrospray ionization (HESI) operated in negative-ion mode. The spray voltage was 4.0 kV, the
vaporizer temp. was 2508, and the cap. temp. 3508. The sheath gas was N2 at a flow rate of 50 (Finnigan
arbitrary units). The auxiliary gas was also N2 at a flow rate of 20 (Finnigan arbitrary units). The events
were either in full-scan mode [80–800], which could be linked with a data-dependent MS/MS of the 1st
most intense ion from scan event 1; collision energy, 25 V; or in single-ion monitoring (SIM) mode based
on the mass that we were interested in ([Mꢀ1]).
When specified, the mass spectrometer for parent-ion scanning experiments was a Thermo Finnigan
TSQ Quantum Ultra (Thermo Fisher Scientific) with HESI operated in negative-mode. The spray voltage
was 4.0 kV, the vaporizer temp. was 4008, and the cap. temp. 3708. The sheath gas was N2 at a flow rate of
50 (Finnigan arbitrary units). The auxiliary gas was also N2 at a flow rate of 15 (Finnigan arbitrary units).
The injections were made in two scan events: first, full Q3MS and, second, full parent-ion scan; product
mass, 96,900; collision energy, 35 V.
Gas Chromatography (GC)/MS. An Agilent-GC-6890 system connected to an Agilent-MSD-5973
quadrupole mass spectrometer (Santa Clara, CA, USA) was operated at 70 eV. He was the carrier gas set
at a constant flow rate of 0.7 ml/min. Separations were performed on fused-silica cap. columns, coated
with SPB-1 (Supelco, 30 mꢂ0.25 mm i.d., 0.25 mm). The temp. program: 508, increased to 2508 at 108/
min, and held for 15 min. MS Interpretation was based on the Wiley MS database.
Synthesis of Steroids. 5a-Androst-16-en-3a-ol (¼a-androstenol; 2) [12]. 3a-Hydroxy-5a-androstan-
17-one (¼androsterone; 1; 11 g, 37.9 mmol) and TsNHNH2 (7.2 g, 37.9 mmol) were heated in dry EtOH
(185 ml) at reflux for 2 h and then concentrated under vacuum to dryness. Et2O (185 ml) was added to the
residue, followed by a 1.6m soln. of MeLi (118 ml, 189 mmol), added dropwise for 1.5 h at 228. After 15 h,
the mixture was poured onto ice-cold aq. NH4Cl, and the org. phase was washed twice with brine, and
then dried (Na2SO4), filtered, and concentrated. The product was purified by FC on SiO2 (CH2Cl2/Et2O
9 :1) to afford odoriferous 2 (6.0 g, 58%). [a]2D0 ¼ þ15.1 (c¼1, CHCl3) [12]. 13C-NMR (125 MHz,
CDCl3): 11.2 (q); 17.1 (q); 20.8 (t); 28.6 (t); 29.0 (t); 32.0 (t); 32.0 (t); 32.1 (t); 34.1 (d); 36.0 (t); 36.4 (s);
39.4 (d); 45.6 (s); 55.2 (d); 56.2 (d); 66.6 (d); 129.3 (d); 144.0 (d).
5a-Androst-16-en-3b-ol (¼ b-Androstenol; 4). The same procedure used for 2 with 3b-hydroxy-5a-
androstan-17-one (¼epiandrosterone; 3; 3 g, 10.33 mmol) afforded 4 (820 mg, 29%). [a]2D0 ¼ þ13.8 (c¼1,
CHCl3) [12]. 13C-NMR (100 MHz, CDCl3): 12.3 (q); 17.1 (q); 21.2 (t); 28.7 (t); 31.5 (t); 32.0 (t); 32.1 (t);
34.1 (d); 35.8 (s); 36.0 (t); 36.8 (t); 38.2 (t); 45.1 (d); 45.6 (s); 55.2 (d); 56.1 (d); 71.3 (d); 129.3 (d); 144.0
(d).
Androsta-5,16-dien-3a-ol (¼a-Androstadienol; 6). Prepared by reduction of 5 as described in [12].
13C-NMR (125 MHz, CDCl3): 16.9 (q); 18.6 (q); 20.6 (t); 28.9 (t); 30.4 (d); 31.8 (t); 32.1 (t); 33.2 (t); 35.9
(t); 37.7 (s); 39.9 (t); 45.4 (s); 51.2 (d); 56.2 (d); 67.1 (d); 123.8 (d); 129.4 (d); 138.9 (s); 143.8 (d).
Androsta-5,16-dien-3b-ol (¼ b-Androstadienol; 8). The same procedure used for 2 with 3b-
hydroxyandrost-5-en-17-one (DHEA; 7; 2 g, 6.93 mmol) furnished 8 (956 mg, 51%). [a]2D0 ¼ ꢀ67.5 (c¼1,
CHCl3) [12]. 13C-NMR (90 MHz, CDCl3): 16.9 (q); 19.4 (q); 20.9 (t); 30.5 (d); 31.7 (t); 31.8 (t); 32.1 (t);
35.9 (t); 36.8 (s); 37.2 (t); 42.4 (t); 45.4 (s); 51.0 (d); 56.2 (d); 71.8 (d); 121.6 (d); 129.4 (d); 141.1 (s); 143.8
(d).
Synthesis and Analysis of Steroid Sulfates. General Procedure [8]. ClSO3H (2 equiv.) was added
dropwise to a soln. of the steroid alcohol (1 equiv.) in pyridine (34 equiv.). The mixture was stirred for 2 h
at 228. The pyridine was removed by washing three times with pentane (5ꢂ the volume of pyridine). The
residue was concentrated, and H2O (2 ml) was added. The pH must be <2; if not, a few drops of 2n
H2SO4 was added. The soln. was loaded on an SPE ENVI-18 cartridge (3 ml for 10–50 mg of product, or
6 ml up to 1 g of product). The cartridge was rinsed with sat. aq. NaHCO3 soln., followed by tBuOMe and
then MeOH. The MeOH fraction was concentrated and then lyophilized to give the corresponding
steroid sulfate in powder form.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
3a-Hydroxy-5a-androstan-17-one (Androsterone) Sodium Sulfate (9). 13C-NMR (125 MHz,
MeOD): 11.9 (q); 14.2 (q); 21.2 (t); 22.7 (t); 27.9 (t); 29.3 (t); 32.1 (t); 32.9 (t); 33.9 (t); 34.6 (t); 36.4
(d); 36.8 (t); 36.9 (s); 40.8 (d); 49.2 (s); 52.8 (d); 55.9 (d); 76.3 (d); 224.2 (s).
5a-Androst-16-en-3a-ol (a-Androstenol) Sodium Sulfate (10). 13C-NMR (125 MHz, MeOD): 11.9
(q); 17.5 (q); 21.9 (t); 27.9 (t); 29.6 (t); 32.9 (t); 33.4 (t); 33.8 (t); 34.7 (t); 35.5 (d); 37.0 (s); 37.2 (t); 41.0
(d); 46.7 (s); 56.8 (d); 57.6 (d); 76.4 (d); 130.2 (d); 145.0 (d).
3b-Hydroxy-5a-androstan-17-one (Epiandrosterone) Sodium Sulfate (11). 13C-NMR (125 MHz,
MeOD): 14.0 (q); 19.8 (q); 21.5 (t); 22.7 (t); 30.0 (t); 31.9 (t); 32.7 (t); 32.8 (d); 36.7 (t); 37.8 (s); 38.4 (t);
40.4 (t); 49.2 (s); 51.8 (d); 53.0 (d); 79.7 (d); 122.8 (d); 141.8 (s); 223.9 (s).
5a-Androst-16-en-3b-ol (b-Androstenol) Sodium Sulfate (12). 13C-NMR (125 MHz, MeOD): 12.7
(q); 17.5 (q); 22.3 (t); 29.8 (t); 29.9 (t); 33.0 (t); 33.4 (t); 35.5 (d); 36.4 (t); 36.8 (s); 37.2 (t); 38.1 (t); 46.6
(d); 46.7 (s); 56.7 (d); 57.6 (d); 79.7 (d); 130.2 (d); 145.0 (d).
3b-Hydroxyandrost-5-en-17-one (DHEA) Sodium Sulfate (13). 13C-NMR (125 MHz, MeOD): 14.0
(q); 19.8 (q); 21.5 (t); 22.7 (t); 30.0 (t); 31.9 (t); 32.7 (t); 32.8 (d); 36.7 (t); 37.8 (s); 38.4 (t); 40.4 (t); 49.2
(s); 51.8 (d); 53.0 (d); 79.7 (d); 122.8 (d); 141.8 (s); 223.9 (s).
Androsta-5,16-dien-3b-ol (b-Androstadienol) Sodium Sulfate (14). 13C-NMR (125 MHz, MeOD):
17.3 (q); 19.7 (q); 22.0 (t); 30.0 (t); 31.8 (d); 32.9 (t); 33.0 (t); 37.1 (t); 38.0 (s); 38.5 (t); 40.5 (t); 46.5 (s);
52.6 (d); 57.7 (d); 79.8 (d); 123.2 (d); 130.3 (d); 142.0 (s); 144.9 (d).
Analysis of Steroid Sulfates in Sterile Sweat. Four solns. of 13 in MeOH at different concentrations
(2.7, 18.9, 40.7, and 81.3 mg/ml) were injected in LC/MS (LXQ) in SIM mode (m/z 366.56–367.56) for the
external calibration curve (RSQ: 0.953). Two sterile sweat samples from men (the same pool as described
in [17]) were injected twice; the result was an average of the two values. The detection limits were
determined by injecting decreasing concentrations of the reference compounds in MeOH in SIM mode:
10: SIM m/z 352.8–353.8, detection limit (signal-to-noise ratio (S/N) 3 (¼0.05 mg/ml)); 12: SIM m/z
352.8–353.8; detection limit S/N 10 (¼0.0125 mg/ml); 14: SIM m/z 350.8–351.8; detection limit S/N 4 (¼
0.025 mg/ml)). The analysis of steroid sulfates in sterile sweat was performed after pre-concentrating the
sweat (27 g) and loading it on a 6-ml SPE ENVI-18 cartridge, previously washed with MeOH (3 ml) and
then conditioned with H2O (7 ml). It was rinsed three times with 2 ml of H2O and then desorbed with
MeOH (5 ml). MeOH was concentrated to obtain dry material (1.2 mg). MeOH was then added
(1.00 ml) to obtain a 27ꢂ concentrated sterile sweat soln.
Synthesis of Steroid Glucuronides. a-Androstenol b-d-glucopyranosiduronic Acid (¼a-(Androstenol
b-glucuronide; 17). Imidate 15 was prepared as described in [16]. The saponification was slightly
modified: a-androstenyl methyl 2,3,4-tri-O-acetyl-b-d-glucopyranosiduronate (16; 63 mg, 0.107 mmol) in
MeOH (1.6 ml) and 5n aq. NaOH (107 ml) were stirred for 1 h. The MeOH was concentrated under
vacuum. The residue was loaded on an SPE cartridge ENVI-18 (6 ml; conditioned as described above).
The SPE was then washed with H2O (2 ml), and the glucuronide was desorbed with MeOH. MeOH was
then evaporated to afford 17 (18 mg), 37%). 13C-NMR (125 MHz, MeOD): 11.9 (q); 17.5 (q); 21.9 (t);
25.9 (t); 29.7 (t); 33.0 (t); 33.4 (t); 33.5 (t); 35.6 (d); 35.6 (t); 37.2 (s); 37.3 (t); 40.8 (d); 46.7 (s); 56.5 (d);
57.8 (d); 73.9 (d); 74.2 (d); 75.1 (d); 76.3 (d); 78.1 (d); 102.0 (d); 130.2 (d); 145.1 (d); 177.1 (s).
a-Androstenol a-Glucuronide (18). A mixture of a-androstenyl methyl 2,3,4-tri-O-acetyl-a- and -b-
d-glucopyranosiduronate 16 (35 mg, 0.059 mmol) in MeOH (0.9 ml) and 5n aq. NaOH (60 ml) was
stirred for 1 h. The same purification as above afforded a 7:3 mixture of a-androstenol b-glucuronide
(17) and a-androstenol-a-glucuronide (18) (9 mg) in 34% yield. 13C-NMR (125 MHz, MeOD): 11.9 (q);
17.5 (q); 21.9 (t); 28.7 (t); 29.8 (t); 33.0 (t); 33.4 (t); 33.5 (t); 34.2 (t); 35.6 (d); 37.2 (t); 37.3 (s); 40.9 (d);
46.7 (s); 56.6 (d); 57.7 (d); 72.1 (d); 73.4 (d); 74.3 (d); 74.6 (d); 74.9 (d); 98.9 (d); 130.2 (d); 145.1 (d);
178.1 (s).
b-Androstenol b-Glucuronide (19) and b-Androstenol a-Glucuronide (20). Prepared as described for
17. Unfortunately, because of the small quantities of starting materials and the poor yield, LC/MS tR and
MS data were obtained for 19/20.
b-Androstadienol b-Glucuronide (21). b-Androstadienyl methyl 2,3,4-tri-O-acetyl-b-d-glucopyrano-
siduronate (16; 90 mg, 0.153 mmol) in MeOH (2.3 ml) and 5n aq. NaOH (153 ml) were stirred for 1 h. The
workup as described above afforded 21 (13 mg, 19%). 13C-NMR (125 MHz, MeOD): 17.3 (q); 19.8 (q);

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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22.0 (t); 30.6 (t); 31.9 (d); 33.0 (t); 33.1 (t); 37.1 (t); 38.2 (s); 38.6 (t); 39.7 (t); 46.5 (s); 52.7 (d); 57.7 (d);
73.8 (d); 75.0 (d); 76.3 (d); 78.0 (d); 79.4 (d); 102.2 (d); 122.6 (d); 130.3 (d); 142.4 (s); 144.9 (d); 177.0 (s).
a-Androstenol b-d-Xylohexofuranosiduronic Acid (¼a-Androstenol b-Glucuronide Furanose
Form; 22). BF3 ·Et2O (10.97 mg, 0.077 mmol) was added at r.t. (22.28) to a mixture of (þ)-d-glucuronic
acid g-lactone (136 mg, 0.77 mmol) and 2 (212 mg, 0.77 mmol) in dioxane (2 ml). The mixture was stirred
for 6 h at 358 and then concentrated under vacuum. The residue was extracted with AcOEt/EtOH 4 :1
and washed twice with brine. The org. phase was then dried (Na2SO4), concentrated under vacuum, and
purified by FC (SiO2; pentane/AcOEt 5 :5) to give a-androstenol b-d-glucurono-3,6-lactone (117 mg,
35%; white solid). The lactone was dissolved in acetone (2.3 ml) and the soln. was cooled in an ice bath.
NaOH (0.5.m; 1 ml) was added dropwise, and the soln. was stirred for 3 h at 228 before concentration
under vacuum. H2O (3 ml) was added, and purification was performed on an SPE ENVI-18 6 ml
cartridge (as described above) to afford 22 (44 mg, 36%). 13C-NMR (125 MHz, MeOD): 12.0 (q); 17.5
(q); 21.9 (t); 25.4 (t); 29.6 (t); 33.0 (t); 33.4 (t); 33.7 (t); 35.3 (t); 35.6 (d); 37.2 (s); 37.3 (t); 41.0 (d); 46.7
(s); 56.8 (d); 57.7 (d); 72.8 (d); 73.5 (d); 78.2 (d); 82.7 (d); 84.2 (d); 106.8 (d); 130.2 (d); 145.0 (d); 177.7
(s).
Analysis of 17. To quantify 17 in sweat, four solns. of 17, prepared in MeOH (0.51, 2.55, 5.1, and
7.65 mg/ml), were injected in to LC/MS (HESIꢀ ) in SIM mode (m/z 448.75–448.75) for the calibration
curve (RSQ: 0.9959). The sweat samples were injected in duplicate after concentrating them by ten
times, as described above.
Analysis of 21. The detection limit of 21 was estimated by injecting decreasing concentrations of 21 in
MeOH and injecting them in to LC/MS (HESIꢀ ) in SIM mode (m/z 446.75–447.75).
GC/MS Quantification of 2. This was performed by using a-androstanol as the internal standard
(I.S.). The response factor between 2 (274 in SIM mode) and the I.S. (276 in SIM mode) was measured in
CH2Cl2 solns., containing 0.1 mg of I.S. and 0.051, 0.102, and 0.205 mg of 2. Prior to injection, the volumes
were reduced to ca. 20 ml under a gentle Ar flow. Each soln. was injected twice in GC/MS (1 ml; in SIM
mode at m/z 274 and 276) and the following equation was applied for the response factor (rf):
Mol: wt: of 2
Area of 2 ꢂ Mol: wt: of I:S:
rf ¼ Area I:S: ꢂ
¼ 0:3845 ꢁ 0:03:
a-Androstenol Formation after Incubation of Sterile Sweat. The I.S. soln. (500 ml, 0.1 mg) was added to
the sterile sweat (500 ml) incubated for one night at 378 with the enzyme b-glucuronidase/arylsulfatase
(from Helix pomatia). The incubated samples were centrifuged at 3260g for 3 min. The lower phase was
removed with a pipette, and the soln. was washed two times with CH2Cl2 (100 ml). The org. layers were
combined, dried (Na2SO4), filtered on cotton, and concentrated under Ar to ca. 20 ml; 1 ml was injected
into the GC/MS in SIM mode at m/z 274 and 276. The concentration was determined from the GC/MS
peak areas corrected by the rf value. The result is the average of three samples from the same pool. In
parallel, human sterile sweat (or a-androstenol conjugates) was incubated with a bacterial soln. to end up
with a final concentration of 108 cfu/ml.
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Received August 26, 2013