D. Barron et al.
FULL PAPER
rated, and the residue was purified by flash chromatography on
silica gel (isohexane/EtOAc, 100:0 to 75:25) to afford the desired
product 10 as white solid (180 mg, 85%). TLC isohexane/EtOAc,
75:25, Rf of 5,7,3Ј,4Ј-tetra-O-benzylcatechin = 0.27. 1H NMR
stirring was continued for 10 min at room temperature. The dark
precipitate was eliminated by filtration through a Pasteur pipette
fitted with cotton wool and rinsed with CH2Cl2 (3ϫ5 mL). The
combined organic layers were directly loaded on the top of a 20 g
(CDCl3, 300 MHz): δ = 7.47–7.25 (m, 20 H), 7.03 (s, 1 H), 6.96 (s, silica cartridge, which was then eluted with isohexane/EtOAc (100:0
2 H), 6.27, 6.21 (ABq, J = 2.2 Hz, 2 H), 5.18 (s, 2 H), 5.17 (narrow
ABq, concealed, 2 H), 5.03 (s, 2 H), 4.99 (s, 2 H), 4.63 (d, J =
8.2 Hz, 1 H), 4.03–3.97 (m, 1 H), 3.11, 2.65 (ABq, J = 16.4, both
parts d with J = 5.7 and 8.8 Hz, 2 H), 1.58 (d, J = 3.7 Hz, 1 H)
ppm.
to 40:60) to afford compound 11 in 76 % yield (108 mg). TLC
isohexane/EtOAc, 7:3, Rf of 5,7,3Ј,4Ј-tetra-O-benzyl-4-(2-ethoxye-
thoxy)epicatechin = 0.45, Rf of 5,7,3Ј,4Ј-tetra-O-benzylepicatechin
= 0.52. 1H NMR (CDCl3, 300 MHz): δ = 7.49–7.26 (m, 20 H), 7.19
(d, J = 1.5 Hz, 1 H), 7.07, 7.00 (ABq, J = 8.2 Hz, A part d with J
= 1.5 Hz, 2 H), 6.29, 6.27 (ABq, J = 2.2 Hz, 2 H), 5.20 (s, 2 H),
5.20 (s, 1 H, concealed), 5.19 (s, 2 H), 5.03 (s, 2 H), 5.09, 5.02
(ABq, partially concealed, J = 11.5 Hz, 2 H), 4.61 (d, J = 2.9 Hz,
1 H), 4.04 (br., 1 H), 3.83–3.78 (m, 2 H), 3.53–3.41 (m, 4 H), 1.55
(d, J = 6.9 Hz, 1 H), 1.17 (t, J = 7.1 Hz, 3 H).
5,7,3Ј,4Ј-Tetra-O-benzylepicatechin (2): Dess–Martin periodinane
(≈ 2 mL, 0.3 in CH2Cl2, 0.608 mmol, 2.2 equiv.) was added in one
batch to a stirred solution of 10 (180 mg, 0.277 mmol) in anhydrous
CH2Cl2 (10 mL) under nnitrogen. The mixture was stirred at room
temperature for about 2 h until TLC (isohexane/EtOAc, 2:1, Rf of
5,7,3Ј,4Ј-tetra-O-benzylcatechin = 0.4, Rf of the ketone = 0.54)
showed the absence of starting material. Subsequently, saturated
aqueous NaHCO3 solution (4.2 mL) and 10% aqueous Na2S2O3
solution (4.2 mL) were added to quench the reaction. The organic
layer was separated, and the aqueous layer was extracted with
CH2Cl2. The combined organic phases were dried (MgSO4) and the
solvents evaporated. The residue was quickly purified by filtration
through a 10 g silica cartridge (eluting with CHCl3/EtOAc, 9:1) to
afford the desired (2R)-5,7,3Ј,4Ј-tetrakis(benzyloxy)flavan-3-one as
a yellow oil (157 mg, 87%). 1H NMR (CDCl3, 300 MHz): δ = 7.45–
7.26 (m, 20 H), 6.96 (s, 1 H), 6.88, 6.86 (ABq, J = 8, B part d with
J = 1.5 Hz, 2 H), 6.35 (narrow ABq, 2 H), 5.24 (s, 1 H), 5.14 (s, 2
H), 5.10 (narrow ABq, 2 H), 5.02 (s, 2 H), 5.02 (s, 2 H), 3.61, 3.45
(ABq, J = 21.5 Hz, 2 H) ppm. Under nitrogen, the ketone (157 mg,
0.242 mmol) was dissolved in dry THF (5 mL), and the solution
was cooled to –78 °C. Then -Selectride® (380 µL, 1.0 solution
in THF, 0.38 mmol, 1.6 equiv.) was added dropwise. The resulting
solution was stirred at –78 °C overnight. When TLC showed the
reaction was complete, saturated NaHCO3 aqueous solution
(≈ 5 mL) was added to quench the reaction. The organic layer was
separated and the aqueous layer was extracted with EtOAc
(3ϫ10 mL). The combined organic phases were dried (MgSO4)
and the solvents evaporated. The residue was purified by flash
chromatography (20 g silica cartridge, eluting with isohexane/
EtOAc, 100:0 to 80:20) to give 2 in 74% yield (116.8 mg). TLC
isohexane/EtOAc, 2:1, Rf of the ketone = 0.54, Rf of 5,7,3Ј,4Ј-tetra-
O-benzylepicatechin = 0.42. 1H NMR (CDCl3, 300 MHz): δ =
7.48–7.25 (m, 20 H), 7.15 (s, 1 H), 7.00, 6.97 (ABq, J = 8.5 Hz, A
part d with J = 1.5 Hz, 2 H), 6.27 (s, 2 H), 5.19 (s, 2 H), 5.18 (s, 2
H), 5.03 (s, 2 H), 5.02 (s, 2 H), 4.92 (s, 1 H), 4.22 (br. s, 1 H), 3.00,
2.92 (ABq, J = 17.5 Hz, both parts d with J = 1.5 and 4 Hz, resp.,
2 H), 1.64 (d, J = 5.5 Hz, 1 H). Part of this material was submitted
to hydrogenation to provide a batch of “synthetic” (–)-epicatechin,
which chiral purity was assessed to be Ͼ92% by HPLC [HPLC
conditions were adapted from the literature:[13] Cyclobond I-2000
RSP 250ϫ4.6 mm column; mobile phase A: 50 mM NaH2PO4 at
pH 3, mobile phase B: 80% acetonitrile in 30 m NaH2PO4 at pH
3; flow 1 mL/min; linear gradient from 10.0 to 13.5% over 45 min,
then increasing to 45% B at 70 min; detection at 210 nm; authentic
samples of (–)-epicatechin and (+/–)-catechin from Biochemika
were used as references].
8-(5,7,3Ј,4Ј-Tetra-O-benzylepicatechin-4β-yl)(5,7,3Ј,4Ј-tetra-O-ben-
zylepicatechin) (13): TMSOTf (68 µL, 0.376 mmol, 1 equiv.) was
added dropwise to a solution of 5,7,3Ј,4Ј-tetra-O-benzylepicatechin
12 (979 mg, 1.504 mmol, 4 equiv.) and 5,7,3Ј,4Ј-tetra-O-benzyl-4-
(2-ethoxy-ethoxy)epicatechin 11 (277.8 mg, 0.376 mmol) in 34 mL
of anhydrous CH2Cl2 at –78 °C. The resulting solution was stirred
at –78 °C for 5 min then the reaction was terminated by addition
of saturated aqueous KHCO3 (0.6 mL). The CH2Cl2 phase was
washed with water (3ϫ56 mL), dried (MgSO4), filtered and con-
centrated to dryness. The residue was purified by column
chromatography (loading with CH2Cl2, 20 g silica cartridge pre-
wetted with isohexane, elution with isohexane/EtOAc, 100:0 to
50:50). Fractions containing 13 were concentrated and further puri-
fied by preparative HPLC [HPLC conditions adapted from the lit-
erature,[7k] using a Phenomenex Gemini C18 column 10µ, 110A,
250ϫ21–20 mm. Conditions: at 20 mL/min; 80 to 100% CH3CN
in water (gradient over 25 min) then CH3CN. UV 280 nm; reten-
tion time of the dimer: ca. 25.6 min]. Pure fractions were concen-
trated, filtered through a 2 g silica cartridge eluting with EtOAc,
and concentrated to dryness to afford 13 as a white foam (139 mg,
28% yield). TLC isohexane/EtOAc, 7:3, Rf of 5,7,3Ј,4Ј-tetra-O-ben-
zyl-4-(2-ethoxyethoxy)epicatechin = 0.43, Rf of 5,7,3Ј,4Ј-tetra-O-
benzylepicatechin = 0.51, Rf of 5,7,3Ј,4Ј-tetra-O-benzylepicatechin-
1
4β,8-(5,7,3Ј,4Ј-tetra-O-benzylepicatechin) = 0.34. H NMR (selec-
tion; 2 rotamers, approx. 3:1, MR = major, mr = minor rotamer)
(CDCl3, 300 MHz): δ = 6.80, 6.44 (ABq, MR, J = 8, B part d with
J = 1.5 Hz, 2 H), 6.36 (s, 1 H, MR), 6.25, 6.09 (ABq, mr, J =
2.5 Hz, 2 H), 6.20 (s, 1 H, mr), 6.02, 5.72 (ABq, MR, J = 2 Hz, 2
H), 5.55 (s, 1 H, MR), 5.29 (s, 1 H, mr), 4.66, 4.38 (ABq, mr, J =
12 Hz, 2 H), 4.63, 4.45 (ABq, MR, J = 11 Hz, 2 H), 4.32 (m, 1 H,
mr), 3.95 (br. d, mr, J = 6 Hz, 1 H), 3.86 (m, 1 H, MR), 3.09–2.84
(m, MR + mr, 2 H), 1.80 (d, MR, J = 6 Hz, 1 H), 1.71 (d, mr, J =
6.5 Hz, 1 H), 1.47 (d, MR, J = 4 Hz, 1 H), 1.30 (d, mr, J = 6.5 Hz,
1 H) ppm.
Procyanidin B2 (1): Epicatechin derivative 13 (50 mg, 0.0385 mmol)
was dissolved in ethyl acetate (5 mL). Water (5 mL) was added,
followed by palladium hydroxide over carbon (15 mg), and the bi-
phasic mixture was hydrogenated (1 atm hydrogen) overnight at
room temperature. Reverse phase TLC analysis (H2O/MeOH, 1:1
+ 1% AcOH) showed completion of the reaction. The reaction
mixture was filtered through a sintered funnel fitted with a glass
fiber prefilter and concentrated to a 4 mL aqueous solution of 1.
This material was compared to an authentic sample of procyanidin
B2 (obtained from Fluka) by reverse-phase TLC (H2O/MeOH, 1:1
+ 1% AcOH).
5,7,3Ј,4Ј-Tetra-O-benzyl-4-(2-ethoxyethoxy)epicatechin (11): 2-
Ethoxyethanol (110 µL, 1.14 mmol, 6 equiv.) and then all at once
with good stirring DDQ (86.3 mg, 0.38 mmol, 2 equiv.) was added
to a solution of 2 (124 mg, 0.19 mmol) in anhydrous CH2Cl2
(5 mL) at room temperature. A black-green color appeared instan-
taneously. After 2 h of vigorous stirring at room temperature under
nitrogen, DMAP (52 mg, 0.42 mmol, 2.2 equiv.) was added and
Synthesis of [2-14C]Procyanidin B2. Preparation of Copper [14C]Cy-
anide: Potassium [14C]cyanide (630 mg, 524 mCi, 9.42 mmol at
6074
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Eur. J. Org. Chem. 2008, 6069–6078