Effect of substitution on the 2+2 cycloaddition reaction of phenylpropanoids

Article abstract of DOI:10.1006/bioo.1999.1143

Formation of the cyclobutane products truxillic and truxinic acid within the plant cell wall could occur by photocycloaddition or possibly by enzyme- mediated radical coupling. Model precursors were synthesized and the mode of formation of the cyclobutane

Full text of DOI:10.1006/bioo.1999.1143

Bioorganic Chemistry 27, 339–350 (1999)
Article ID bioo.1999.1143, available online at http://www.idealibrary.com on
Effect of Substitution on the 2ϩ2 Cycloaddition Reaction of
Phenylpropanoids
Wendy R. Russell,* A. Bryan Hanley,† Mark J. Burkitt,‡ and Andrew Chesson*
*Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, United Kingdom;
†Central Science Laboratory, Norwich Research Park, Norwich NR4 7UQ, United Kingdom;
and ‡Molecular Mechanisms Group, Gray Laboratory Cancer Research Trust, P.O. Box 100,
Mount Vernon Hospital, Northwood, Middlesex, HA6 2JR, United Kingdom
Received March 9, 1999
Formation of the cyclobutane products truxillic and truxinic acid within the plant cell wall
could occur by photocycloaddition or possibly by enzyme-mediated radical coupling. Model
precursors were synthesized and the mode of formation of the cyclobutane products was
investigated. Modeling studies suggested that all the model compounds with the exception of
the 2-hydroxylated diester contained the correct geometry for photocyclization and this was
confirmed by irradiation. Delocalization of the unpaired electron was measured by ESR and
the photochemical reaction rates were compared with the partial atomic orbital population of
the singly occupied molecular orbital. It was observed that the 3-hydroxylated derivative had
almost no delocalization to C8 and the lowest reaction rate. The rate of reaction increased with
increasing extent of methoxylation. Incubation with silver oxide or with peroxidase failed to
induce intramolecular dimerization. These results suggest that the biological mode of formation
of truxillic and truxinic acid is the most likely via light-catalyzed 2ϩ2 cycloaddition.
᭧ 1999
Academic Press
INTRODUCTION
Photochemical cyclodimerization of crystalline cinnamic acid (1) (Fig. 1) has been
extensively studied and the efficiency of the reaction in the solid state has been shown
to be determined entirely by the dimensions of the crystal lattice and hence by the
separation and relative orientation of the exocyclic double bonds (1). The upper limit
˚
for the separation of the double bonds is of the order of 4.0 A and the relative
orientation must be parallel (2). This has prompted interest in the mechanism of
formation of the widely reported cyclobutane derivatives truxillic and truxinic acids
(7 and 8) found within and contributing to the cross-linking of the polymer network
of the plant cell wall (3,4).
Because of the strict steric requirements for photocyclization, irradiation of substi-
tuted cinnamic acids in solution produces only insignificant amounts of cycloaddition
products and therefore topochemical assistance is required in order to probe the
mechanism (5–7). It has been demonstrated that by varying the length of the methylene
˚
spacer group linking two cinnamic acid moieties, optimum separation of Ͻ4.0 A
with parallel alignment of the ␣–␤ unsaturated double bond can be satisfied by the
339
0045-2068/99 $30.00
Copyright ᭧ 1999 by Academic Press
All rights of reproduction in any form reserved.

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RUSSELL ET AL.

2ϩ2 CYCLOADDITION OF PHENYLPROPANOIDS
341
preparation of dicinnamyl esters of propane 1,3-diol and butane 1,4-diol (8). In the
case of a radical coupling mechanism, similar molecular separation and alignment of
the exocyclic double bonds are required; however, in addition a 4-hydroxyl substituent
is a prerequisite as it facilitates formation of the quinone methide and concomitant
stabilization of the ␤ alkenyl radical.
In this study butane 1,4-diol diesters have been used as model precursors of 7 and
8. These were prepared by reacting butane-1,4-diol with 4-hydroxycinnamic acid (2),
4-hydroxy-3-methoxycinnamic acid (5), and 4-hydroxy-3, 5-dimethoxycinnamic acid
(6). Although not naturally occurring within the plant cell wall, the 2- and 3-hydroxyl-
ated derivatives (4 and 3) have also been included. Of these latter two, delocalization
of the unpaired electron to C8, facilitating cyclodimerization involving a ␤ alkenyl
radical, can be achieved only by the 2-hydroxycinnamate diester (4a). The 3-hydroxyl-
ated compound (3a) is unable to undergo cyclization by this mechanism but may
photodimerize. Competing reactions, for example, the intramolecular bond formation
found in coumarin biosynthesis (9) and also the steric effect of substitution at position
2, can be investigated by the 2-hydroxylated compound (4a). Another reaction compet-
ing with photocycloaddition is E–Z isomerization (10). However, since the favorable
conformation for high quantum efficiency of cyclobutane formation is when both
cinnamic moieties are in the E conformation, removal of the E–E confomer by
dimerization should therefore shift the equilibrium in favor of further production of
the E isomer (11).
The distribution of the unpaired electron has important consequences not only for
the enzymatic formation of the cyclobutane derivative but also for the alternative
mechanism viz. the photocycloaddition reaction (12). In general photochemical reac-
tivity can be described reasonably on the basis of the partial atomic orbital population
on the SOMO (singly occupied molecular orbital) (13) and it has been demonstrated
that cycloaddition by direct irradiation can involve both the triplet (t1) and the singlet
(s1) excited states (14). It should therefore be possible to correlate the substituent
effect on reactivity for cycloaddition on the basis of unpaired electron density, as high
efficiency is observed with high electron density of the lowest ␲ → ␲* excited state.
In this investigation we sought to determine the effects of ring substitution on the
efficiency of photodimerization of the model compounds and to correlate this with
unpaired electron spin density at the exocyclic double bond positions. Finally, by
determining which mechanism is operating and by correlating that with the steric and
electronic requirements, this study is designed to shed light on the likely mechanism
of formation of truxillic and truxinic acids in vivo and thereby to contribute to the
understanding of their role within the cell wall structure.
FIG. 1. Structures of cinnamic acid 1, 4-hydroxycinnamic acid 2, 3-hydroxycinnamic acid 3, 2-
hydroxycinnamic acid 4, 4-hydroxy-3-methoxycinnamic acid 5, and 3,5-dimethoxy-4-hydroxycinnamic
acid 6 and butane-1,4-diol diesters containing 4-hydroxycinnamic acid 2a (R3 ϭ OH; R1, R2, and
R4 ϭ H), 3-hydroxycinnamic acid 3a (R2 ϭ OH; R1, R3, and R4 ϭ H), 2-hydroxycinnamic acid 4a
(R1 ϭ OH; R2, R3, and R4 ϭ H), 4-hydroxy-3-methoxycinnamic acid 5a (R3 ϭ OH; R2 ϭ OCH3; R1
and R4 ϭ H), and 3,5-dimethoxy-4-hydroxycinnamic acid 6a (R3 ϭ OH; R1 ϭ H; R2 and R4 ϭ OCH3)
and truxillic acid 7 and truxinic acid 8.

342
RUSSELL ET AL.
RESULTS AND DISCUSSION
The distances between C7–C7Ј and C8–C8Ј for the energy-minimized structures
of the various diesters calculated using the program Cerius2 are shown in Table 1.
˚
In each case the distances are less than 4.0 A with the exception of the 2-hydroxycinna-
mate diester (4a). In addition modeling suggests that the parallel orientation of the
exocyclic double bonds can be achieved on minimization in all cases except that of 4a.
ESR spectra were recorded following the one-electron oxidation of the substituted
cinnamic acids (Fig. 2) and the hyperfine coupling constants were determined by
computer simulation (Table 2, Fig. 2). Applying the McConnell (15) relationship with
a Q value of 22.5 Gauss (considered the best estimate, assuming the value to be
constant for all C–H bonds (16)), values were estimated for the unpaired electron
spin density (Fig. 3). Polarization was accounted for by an Austin Model 1 (AM1)
calculation on the energy-minimized structures (12). For all but 3-hydroxycinnamic
acid (3) a significant amount of unpaired electron density was found on C8, indicating
the possible participation of this position in radical-coupling reactions.
The predictions made on the basis of modeling were tested experimentally by
˚
irradiation at 3500 A and the rate constants for the reaction calculated (Table 3, Fig.
4). As expected no product was obtained for the 2-hydroxy-substituted model (4a),
although some E–Z isomerization was observed. All other diesters gave two products
in varying concentrations (Table 4, A and B). It was not possible to characterize the
exact stereochemistry of the products obtained, due to the attachment of the methylene
side-chain (17). In an attempt to estimate the relative orientation of the cyclobutane
substituents, dihedral angles were estimated from the observed cyclobutane proton
coupling constants via the Karplus equation and these were compared to values
obtained computationally from energy-minimized structures. The models constructed
by head-to-tail dimerization, with the tetramethylene chain extending from the same
side of the cyclobutane ring, generally gave minimized structures with the lowest
energy. The structures containing the E–E isomers (dihedral angles of around 20Њ
and 120Њ) and the E–Z isomers (dihedral angles of around 124Њ and 125Њ) gave the
best comparison with the cycloaddition products A and B obtained on irradiation
(Table 4).
TABLE 1
˚
Distance (A) between Positions C7–7Ј and C8–8Ј Calculated for Energy-Minimized Structures of
the Cinnamate Diesters of (a) Cinnamic Acid 1, (b) 4-Hydroxycinnamic Acid 2, (c)
3-Hydroxycinnamic Acid 3, (d) 2-Hydroxycinnamic Acid 4, (e) 4-Hydroxy-3-Methoxycinnamic
Acid 5, and (f) 3,5-Dimethoxy-4-hydroxycinnamic Acid 6
C7–7Ј
C8–8Ј
a
b
c
d
e
f
3.550
3.564
3.544
4.127
3.596
3.614
3.552
3.561
3.564
3.390
3.561
3.590

2ϩ2 CYCLOADDITION OF PHENYLPROPANOIDS
343
FIG. 2. ESR spectra and computer simulation of radicals obtained from (a) 4-hydroxycinnamic acid
2, (b) 3-hydroxycinnamic acid 3, (c) 2-hydroxycinnamic acid 4, (d) 4-hydroxy-3-methoxycinnamic acid
5, and (e) 3,5-dimethoxy-4-hydroxycinnamic acid 6.

344
RUSSELL ET AL.
TABLE 2
ESR Hyperfine Coupling Constants (aH ) for the Radicals Generated from (a) 4-Hydroxycinnamic
Acid 2, (b) 3-Hydroxycinnamic Acid 3, (c) 2-Hydroxycinnamic Acid 4, (d) 4-Hydroxy-3-
methoxycinnamic Acid 5, and (e) 3,5-Dimethoxy-4-hydroxycinnamic Acid 6
aH(2)
aH(3)
aH(4)
aH(5)
aH(6)
aH(7)
aH(8)
aH(OCH3)
a
b
c
d
e
1.68
6.21
—
1.53
0.95
5.26
—
3.74
—
—
6.22
2.56
—
5.65
1.08
9.08
5.28
—
1.68
9.29
1.64
1.70
1.26
3.06
0.56
2.06
2.64
1.69
6.36
2.22
5.50
5.56
4.82
—
—
—
1.69
1.33
—
—
FIG. 3. Unpaired electron density calculated from ESR hyperfine splittings via the McConnell
equation for (a) 4-hydroxycinnamic acid 2, (b) 3-hydroxycinnamic acid 3, (c) 2-hydroxycinnamic acid
4, (d) 4-hydroxy-3-methoxycinnamic acid 5, and (e) 3,5-dimethoxy-4-hydroxycinnamic acid 6.

2ϩ2 CYCLOADDITION OF PHENYLPROPANOIDS
345
TABLE 3
Rate Constants (molϪ1 dm3 sϪ1) and r2 Values for the Diesters of (a) 4-Hydroxycinnamic Acid 2,
(b) 3-Hydroxycinnamic Acid 3, (c) 2-Hydroxycinnamic Acid 4, (d) 4-Hydroxy-3-
methoxycinnamic Acid 5, and (e) 3,5-Dimethoxy-4-hydroxycinnamic Acid 6
a
b
c
d
e
Rate constant
2.77 ϫ 10Ϫ4
0.81 ϫ 10Ϫ4
—
—
3.28 ϫ 10Ϫ4
7.06 ϫ 10Ϫ4
r2
0.986
0.911
0.954
0.995
Cyclization of the 4-hydroxycinnamate diester (2a) had a significantly higher rate
constant than the 3-hydroxylated model (3a) and the rate constant was further increased
by methoxyl substitution (5a and 6a) (Table 3). The limited delocalization of the
unpaired electron to C8 for the 3-hydroxylated diester may explain the low efficiency
of this reaction. The similar values obtained for spin density at C8 for the 4-hydroxyl-
ated, mono- and dimethoxylated models, however, could not explain the apparent
increase in reaction rate with increasing methoxyl substitution on the basis of spin
density at this position alone. A more likely explanation of the enhanced rate of
photodimerization with methoxyl substitution is the overall ␲ electron density ob-
served to increase with increasing methoxylation.
Incubation of all the models with peroxidase at various pH values failed to yield
FIG. 4. Graph of ln [concentration (mol dmϪ3)] against time (minutes) with best fit line (Table 2)
for the diesters of ᭡, 4-hydroxycinnamic acid 2; ⅷ, 3-hydroxycinnamic acid 3; ࡗ, 2-hydroxycinnamic
acid 4; ϫ, 4-hydroxy-3-methoxycinnamic acid 5; and ϫ, 3,5-dimethoxy-4-hydroxycinnamic acid 6.

346
RUSSELL ET AL.
TABLE 4
Chemical Shift, Coupling Constants, and Estimated Dihedral Angles for the Cyclobutane Protons
of Products A and B for the Photodimerized Diesters of (a) 4-Hydroxycinnamic Acid 2, (b)
3-Hydroxycinnamic Acid 3, (c) 2-Hydroxycinnamic Acid 4, (d) 4-Hydroxy-3-Methoxycinnamic
Acid 5, and (e) 3,5-Dimethoxy-4-Hydroxycinnamic Acid 6
Ratio
A:B
Product A
(␦ 3.0 and 3.6 ppm)
Estimated
Product B
(␦ 3.0 and 3.6 ppm)
Estimated
␸
␸
a
b
c
d
e
1.5:1.0
0.8:1.0
—
1.0:1.0
1.0:1.0
(J ϭ 10.1 and 3.8 Hz)
(J ϭ 9.8 and 3.7 Hz)
—
(J ϭ 10.1 and 3.8 Hz)
(J ϭ 10.4 and 4.0 Hz)
21Њ 123Њ
23Њ 122Њ
—
21Њ 123Њ
19Њ 124Њ
(J ϭ 6.2 and 2.4 Hz)
(J ϭ 6.6 and 2.2 Hz)
—
(J ϭ 5.8 and 1.9 Hz)
(J ϭ 6.0 and 2.3 Hz)
133Њ 115Њ
135Њ 114Њ
—
131Њ 112Њ
132Њ 115Њ
cycloaddition products. This was as expected for the 3-hydroxylated model for which
only starting material was recovered after incubation. Delocalization of the unpaired
electron to C8 is a requirement for oxidative coupling of phenylpropanoids which
was not possible in the case of the 3-hydroxylated compound (3a). The recovery of
virtually all of the 2-hydroxylated derivative (4a) and most of the 4-hydroxylated
model, normally considered substrates for peroxidase-catalyzed oxidation, was proba-
bly due to the decreased electron density on the phenolic hydroxyl, in comparison
with the methoxylated compounds (12) and their decreased ability to reduce peroxidase
compound I (18). Products of oxidative coupling, normally observed for both the
mono- and the dimethoxylated substrates, were present as characterized previously
(12). This indicates that the phenoxyl radical was generated. However, no signals
indicative of cycloaddition products were detected for either substrate.
It is possible that an enzyme-catalyzed mechanism may operate in vivo and that
the experimental conditions used in the incubations were inappropriate. Therefore,
incubation of with silver(I) oxide, a known one-electron oxidant of phenylpropanoids,
was performed. Again, there was no cyclodimer formation observed. These results
and the recent discovery of a unsubstituted cyclobutane derivative from a plant source
(19) appear to support the hypothesis that light-catalyzed cyclodimerization is the
most likely mechanism for the formation of truxillic and truxinic acids in plant cell
walls. This may argue for a role for cycloaddition products in the trophic response
of the growing plant to light.
EXPERIMENTAL
General laboratory reagents and substituted 4-hydroxycinnamic acids were from
Aldrich (UK). Evaporations were performed under reduced pressure at temperatures
less than 40ЊC. NMR spectra were recorded using a Jeol LA-300 spectrometer, fitted
with a 5-mm multinuclear, normal-geometry TH5 probe. Operating field for 1H NMR
was 300.4 MHz. Tetramethylsilane (0.03% v/v) was used as an internal reference and
samples were run with a probe temperature of 25ЊC. J values are given in Hz.
Electron spin resonance and computer simulation. Radicals were generated in a
two-stream ESR flow cell, positioned in the cavity of a Bruker E106 spectrometer,

2ϩ2 CYCLOADDITION OF PHENYLPROPANOIDS
347
via the continuous mixing of substrate (10Ϫ2 mol dmϪ3 in 50% v/v methanol) and
ammonium cerium(IV) nitrate (10Ϫ3 mol dmϪ3 in 0.25 mol dmϪ3 sulfuric acid)
solutions. Flow was maintained using a Watson–Marlow Flow 505S/RL pump placed
before the cell which produced a combined flow rate of 4.8 cm3 sϪ1. Spectra (X-
band) were recorded using the instrument settings modulation frequency 100 KHz,
center field 3479 Gauss, sweep width 40 Gauss, time constant 41 ms, sweep time 90
s, and power 20 mW and a suitable receiver gain setting (typically 6.3 ϫ 104).
Computer simulations of spectra, giving the hydrogen hyperfine coupling constants
(aH), were performed using the SIMEPR program (20), which sequentially varies all
the parameters for each radical species until a minimum in the error surface is located.
Goodness of fit was determined by visual comparison and as a minimum in the sum
of the squared residuals. The density of the unpaired electron (␳) at each carbon
atom was calculated from the hyperfine coupling constants using the McConnell
relationship, aH ϭ ␳Q, in which Q is the proportionality factor. (16).
Modeling. Theoretical values for the spin density were obtained by the AM1
method using the HyperChem molecular simulation program. Compensation for the
radical center was achieved by deleting the phenoxyl hydrogen and setting the specific
charge to zero and spin multiplicity to 2. An Unrestricted Hartree-Fock wave function
was then selected to allow for the unpaired electron. Once optimized, a single point
calculation employing AM1 was performed to give distribution of spin density. Energy
minimization calculations were performed using the Universal 1.01 force field using
the program Cerius2 developed by BIOSYM/Molecular Simulations on an SGI Indigo
2 workstation. Initially most molecular systems were minimized by the steepest-
descent method for approximately 100 steps. Conjugate gradient minimization was
Ϫ1
˚
A
then used until a convergence criteria of 0.1 kcal molϪ1
was reached.
Preparation of the methoxycarbonyloxycinnamic acids. To a solution of NaOH (3
g, 75 mmol) in water (80 cm3), cooled to Ϫ10ЊC, the respective hydroxycinnamic
acids (26 mmol) were added and vigorously stirred. ClCO2CH3 (4 g, 36 mmol) was
then added dropwise and the mixture maintained at Ϫ5ЊC for a further 4 h. The pH
was reduced to pH 5 with HCl/water (1/1) and the resulting precipitate was filtered.
Crystallization from ethanol afforded the corresponding methoxycarbonyloxycinnamic
acids as white needles. 4-Methoxy carbonyloxycinnamic acid (70%): mp 194–196ЊC,
␦H (CD3OD) 3.88 (3H, s, OCH3), 6.47 (1H, d, J ϭ 16, C(8)H), 7.23 (2H, dt, J ϭ
8.7 and 2.7, C(3,5)H), 7.64 (2H, dt, J ϭ 8.7 and 2.7, C(2,6)H), 7.66 (1H, d, J ϭ 16,
C(7)H) ppm, m/z 221. 3-Methoxy carbonyloxycinnamic acid (72%): mp 138–141ЊC,
␦H (CD3OD) 3.88 (3H, s, OCH3), 6.51 (1H, d, J ϭ 16, C(8)H), 7.22 (1H, ddd, J ϭ
8 and 2.2, C(4)H), 7.44 (1H, t, J ϭ 8, C(5)H), 7.44 (1H, t, J ϭ 2.2, C(2)H), 7.51
(1H, ddd, J ϭ 8 and 2.2, C(6)H), 7.65 (1H, d, J ϭ 16 C(7)H) ppm, m/z 221. 2-
Methoxy carbonyloxycinnamic acid (68%): mp 174–176ЊC, ␦H (CD3OD) 3.89 (3H,
s, OCH3), 6.52 (1H, d, J ϭ 16, C(8)H), 7.25 (1H, dd, J ϭ 8 and 1.7 C(6)H), 7.32
(1H, dt, J ϭ 8 and 1.7, C(5)H), 7.45 (1H, dt, J ϭ 8 and 1.7, C(4)H), 7.74 (1H, d,
J ϭ 16, C(7)H), 7.8 (1H, dd, J ϭ 8 and 1.7, C(3)H) ppm, m/z 221. 3-Methoxy-4-
methoxy carbonyloxycinnamic acid (50%): mp 170–173ЊC, ␦H ((CD3)2CO) 3.87 (3H,
s, OCH3), 3.94 (3H, s, CO2CH3), 6.58 (1H, d, J ϭ 16, C(8)H), 7.22 (1H, d, J ϭ 8.2,
C(2)H), 7.30 (1H, dd, J ϭ 8.2 and 1.8, C(6)H), 7.51 (1H, d, J ϭ 1.8, C(5)H), 7.67
(1H, d, J ϭ 16, C(7)H) ppm, m/z 251. 3,5-Dimethoxy-4-methoxy carbonyloxycinnamic

348
RUSSELL ET AL.
acid (70%): mp 156–159ЊC, ␦H ((CD3)2CO) 3.86 (3H, s, OCH3), 6.60 (1H, d, J ϭ
15.8, C(8)H), 7.13 (2H, s, C(2,6)H), 7.65 (1H, d, J ϭ 15.8, C(7)H) ppm, m/z 281.
Preparation of methoxycarbonyloxy protected substituted butane-1,4-dicinna-
mates. 4-Methoxycarbonyloxycinnamic acid (1.11 g, 5 mmol) was dissolved in tolu-
ene (150 cm3) under reflux. SOCl2 (0.41 cm3, 5.5 mmol) was added and the mixture
maintained under reflux for 90 min. The solution was cooled and the solvent removed
under reduced pressure to yield a white solid. This product was suspended in pyridine
(75 cm3) under nitrogen, and butane-1,4-diol (0.2 cm3, 2 mmol) was added. This
mixture was left with stirring at room temperature for 24 h. The pyridine was removed
by coevaporation with toluene under reduced pressure and the product redissolved
in CHCl3. This was extracted with NaHCO3 (3% w/v in water) and the organic layer
dried over anhydrous Na2SO4. The solvent was then removed in vacuo. Crystallization
from chloroform afforded butane 1,4-di-(4-methoxycarbonyloxy) cinnamate as white
crystals (35%): mp 171–173ЊC, ␦H (CDCl3) 1.85 (4H, br signal, C(CH2)2C), 3.91
(6H, s, CO2CH3), 4.28 (4H, br signal, OCH2), 6.4, (2H, d, J-16, C(8)H), 7.21 (4H
dt, J ϭ 8.8 and 1.9, C(2,6)H), 7.54 (4H, dt, J ϭ 8.8 and 1.9, C(3,5)H), 7.66 (2H, d,
J ϭ 16, C(7)H) ppm, m/z 497. All other methoxycarbonyloxy-protected butane 1,4-
diol dicinnamates were prepared by the same method. Butane 1,4-di-(3-methoxycar-
bonyloxy)cinnamate was crystallized from methanol as white plates (30%): mp 109–
111ЊC, ␦H (CDCl3) 1.84 (4H, br signal, C(CH2)2C), 3.91 (6H, s, CO2CH3), 4.28 (4H,
br signal, OCH2), 6.44, (2H d, J ϭ 16, C(8)H), 7.2 (2H, t, J ϭ 4.6, C(5)H), 7.3 (2H,
d, J ϭ 1.2, C(2)H), 7.4 (4H, dt, J ϭ 4.6 and 1.2, C(4,6)H), 7.65 (1H, d, J ϭ 16, C(7)H)
ppm, m/z 497. Butane 1,4-di-(2-methoxycarbonyloxy)cinnamate was crystallized from
a large volume of methanol as off-white plates (49%): mp 119–122ЊC, ␦H (CDCl3)
1.8 (4H, br signal, C(CH2)2C), 3.90 (6H, s, CO2CH3), 4.3 (4H, br signal, OCH2),
6.48, (2H, d, J ϭ 16, C(8)H), 7.22 (2H, dd, J ϭ 8.0 and 1.0, C(6)H), 7.28 (2H, dt,
J ϭ 8.0 and 1.0, C(5)H), 7.41 (2H, dt, J ϭ 8.0 and 1.0, C(4)H), 7.63 (2H, dd, J ϭ
8.0 and 1.0, C(3)H), 7.65 (2H, d, J ϭ 16, C(7)H) ppm, m/z 497. Butane 1,4-di-(4-
methoxycarbonyloxy-3-methoxy)cinnamate was obtained as an oil (65%): ␦H
((CD3)2CO) 1.83 (4H, br signal, C(CH2)2C), 3.88 (6H, s, OCH3), 3.90 (6H, s, CO2CH3),
4.26 (4H, br signal, OCH2), 6.75 (2H, d, J ϭ 16, C(8)H), 7.28 (2H, d, J ϭ 8.01,
C(6)H), 7.34 (2H, d, J ϭ 8.01, C(5)H), 7.58 (2H, s, C(2)H), 7.69 (2H, d, J ϭ 16,
C(8)H) ppm. Butane 1,4-di-(4-methoxycarbonyloxy-3,5-dimethoxy)cinnamate was
obtained as a brown solid (60%): mp 87–88ЊC, ␦H ((CD3)2CO) 1.77 (4H, br signal,
C(CH2)2C), 3.81 (6H, s, OCH3), 3.84 (6H, s, CO2CH3), 4.26 (4H, br signal, OCH2),
6.74 (2H, d, J ϭ 16, C(8)H), 7.26 (4H, s, C(2,6)H), 7.62 (2H, d, J ϭ 16, C(7)H) ppm.
Preparation of the substituted butane 1,4-dicinnamates. Butane 1,4-di-(4-methoxy-
carbonyloxy)cinnamate (0.65 g, 1.3 mmol) was suspended in ethanol (288 cm3).
Ammonia solution (112 cm3, 35% w/v in water) was added and the reaction left for
16 h in which time the solution became clear. The solution was then neutralized by
dropwise addition of acetic acid and the solvent removed under reduced pressure.
The crude product was then dissolved in ethyl acetate and extracted with NaHCO3
(3% w/v in water). The organic layer was then dried over anhydrous Na2SO4 and
filtered and the solvent evaporated under reduced pressure. Crystallization from metha-
nol afforded butane 1,4-di-(4-hydroxy)cinnamate as white crystals (71%): mp 176–
177ЊC, ␦H (CD3OD) 1.83 (4H, br signal, C(CH2)2C), 4.23 (4H, br signal, OCH2),

2ϩ2 CYCLOADDITION OF PHENYLPROPANOIDS
349
6.32, (2H, d, J ϭ 16, C(7)H), 6.78 (4H, d, J ϭ 8.69, C(2,6)H), 7.44 (4H, d, J ϭ 8.6,
C(3,5)H), 7.60 (2H, d, J ϭ 16, C(8)H) ppm, m/z 381. Calcd for C22H22O6: C, 69.10;
H, 5.80%. Found: C, 68.65; H, 5.68%. All other substituted butane 1,4-dicinnamates
were produced by the same method. Butane 1,4-di-(3-hydroxy)cinnamate was crystal-
lized from methanol as brown crystals (98%): mp 172–174ЊC, ␦H (CD3OD) 1.84 (4H,
br signal, C(CH2)2C), 4.26 (4H, br signal, OCH2), 6.45, (2H, d, J-16, C(8)H), 6.82
(2H, dd, J ϭ 7.8 and 1.9, C(6)H), 6.99 (2H, d, J ϭ 1.9, C(2)H), 7.03 (2H, dd, J ϭ
7.9 and 1.9, C(4)H), 7.2 (2H, t, J ϭ 7.8, C(5)H) 7.59 (2H, d, J ϭ 16, C(7)H) ppm,
m/z 381. Calcd for C22H22O6: C, 69.10; H, 5.80%. Found: C, 69.14; H, 5.85%. Butane
1,4-di-(2-hydroxy)cinnamate was crystallized from methanol as light brown crystals
(89%): mp 200–202ЊC, ␦H (CD3OD) 1.85 (4H, br signal, C(CH2)2C), 4.25 (4H, br
signal, OCH2), 6.60, (2H, d, J ϭ 16, C(8)H), 6.82 (2H, dt, J ϭ 8.1 and 1.4, C(5)H),
6.83 (2H, dd, J ϭ 8.1 and 1.4, C(6)H), 7.28 (2H, dt, J ϭ 8.1 and 1.4, C(4)H), 7.48
(2H, dd, J ϭ 8.1 and 1.4, C(3)H), 7.65 (2H, d, J ϭ 16, C(7)H) ppm, m/z 381. Calcd
for C22H22O6: C, 69.10; H, 5.80%. Found: C, 68.45; H, 6.19%. Butane 1,4-di-(3-
methoxy-4-hydroxy)cinnamate was crystallized from methanol/water as a yellow-
brown crystalline solid (36%): mp 157–159ЊC, ␦H ((CD3)2CO) 1.82 (4H, br signal,
C(CH2)2C), 3.91 (6H, s, OCH3), 4.23 (4H, br signal, OCH2), 6.42 (2H, d, J ϭ 15.8,
C(8)H), 6.87 (2H, d, J ϭ 8.2, C(5)H), 7.15 (2H, d, J ϭ 8.2 and 1.88, C(6)H), 7.35
(2H, d, J ϭ 1.88, C(2)H), 7.61 (2H, d, J ϭ 15.8, C(7)H) ppm, m/z 441. Calcd for
C24H26O8: C, 65.15; H, 5.92%. Found: C, 64.71; H, 6.04%. Butane 1,4-di-(3,5-
dimethoxy-4-hydroxy)cinnamate was crystallized from methanol/water as yellow
crystals (34%): mp 155–156ЊC, ␦H ((CD3)2CO) 1.83 (4H, br signal, C(CH2)2C), 3.89
(6H, s, OCH3), 4.23 (4H, br signal, OCH2), 6.45 (2H, d, J ϭ 16, C(8)H), 7.03 (4H,
s, C(2,6)H), 7.60 (2H, d, J ϭ 16, C(7)H) ppm, m/z 501. Calcd for C26H30O10: C,
62.14; H, 6.02%. Found: C, 61.45; H, 6.12%.
Photodimerization. De-aerated solutions in (CD3)2CO were irradiated in a Hanova
3
˚
irradiation cell at 3500 A. Samples (0.5 cm ) were taken at 2.5, 5.0, 10.0, 30.0, and
60.0 min and the products observed immediately by NMR.
Reaction with peroxidase. The substituted butane 1,4-dicinnamates (64 mmol) were
dissolved in buffer/methanol (1:1) at pH 5, pH 7, and pH 9. Solutions were individually
added with H2O2 (30 ␮l, 30%) in the same buffer to peroxidase (Type I ex horseradish;
Sigma) (1 mg, 116 U) at a rate of 1 cm3 hϪ1 under nitrogen and with light excluded.
After 24 h the products were extracted into ethyl acetate. The organic layer was then
washed with water and dried over anhydrous Na2SO4 and the solvent was removed
by evaporation at reduced pressure.
Reaction with silver(I) oxide. The substituted butane 1,4-dicinnamates (32 mmol)
were dissolved in CH2Cl2 (25 cm3) under nitrogen and light was excluded. Ag2O (1.5
eq) was added. After 24 h the product was isolated by filtration through a bed of
magnesium sulfate and removal of the solvent by evaporation at reduced pressure.
ACKNOWLEDGMENTS
Financial support for this work was provided by the Scottish Office, Agriculture, Environment and
Fisheries Department. The authors are grateful to David R. Duling (Laboratory of Molecular Biophysics,
National Institute of Environmental Health Sciences, NC) for the gift of the ESR simulation software.

350
RUSSELL ET AL.
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