Epicatechin B-ring conjugates: First enantioselective synthesis and evidence for their occurrence in human biological fluids

Article abstract of DOI:10.1021/ol3016463

Herein, the first enantioselective total synthesis of a number of biologically relevant (-)-epicatechin conjugates is described. The success of this synthesis relied on (i) optimized conditions for the stereospecific cyclization step leading to the catechin C ring; on (ii) efficient conjugation reactions; and on (iii) optimized deprotection sequences. These standard compounds have been subsequently used to elucidate for the first time the pattern of (-)-epicatechin conjugates present in four different human biological fluids following (-)-epicatechin absorption.

Full text of DOI:10.1021/ol3016463

ORGANIC
LETTERS
2012
Vol. 14, No. 15
3902–3905
Epicatechin B-Ring Conjugates: First
Enantioselective Synthesis and Evidence
for Their Occurrence in Human Biological
Fluids
ꢀ
ꢀ ꢁ
Fedor Romanov-Michailidis, Florian Viton, Rene Fumeaux, Antoine Leveques,
Lucas Actis-Goretta, Maarit Rein, Gary Williamson, and Denis Barron*,†
Nestle Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland
Denis.Barron@rd.nestle.com
Received June 15, 2012
ABSTRACT
Herein, the first enantioselective total synthesis of a number of biologically relevant (ꢀ)-epicatechin conjugates is described. The success of
this synthesis relied on (i) optimized conditions for the stereospecific cyclization step leading to the catechin C ring; on (ii) efficient
conjugation reactions; and on (iii) optimized deprotection sequences. These standard compounds have been subsequently used to elucidate
for the first time the pattern of (ꢀ)-epicatechin conjugates present in four different human biological fluids following (ꢀ)-epicatechin
absorption.
Glucuronidated, sulfated, and/or methylated forms
of (ꢀ)-epicatechin are released in the human blood-
stream following consumption of (ꢀ)-epicatechin-rich
products such as cocoa, red wines, and tea.1 However,
since (ꢀ)-epicatechin metabolites are not commercially
available, they have often been only partly character-
ized, and reports studying their biological activities are
scarce.2 Consequently, there is a recurrent need for
access to pure analytical standards of (ꢀ)-epicatechin
conjugates. Over the past years, numerous efforts aimed
at developing a scalable (hemi)synthetic approach to
produce (ꢀ)-epicatechin and (þ)-catechin conjugates
have been reported. Among these methodologies, direct
derivatization3 and hemisynthetic4 approaches were
prevalent. Unfortunately, these methods were plagued
by low chemical yields, poor selectivities, and tedious
purification steps. Despite this, few studies making use
of synthetic conjugated (epi)catechin standards have
been recently published. However, the details of the
chemical synthesis involved were not provided,5,6 except
for the preparation of epicatechin glucuronides conju-
gated at the A-ring.7
(4) Cren-Olive, C.; Lebrun, S.; Rolando, C. J. Chem. Soc., Perkin
Trans. 1 2002, 6, 821–830.
(5) Schroeter, H.; Heiss, C.; Balzer, J.; Kleinbongard, P.; Keen, C. L.;
Hollenberg, N. K.; Sies, H.; Kwik-Uribe, C.; Schmitz, H. H.; Kelm, M.
Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1024–1029.
(6) Ottaviani, J. I.; Momma, T. Y.; Kuhnle, G. K.; Keen, C. L.;
Schroeter, H. Free Radical Biol. Med. 2012, 52, 1403–1412.
(7) Mull, E. S.; Van Zandt, M.; Golebiowski, A.; Beckett, R. P.;
Sharma, P. K.; Schroeter, H. Tetrahedron Lett. 2012, 53, 1501–1503.
†
ꢀ
Present address: Nestle Institute of Health Sciences, EPFL innovation
square, 1015 Lausanne, Switzerland.
(1) Scalbert, A.; Williamson, G. J. Nutr. 2000, 130, 2073S–2085S.
(2) Barron, D. Recent Adv. Polyphenol Res. 2008, 1, 317–358.
(3) Gonzalez-Manzano, S.; Gonzalez-Paramas, A.; Santos-Buelga,
C.; Duenas, M. J. Agric. Food Chem. 2009, 57, 1231–1238.
r
10.1021/ol3016463
Published on Web 07/17/2012
2012 American Chemical Society

Figure 1. Structures of the synthesized (ꢀ)-epicatechin conju-
gates: 30-O-glucuronide 1, 40-O-glucuronide 2, 30-O-sulfonate 3,
30-O-methyl-40-O-sulfonate 4.
Herein, we describe the first fully documented enan-
tioselective total synthesis of four biologically relevant
(ꢀ)-epicatechin B-ring conjugates (Figure 1). These
fully characterized molecules were subsequently used
as analytical-grade standards to elucidate the distribu-
tion pattern of (ꢀ)-epicatechin conjugates present in
four human biological fluids after (ꢀ)-epicatechin con-
sumption. The retrosynthetic analysis scheme leading to
the pivotal (ꢀ)-epicatechin core A, ready for regioselec-
tive glucuronide or sulfate conjugation, is displayed in
Figure 2.
The precursor of ring A of tribenzylated (ꢀ)-epica-
techin (A) is the phloroglucinol derivative E, while the
cinnamic alcohol derivative D, itself prepared from the
benzaldehyde derivative F, is the precursor of rings B
and C. A FriedelꢀCrafts alkylation of E by D would
lead to diphenol C, which could then be transformed
into the enantiomerically enriched tetraol B follow-
ing a standard Sharpless asymmetric dihydroxylation
protocol. Finally, a stereospecific cyclization of B
would give rise to a catechin derivative, eventually
converted to A following an inversion of configuration
at C3.
Figure 2. Retrosynthetic analysis.
Starting from enantiomerically pure tetraols 5ꢀ7
(Figure 3; synthesis details to be found in the Supporting
Information), the key step of the synthesis of the epica-
techin core A was the stereospecific cyclization. The
success of the cyclization relied on emphasizing the
kinetic SN2 pathway for cyclization by applying milder
reaction conditions as compared to the previously re-
ported ones.8 Namely, (i) triethyl orthoformate was
replaced by the less reactive triethyl orthoacetate, (ii)
the PPTS catalyst loading was lowered from 60 to 10 mol %,
(iii) the reaction temperature was dropped down from
60 to 45 °C, and (iv) the reaction time was shortened
from 6 to 4 h. On the other hand, in order to avoid
potential base-induced epimerization problems,9 the
classical alkaline methanolysis step (K2CO3/MeOH)
was replaced by a reductive step involving DIBAL-H
Figure 3. Synthesis of the protected (R,R)-epicatechin derivatives 20ꢀ22.
Org. Lett., Vol. 14, No. 15, 2012
3903

Figure 4. Syntheses of (ꢀ)-epicatechin 30- and 40-O-β-D-
glucuronides.
Figure 5. Syntheses of (ꢀ)-epicatechin 30-sulfate and 30-methyl-
40-sulfate.
as a reagent. Thus tetraols 5ꢀ7 were first cyclized cleanly
to the corresponding 3-acylcatechins 8ꢀ10 with com-
plete trans-selectivity (>98% inversion according to
1H NMR analysis of the crude reaction mixture). Sub-
sequent TBS-protection of the sole bare hydroxyl group,
followed by DIBAL-promoted deacetylation afforded
the desired (R,S)-catechin skeletons 14ꢀ16 in excel-
lent yields. These were then subjected to a two-step
C3-inversion protocol (DMP-oxidation followed by dia-
stereoselective L-selectride reduction), yielding epicate-
chin derivatives 20ꢀ22.
Regiospecific O-sulfonations were carried out on pro-
tected scaffolds 20 and 22 (Figure 5). Neopentyl chloro-
sulfate (34) was chosen as the optimal sulfonating rea-
gent.10 In the preparation of compound 3 from compound
35, the neopentyl protecting group was first removed with
NaN3, followed by hydrogenative debenzylation over
Pd(OH)2/C.
However, partial cleavage of the free sulfate moiety took
place duringhydrogenolysis, and compound3 was isolated
in only small amounts (9% of 3 from 35 over two steps).
Thus, in the synthesis of 30-O-methyl-(ꢀ)-epicatechin 40-O-
sulfonate (4) from compound 36, we adopted a reverse
deprotection sequence (first hydrogenation, and then
treatment with NaN3). This markedly increased the iso-
lated yield (66% of 4 from 36 over two steps).
The synthesized compounds were subsequently used
as analytical-grade standards to elucidate the pattern of
(ꢀ)-epicatechin conjugates present in human biological
samples. Blood, urine, intestinal fluids, and bile samples
were collected after human volunteers receiced 50 mg of
(ꢀ)-epicatechin.11 (ꢀ)-Epicatechin metabolites were ex-
tracted (see Supporting Information) and analyzed by
UPLC-MS/MS on reversed-phase. The UPLC-MS/MS
analysis demonstrated the presence of nine conjugates
En route to the synthesis of (ꢀ)-epicatechin 30- and 40-
glucuronides, compounds 20 and 21 were acetylated at
carbon C3 to compounds 23 and 24 (Figure 4). Removal
of the TBS protecting groups afforded compounds 25
and 26, ready for regiospecific O-glucuronidation at
positions 30 and 40, respectively. Thus, 25 and 26 were
coupled with glucuronic acid donor 27, under BF3•OEt2
catalysis. Mild alkaline hydrolysis, followed by hydro-
genolysis over Pd(OH)2/C, yielded the desired glucuro-
nides 1 and 2.
(8) (a) Li, L.; Chan, T. H. Org. Lett. 2001, 3, 739–741. (b) Wan, S. B.;
Chen, D.; Dou, Q. P.; Chan, T. H. Bioorg. Med. Chem. 2004, 12, 3521–
3527. (c) Viton, F.; Landreau, C.; Rustidge, D.; Robert, F.; Williamson,
G; Barron, D. Eur. J. Org. Chem. 2008, 36, 6069–6078. (d) Sharma,
P. K.; He, M.; Romanczyk, L. J., Jr; Schroeter, H. J. Label. Compd.
Radiopharm. 2010, 53, 605–612.
(10) Simpson, L. S.; Widlanski, T. S. J. Am. Chem. Soc. 2006, 128,
1605–1610.
(11) Eudra-CT-number: 2006-006008-12.
(9) Kiatgrajai, P.; Wellons, J. D.; Gollob, L.; White, J. D. J. Org.
Chem. 1982, 47, 2910–2912.
3904
Org. Lett., Vol. 14, No. 15, 2012

epicatechin-O-sulfonates. Comparison with the LC reten-
tion times and MS data of our authentic synthetic stan-
dards allowed us to unambiguously assign four out of the
nine detected conjugates (Table 1).
Table 1. Comparative UPLC and MS Data for Authentic
Standards of (ꢀ)-Epicatechin Glucuronides/Sulfates, and (ꢀ)-
Epicatechin Conjugates Present in Human Biological Fluids
found
MS/MS
main
epicatechin
metabolites
peak
exact
mass
elemental
tR
composition fragment
Table 2. Occurrence of Conjugates 1ꢀ4 in Human Biological
Fluids
UPLC peak
(1) EC-O-
1.70 465.1051
C
21H21O12
289.0719
compd
intestinal fluid
plasma
bile
urine
glucuronide
1
2
3
4
þ
þ
þþþ
þ
þþþ
þ
þþþ
þ
(2) EC-O-sulfonate 1.84 369.0286
(3) EC-O-sulfonate 2.38 369.0292
C15H13O9S
289.0722
289.0729
289.0719
C
C
15H13O9S
21H21O12
þþþ
þ
þþþ
þ
þþþ
þ
þþ
þ
(4) EC-O-
2.41 465.1051
2.53 383.0444
3.36 383.0445
3.61 383.0446
4.08 383.0445
4.43 383.0443
glucuronide
(5) O-Methyl-EC-
O-sulfonate
C
16H15O9S
303.0872
303.0865
303.0874
303.0862
303.0869
Thus peak 1 was (ꢀ)-epicatechin 40-O-β-D-glucuronide,
peak 3 was (ꢀ)-epicatechin 30-O-sulfonate, peak 4 was (ꢀ)-
epicatechin 30-O-β-D-glucuronide, and peak 5 was 30-O-
methyl-(ꢀ)-epicatechin-40-O-sulfonate.
(6) O-Methyl-EC-
O-sulfonate
C16H15O9S
(7) O-Methyl-EC-
O-sulfonate
C
C
C
16H15O9S
16H15O9S
16H15O9S
(8) O-Methyl-EC-
O-sulfonate
These molecules were shown to be present in human
intestinal fluid, plasma, bile, and urine following ex-
posure to (ꢀ)-epicatechin (Table 2). Among these four
conjugates (ꢀ)-epicatechin 30-O-sulfonate and (ꢀ)-
epicatechin 30-O-β-D-glucuronide were shown to be
the major circulating forms in biological samples. Fur-
ther details concerning the quantification and the
biological activity of these conjugates will be reported
in a separate paper.
(9) O-Methyl-EC-
O-sulfonate
synthetic standards
(ꢀ)-epicatechin-40-
O-glucuronide
(ꢀ)-epicatechin-30-
O-sulfonate
(ꢀ)-epicatechin-30-
O-glucuronide
30-O-methyl-(ꢀ)-
epicatechin-40-O-
sulfonate
1.68 465.1035
2.42 369.0289
2.45 465.1017
2.58 383.0445
C21H21O12
C15H13O9S
C21H21O12
C16H15O9S
289.0712
289.0719
289.0702
303.0872
Supporting Information Available. Detailed experimen-
tal procedures and complete spectroscopic characteriza-
tion of all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
(Table 1), which were two (ꢀ)-epicatechin O-glucuronides,
two (ꢀ)-epicatechin O-sulfonates, and five O-methyl-(ꢀ)-
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 15, 2012
3905