È
K. Syrjanen, G. Brunow / Tetrahedron 57 (2001) 365±370
369
described by Bailey and Dence.23 Synthesis and NMR for
compounds 11 and 16 see Ref. 17.
2.34 (3H, s OCOCH3), 3.79 (3H, s, OCH3), 3.86 (3H, s,
OCH3), 3.94 (3H, s, OCH3), 4.05±4.21 (2H, m, Hg0),
4.54, (1H, dd, J11.9 and 3.5 Hz, Hb0) 4.90 (1H, d, J
9.6 Hz, Ha0), 5.89±6.04 (2H, m, Ha), 6.91±7.09 (7H,
arom.). dC (300 MHz; CDCl3): 21.4, 21.5, 22.1, 22.6, 23.2
(CH3, OCOCH3), 56.7 (OCH3), 64.5 (g0), 72.8 (a), 73.0 (a),
83.3 (b0), 85.0 (a0), 110.4, 111.1, 111.9, 119.3, 119.8, 120.1,
120.5, 123.3, 133.1, 133.6, 137.6, 138.9, 140.5, 146.8,
147.2, 151.9, 153.1 (arom. C), 169.5, 171.0, 171.4
(CvO). The assignments were done with the aid of
HSQC-TOCSY spectra.
3.3. Synthesis of dibenzodioxocin compound 17
3.3.1. Dehydrodiacetovanillone. Acetovanillone (20 g;
120 mmol) and horseradish peroxidase (40 mg) were
dissolved in ethanol (200 ml)±water (200 ml). Hydrogen
peroxide (8.8 ml; 8 M; 70 mmol) diluted to 60 ml ethanol±
water was added slowly to the solution with syringe pump
during 6 h 20 min. The mixture was then stirred for an addi-
tional 10 h. The precipitate was ®ltered and washed with
water and acetone. The yield of dehydrodiacetovanillone
was 55% (10.78 g; 32.7 mmol).
3.4. Carbohydrate gel experiment
Carrageenan (0.13 g, Type 1, Commercial Grade, [9000-07-1],
Sigma) was added to the buffer (48 ml, pH3.5) and the
mixture was stirred at 508C untill clear homogeneous 0.25%
gel was formed. The solution was cooled to 308C and
apocynol (0.17 g; 1 mmol) in acetone (2 ml) and horse
radish peroxidase (10 mg) in buffer (1 ml; pH3.5) were
added. The gel was allowed to cool. Coniferyl alcohol
(0.18 g; 1 mmol) and hydrogen peroxide (0.65 ml;
0.5 mmol) in acetone (1 ml)±buffer (19 ml) were added on
top of the gel. The gel was quite soft and added solution
started to immediately diffuse into the gel. The gel was left
in room temperature for 20 h. The whole mixture was
extracted with ethyl acetate, the organic layer dried with
NaSO4, evaporated and acetylated. The main products
were b-O-4-, b-5- and b-b-dimers (4±6) from coniferyl
alcohol and two unexpected cross products (13 and 14)
from coniferyl alcohol and apocynol. Some b-O-4-cross-
coupling product 11 was also detected. Both erythro- and
threo-isomers of 14 were formed. Yields were not repro-
ducible, but more compound 13 than compound 14 was
formed each time. The yields of the dimers of coniferyl
alcohol were similar to those observed in homogeneous
solutions.17
3.3.2. Dehydrodiapocynol (15). Dehydrodiacetovanillone
(2 g; 6.1 mmol) was added to the ethanol (30 ml)±water
(30 ml) solution. The starting material dissolved after
NaBH4 (0.45 g; 12 mmol) was added. The mixture was
stirred at room temperature for 6 days and then neutralized
with 1N HCl. Water was then added and the whole mixture
was extracted with ethyl acetate, dried with NaSO4 and the
solvent was evaporated. The crude yield of compound 15
was 1.8 g (88%). The synthesis made according to earlier
published method24 gave poor yield but by using ethanol±
water as a solvent instead of 1N sodium hydroxide, the yield
was raised and the product was pure according to NMR-
spectra. Reaction time was not optimized, a shorter time is
probably suf®cient.
Compound 15: dH (200 MHz; CDCl3): 1.49 (6H, d, J
6.4 Hz, CH3), 3.89 (6H, s, OCH3), 4.83 (2H, q, J6.4 Hz,
CHCH3), 6.35 (arom. OH), 6.89 (2H, d, J2.2 Hz, arom.),
6.95 (2H, d, J2.2 Hz, arom.). dC (200 MHz; CDCl3): 25.6
(CH3), 56.7 (OCH3), 70.8 (CH), 108.2,121.0, 124.8, 138.5,
142.5, 148.0 (arom. C).
3.3.3. Dibenzodioxocin compound (17). Dehydrodiapo-
cynol (0.33 g; 1 mmol) and horseradish peroxidase
(20 mg; 529 U/mg) were dissolved in acetone (1 ml)Ð
buffer (4 ml; pH6). Coniferyl alcohol (0.36 g, 2 mmol)
dissolved in acetone (8 ml)Ðbuffer (12 ml; pH6) and
hydrogen peroxide (1.25 ml; 1 mmol) dissolved in 20 ml
of buffer (pH6) were added slowly with the aid of syringe
pump to the mixture of HRP and dehydrodiapocynol over
6 h. During the ®rst 10 min the addition was faster and 3 ml
of both coniferyl alcohol and hydrogen peroxide were added
during that time. The mixture was stirred at room tempera-
ture for additional 14 h and then extracted with ethyl
acetate, dried with NaSO4 and the solvent evaporated. The
whole product was acetylated for puri®cation and analysis.
Dibenzodioxocin tetra-acetate was separated by ¯ash chro-
matography (eluent: ethyl acetate/hexane 9:11) and the
yield of trans-isomer was 0.13 g (25%). The cis-isomer
was not detected. For HRMS and NMR analysis further
puri®cation was done by preparative HPLC (same eluent
as in ¯ash chromatography).
Triacetate of trimer 13: m/z 634 (M1, 14%), 472 (2), 440
(13), 424 (13), 338 (33), 324 (20), 168 (37), 150 (100), 135
(32). (Found: M1, 634.2404. C35H38O11 requires M,
634.2414). dH (300 MHz; CDCl3): 1.51 (3H, d, J6.5 Hz,
CH3), 2.09 (3H, s, OCOCH3), 2.34 (3H, s, arom. OCOCH3),
2.36 (3H, s, arom. OCOCH3), 3.78±3.91 (1H, m, Hb), 3.85
(3H, s, OCH3), 3.88 (3H, s, OCH3), 3.95 (3H, s, OCH3), 3.99
(1H, ddd, J1.2, 6.6 and 12.4 Hz, Hg01), 4.11 (1H, ddd,
J1.3, 5.7 and 12.3 Hz, Hg02), 4.33 (1H, dd, J7.8 and
11.2 Hz, Hg1) 4.49 (1H, dd, J5.4 and 11.2 Hz, Hg2),
4.54 (1H, q, J6.4 Hz, CHCH3), 5.57 (1H, d, J6.8 Hz,
Ha), 6.18 (1H, dt, J5.6 and 15.9 Hz, Hb0), 6.53 (1H, d,
J15.9 Hz, Ha0), 6.78±7.11 (8H, arom.). dC (300 MHz;
CDCl3): 21.4 (OCOCH3), 25.0 (CH3), 51.2 (b), 56.6
(OCH3), 66.1 (g), 69.9 (g0), 88.7 (a), 125.0 (a0), 132.8
(b0), 110.5±151.8 (arom. C), 169.5, 171.4 (CvO). The
assignments were done with the aid of HSQC-TOCSY
spectra.
Tetra-acetate of erythro-isomer of compound 14: m/z 694
(M1, 1%), 662 (1), 323 (3), 221 (6), 192 (10), 150 (100), 135
(36). (Found: M1, 694.2615. C37H42O13 requires M,
694.2625). dH (200 MHz; CDCl3): 1.51 (3H, d, J6.4 Hz,
CH3), 2.05 (3H, s, OCOCH3), 2.13 (3H, s, OCOCH3), 2.34
(3H, s, arom. OCOCH3), 2.36 (3H, s, arom. OCOCH3), 3.83
Tetra-acetate of compound 17: m/z 680 (M1, 6%), 620 (8),
560 (70), 500 (53), 458 (65), 341 (14), 296 (100). (Found:
M1, 680.2451. C36H40O13 requires M, 680.2469). dH
(300 MHz; CDCl3): 1.63 (6H, t, J6.9 Hz, CH3), 1.99
(3H, d, J2.7 Hz, OCOCH3), 2.11±2.17 (6H, m, OCOCH3),