Synthesis of tritium-labeled hydroxytyrosol, a phenolic compound found in olive oil

Article abstract of DOI:10.1021/jf000468l

(3,4-Dihydroxyphenyl)ethanol, commonly known as hydroxytyrosol (1), is the major phenolic antioxidant compound in olive oil, and it contributes to the beneficial properties of olive oil. Bioavailability and metabolism studies of this compound are extremely limited, in part, related to unavailability of radiolabeled compound. Studies with radiolabeled compounds enable use of sensitive radiometric analytical methods as well as aiding elucidation of metabolic and elimination pathways. In the present study a route for the formation of hydroxytyrosol (1), by reduction of the corresponding acid 2 with tetrabutylammonium boronate, was found. Methods for the incorporation of a tritium label in 1 were investigated and successfully accomplished. Tritiated hydroxytyrosol (1t) was synthesized with a specific activity of 66 Ci/mol. The stability of unlabeled and labeled hydroxytyrosol was also investigated.

Full text of DOI:10.1021/jf000468l

J. Agric. Food Chem. 2000, 48, 4087−4090
4087
Syn th esis of Tr itiu m -La beled Hyd r oxytyr osol, a P h en olic Com p ou n d
F ou n d in Olive Oil
Kellie L. Tuck,* Hai-Wei Tan, and Peter J . Hayball*
Centre for Pharmaceutical Research, School of Pharmacy and Medical Sciences, University of South
Australia, Adelaide, 5000, Australia
(3,4-Dihydroxyphenyl)ethanol, commonly known as hydroxytyrosol (1), is the major phenolic
antioxidant compound in olive oil, and it contributes to the beneficial properties of olive oil.
Bioavailability and metabolism studies of this compound are extremely limited, in part, related to
unavailability of radiolabeled compound. Studies with radiolabeled compounds enable use of sensitive
radiometric analytical methods as well as aiding elucidation of metabolic and elimination pathways.
In the present study a route for the formation of hydroxytyrosol (1), by reduction of the corresponding
acid 2 with tetrabutylammonium boronate, was found. Methods for the incorporation of a tritium
label in 1 were investigated and successfully accomplished. Tritiated hydroxytyrosol (1t) was
synthesized with a specific activity of 66 Ci/mol. The stability of unlabeled and labeled hydroxytyrosol
was also investigated.
Keyw or d s: Hydroxytyrosol; olive oil; deuteriation; tritiation; tetrabutylammonium boronate
INTRODUCTION
acid can be reduced with lithium aluminum hydride to
directly give 1 (Verhe et al, 1992). However, all of these
reactions, except one (Bai et al, 1998), give poor yields
of 1 and none describe the introduction of a tritium
label.
In an effort to investigate the biological properties of
1, we examined two different syntheses for its facile
formation from 2. Specifically, we wish to examine the
bioavailability and the pharmacokinetic properties of 1
after oral and parenteral administration. We also plan
to investigate the metabolic profile of this pivotal
compound found in olive oil. Two routes for incorpora-
tion of a tritium label in 1 were explored, and we would
like to disclose a successful procedure for the synthesis
of tritiated hydroxytyrosol (1t).
(3,4-Dihydroxyphenyl)ethanol (1), also known as hy-
droxytyrosol, is one of the major phenolic compounds
present in olive oil (Fedeli, 1979). Epidemiological
studies have linked the high dietary intake of natural
antioxidants, found in olive oil with a lower incidence
of coronary heart disease (Keys, 1970; Keys, 1995) and
certain cancers, namely prostate and colon cancers
(Martin-Moreno et al, 1994; Lipworth et al, 1997).
Hydroxytyrosol (1), a phenol, is one of the major
antioxidants present in olive oil (Montedoro, 1972).
However, despite the reported importance of the biologi-
cal properties of phenols and polyphenols, published
data on the absorption and disposition of 1 are scarce
(Visioli et al, 1998; Visioli et al, 2000). This lack of data
is mainly because 1, which is easily oxidized, is not
available commercially and because a sensitive method
for the determination of 1 in biological matrixes is
limited. A GC-MS quantitative analysis procedure has
been used to measure the concentration of 1 in rat
plasma after it was orally administered (Bai et al, 1998).
However, GC-MS was not available in our laboratory
and we investigated an alternative technique to analysis
by GC-MS. One method of increasing the sensitivity of
detection is with the use of radiolabeled compounds.
Tritium-labeled compounds often have high specific
activities and can be introduced postsynthetically, for
example with the use of isotopic exchange reactions.
Hydroxytyrosol has been previously synthesized from
(3,4-dihydroxyphenyl)acetic acid (2) by two routes: di-
rect reduction with lithium aluminum hydride (Baraldi
et al, 1983) and reduction with (trimethylsilyl)diaz-
omethane and sodium borohydride (Bai et al, 1998).
Also, the methyl ester of (3,4-dihydroxyphenyl)acetic
MATERIALS AND METHODS
Rea gen ts. (3,4-Dihydroxyphenyl)acetic acid (2), NaBH4,
NaBD4 (98%), deuterium oxide (99.9%), and LiAlH4, 1.0 M
solution in THF, were obtained from Aldrich (Sydney, Aus-
tralia). THF was freshly distilled from sodium/benzophenone
prior to use. All other chemicals and solvents were of analytical
grade or higher and used without purification. Organic solu-
tions were dried over MgSO4, unless otherwise stated. Chro-
matography was performed with silica gel (230-400 mesh).
TLC was performed with Merck DC Alufolien Kieselgel 60 F254
,
which was visualized either with UV light or by immersion in
acidic ammonium molybdate solution.
Ap p a r a tu s. NMR spectra were measured with a Varian
spectrometer with an operating frequency of 200 MHz; acetone-
d6 or water-d2 was used as the solvent. HPLC analysis and
sample collection were performed on a Hewlett Packard 1100
Series system consisting of a 1100 series isocratic pump, 1100
series autosampler, and 1100 series variable wavelength
detector and with a semipreparative SymmetryPrep C18 7 µm
(7.8 mm × 300 mm) column or an analytical DuPont Zorbax
phenyl (4.6 mm × 25 cm) column. The compounds were
detected at 281 nm. Specific activities were measured by
preparing solutions of hydroxytyrosol (1) of known concentra-
tion, in scintillant, and radiometrically counting the aliquots
using a PACKARD Tri-Carb 2000CA liquid scintillation
* Corresponding authors: K.L.T., Ph 61 8 83022301, fax 61
8 83022389, e-mail Kellie.Tuck@unisa.edu.au; P.J .H., Ph 61
8 83021646, fax 61 8 83022389, e-mail Peter.Hayball@
unisa.edu.au.
10.1021/jf000468l CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/09/2000

4088 J. Agric. Food Chem., Vol. 48, No. 9, 2000
Tuck et al.
counter. HPLC radiometric analysis was performed with a
Radiomatic 150TR-flow scintillation analyzer (scintillant flow
2.5 mL/min and HPLC flow 1 mL/min).
Sch em e 1
P r ep a r a tion of Hyd r oxytyr osol w ith LiAlH4. (3,4-Di-
hydroxyphenyl)acetic acid (2) (0.5 g, 3.0 mmols) dissolved in
THF (2.0 mL) was added to an ice-cooled solution of 1.0 M
LiAlH4 in THF (4.5 mL, 4.5 mmol). After the addition was
complete, the suspension was heated under reflux for 3 h and
cooled in an ice bath, and excess LiAlH4 was destroyed by the
careful addition of H2O (10 mL) and 1.0 M HCl (10 mL). The
mixture was extracted with ethyl acetate (3 × 30 mL). The
combined organic extracts were dried and concentrated in
vacuo. Careful flash chromatography (ethyl acetate/hexanes,
50/50 v/v) gave 1 as a colorless oil (45%). δH: 2.70 (2H, t, J )
6.8 Hz, H1′), 3.75 (2H, t, J ) 6.8 Hz, H2′), 6.70 (1H, dm, J )
8.0 and 2.0 Hz, H6), 6.80 (1H, d, J ) 2.0 Hz, H2), 6.84 (1H, d,
J ) 8.0 Hz, H5).
Tr itia tion of 1 w ith Am ber lyst 15. 1 (46.4 mg), Amberlyst
15 (51.1 mg), T2O (5 mCi/mL, 0.2 mL, 1 Ci), and a magnetic
stirrer (7 mm) were introduced into a Hewlett-Packard HPLC
vial (2 mL capacity). The flask was evacuated with N2, the
top was screwed on, and the flask was placed into an oil bath.
The reaction was heated at 90 °C, with continuous stirring,
for 24 h. On completion the tube was cooled, the solution
removed, and H2O (1 mL) added to the flask to rinse the resin;
this solution was removed and added to the initial solution.
This procedure was repeated three times, to ensure complete
removal of the substrate. Freeze-drying of the solution gave
tritiated hydroxytyrosol (1t) in a recovery yield of 97%. The
residue was dissolved in H2O (2 mL) and purified by prepara-
tive HPLC (95 v/v H2O (containing 2/5 v/v acetic acid/MeOH,
2 mL/min). The eluted substrate in mobile phase (85 mL) was
extracted with ethyl acetate (4 × 80 mL). The combined
organic phases were dried (Na2SO4), and solvent was removed
in vacuo. The specific activity of 1t was found to be 66 Ci/mol.
Sta bility of 1 a t p H 8.85. 1 (1.8 mg) was added to colonic
lavage buffer (5 mL) (Davies et al, 1980). The resulting solution
had a pH of 8.85. Aliquots (100 µL) were added to ependorf
tubes and incubated at 37 °C. At time intervals of 0, 5, 10, 15,
20, 30, 40, 50, 60, 80, 100, and 120 min the ependorf solutions
were diluted with mobile phase (99.5 v/v H2O (containing 0.2/
0.5 v/v acetic acid)/MeOH, 1 mL/min). These solutions were
further diluted (10×) with mobile phase and analyzed by
HPLC. The results are summarized in Figure 3.
P r ep a r a tion of Hyd r oxytyr osol (1) w ith Tetr a bu ty-
la m m on iu m Bor on a te. Preparation of Tetrabutylammonium
Boronate. Tetrabutylammonium hydrogen sulfate (0.34 g, 1.0
mmols) dissolved in H2O (0.2 mL) was added to 5.0 M NaOH
(0.25 mL). To the mixture, cooled in a slurry of ice, was added
NaBH4 (0.04 g, 1.1 mmol) dissolved in H2O (0.1 mL). After
stirring of the reaction mixture for 5 min, it was extracted with
CH2Cl2 (3 × 5 mL). The combined organic extracts were dried
1
and concentrated in vacuo to approximately /3 of the original
volume. The solution was then used directly in the next step.
Preparation of 1 with Tetrabutylammonium Boronate. To a
round-bottom flask containing the above solution of tetrabu-
tylammonium boronate (1.0 mmols) was added (3,4-dihydrox-
yphenyl)acetic acid (2) (0.84 g, 0.5 mmol) dissolved in THF
(0.2 mL). The flask was cooled in an ice bath, and MeI (0.15
mL, 2.0 mmol) was carefully added, so to avoid the excess
evolution of methane. After being stirred for 2 h at room
temperature, the excess hydride was destroyed by the careful
addition of ethanol (10 mL) and 1.0 M HCl (10 mL) to the ice-
cooled solution. The solution was extracted with ethyl acetate
(3 × 30 mL), and the combined organic extracts were dried,
and concentrated in vacuo. Column chromatography (ethyl
acetate/hexanes, 50/50 v/v) gave the product as a light yellow
oil (99%). δH: 2.70 (2H, t, J ) 6.8 Hz, H1′), 3.75 (2H, t, J ) 6.8
Hz, H2′), 6.70 (1H, dm, J ) 8.0 and 2.0 Hz, H6), 6.80 (1H, d,
J ) 2.0 Hz, H2), 6.84 (1H, d, J ) 8.0 Hz, H5).
RESULTS AND DISCUSSION
P r ep a r a tion of Deu ter a ted Hyd r oxytyr osol (1a ) w ith
Deu ter a ted Tetr a bu tyla m m on iu m Bor on a te. The above
procedure was repeated with tetrabutylammonium hydrogen
A sample of hydroxytyrosol (1) was obtained by
reduction of (3,4-dihydroxyphenyl)acetic acid (2) with
lithium aluminum hydride; spectral data were compa-
rable to that which had previously been published
(Montedoro et al, 1993). However, optimization of this
reaction gave 1 in only a 45% yield after purification
by chromatography. It must be noted that care needs
to be taken during the chromatography as 1 is not stable
on silica. Consequently chromatographic purification
must be performed quickly so that the yield of 1 is
optimized.
In an effort to improve the yield of 1 from its
corresponding acid 2 an alternative reduction reaction
was investigated. Tetrabutylammonium boronate is a
reducing reagent of esters (Bra¨ndstro¨m et al, 1972) and
gives the corresponding alcohols in yields of 80-98%.
Tetrabutylammonium boronate, formed by reaction of
tetrabutylammonium hydrogen sulfate with sodium
borohydride, does reduce 2 to give 1 in a 99% yield
(Scheme 1); spectral data correspond to that of 1
synthesized previously. It was recognized at this stage,
with the use of deuterated or tritiated sodium borohy-
dride, that it was possible to incorporate a label at the
2′ position in 1. Indeed, reaction of 3,4-dihydroxyphe-
nylacetic acid with tetrabutylammonium boronate,
formed by reaction of tetrabutylammonium hydrogen
sulfate with sodium borodeuteride, gave the correspond-
ing deuterated hydroxytyrosol (1a ) in a 56% yield. The
sulfate (3.4 g, 10.0 mmol), D2O (2.0 mL), 5.0 M NaOH (2.5
1
mL), NaBD4 (0.4 g, 11.0 mmol), and D2O (2.0 mL). A
/
10
amount of this mixture was reacted with 2 (0.84 g, 0.5 mmols)
dissolved in THF (0.2 mL). After workup and concentration
the deuterated compound 1a was obtained as a yellow oil in a
56% yield. δH: 2.70 (2H, s, H1′), 6.70 (1H, dm, J ) 8.0 and 2.0
Hz, H6), 6.80 (1H, d, J ) 2.0 Hz, H2), 6.84 (1H, d, J ) 8.0 Hz,
H5).
Deu ter ia tion of 1 w ith Am ber lyst 15. (i) Reaction with
D2O. 1 (24.5 mg), Amberlyst 15 (25.2 mg), D2O (0.2 mL), and
a magnetic stirrer (7 mm) were introduced into a Hewlett
Packard HPLC vial (2 mL capacity). The flask was evacuated
with N2, the top was screwed on, and the flask was placed into
an oil bath. The reaction was heated at 90 °C, with continuous
stirring, for 24 h. On completion the tube was cooled, the
solution removed, and H2O (3 × 0.2 mL) added to the flask to
rinse the resin; this solution was removed and added to the
initial solution. Freeze-drying of the solution gave deuterated
hydroxytyrosol (1d ) in a recovery yield of 98%. Analysis by
1H NMR spectroscopy showed complete disappearance of the
aromatic protons. δH: 2.70 (2H, t, J ) 6.8 Hz, H1′), 3.75 (2H,
t, J ) 6.8 Hz, H2′).
(ii) Reaction with 50% D2O/ H2O. The above procedure was
repeated with 1 (10.9 mg), Amberlyst 15 (12.0 mg), D2O (0.1
mL), and H2O (0.1 mL). The substrate 1d was obtained in a
66% recovery yield. δH: 2.70 (2H, t, J ) 6.8 Hz, H1′), 3.75 (2H,
t, J ) 6.8 Hz, H2′), 6.70 (0.67H, dm, J ) 8.0 and 2.0 Hz, H6),
6.80 (0.67H, d, J ) 2.0 Hz, H2), 6.84 (0.67H, d, J ) 8.0 Hz,
H5).

Synthesis of Tritium-Labeled Hydroxytyrosol
J. Agric. Food Chem., Vol. 48, No. 9, 2000 4089
Sch em e 2
1H NMR spectrum of 1a showed disappearance of the
triplet at 3.75, and the triplet previously at 2.70 was
now a singlet. Hence, a deuterium had been successfully
incorporated at the 2′ position.
Consequently, there now exists a procedure for the
introduction of a deuterium at the 2′ position of 1 and
this procedure can be applied to the tritiation of 1.
Unfortunately, sodium borotritide is very expensive, and
hence, an alternative cheaper route for the tritiation of
1 was investigated. Nevertheless, an alternative, ef-
ficient route for the synthesis of 1 from its corresponding
acid 2 has been found.
There have been recent reports in the literature of
the use of ion-exchange resins, in the acid form, to
deuterate and tritiate a large number of molecules
(Brewer et al, 1994). We have recently modified this
technique and have applied it, with the use of the
polymer supported acid catalyst Amberlyst 15, to deu-
terate a large number of phenolic compounds (Tuck et
al, 2000). We now wish to report the synthesis of
tritiated hydroxytyrosol (1t) using Amberlyst 15.
We initially deuterated rather than tritiated 1 due
to the wide availability, high isotopic purity, and af-
fordability of deuterated water and the fact that the
F igu r e 1. Radiometric chromatogram of 1t before purification
by preparative HPLC.
1
reaction could be monitored by H NMR spectroscopy.
Reaction of 1 with Amberlyst 15 and deuterium oxide,
after heating for 24 h, resulted in full exchange of the
aromatic protons at the 2, 5, and 6 positions of the
aromatic ring (Scheme 2). However, when the reaction
was stopped after 15 h the incorporation was 100% at
C6, 70% at C2, and 15% at C5. The difference in the
rate of deuteriation is due to the relationship of the
proton to the hydroxyl groups present in the substrate.
In these compounds the ortho and para positions are
mesomerically activated and the protons in these posi-
tions will exchange preferentially; this is in accord with
an electrophilic substitution type mechanism. Reaction
of 1 with a solution of H2O/D2O (1:1), 24 h of heating,
gave a 33% incorporation of a deuterium at C6, C2, and
C5.
It was discovered that up to 50 mg of substrate could
be effectively deuterated with only 0.2 mL of deuterium
oxide and by optimization of the extraction procedure
recovery yields as high as 98% were obtained.
With optimization of the deuteriation conditions, the
reaction was attempted with tritiated water. Reaction
of 1 with tritiated water (5 mCi/mL) gave tritiated 1t
in a recovery yield of 97%. Radiolabeled 1t was purified
by preparative HPLC; after extraction and concentra-
tion the specific activity was found to be 66 Ci/mol.
Figures 1 and 2 show the radiometric chromatograms
of 1t before and after purification; the peak at 3.5 is
due to residual tritiated water, and the peak at 6.5 is
due to 1t. Figure 2 shows the radiometric chromatogram
of 1t after its purification by preparative HPLC.
The stability of unlabeled 1 was investigated in a
number of solutions. An aqueous solution of 1 (50 µg/
mL) showed no degradation products by HPLC, even
after storage at 4 °C for 5 months. 1 was also stable in
F igu r e 2. Radiometric chromatogram of 1t after purification
by preparative HPLC.
this aqueous solution when heated at 37 °C for 2 h. The
stability of 1 (50 µg /mL) was also examined in an acidic
solution (pH 3.1); after several months storage at 4 °C
no degradation was seen by HPLC. However, in a basic
solution (pH 8.85), at 37 °C, 1 (36 µg/mL) quickly
degrades (t1/2 ) 92 min) to an unknown product. The
results are represented in Figure 3.
The stability of the tritium label in 1t was also
investigated. Tritiated 1t was stable in aqueous and
acidic (pH 3.1) solutions, at 4 °C, for a number of weeks.
No exchange of the label was observed by radiometric
HPLC. Also, no change in the specific activity of 1t was

4090 J. Agric. Food Chem., Vol. 48, No. 9, 2000
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In conclusion, a facile route for the synthesis of 1 from
its corresponding acid 2 with the use tetrabutylammo-
nium boronate has been found. Tritiated 1t, with a high
specific activity, 66 Ci/mol, can be synthesized from
unlabeled 1 with the use of Amberlyst 15 as a polymer
supported acid catalyst. Elucidation of the absorption,
metabolism, and disposition of hydroxytyrosol should
now be possible. Numerous studies (Manna et al, 1997;
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important dietary antioxidants such as vitamin C and
R-tocopherol.
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Received for review April 12, 2000. Revised manuscript
received J une 6, 2000. Accepted J une 7, 2000.
J F000468L