Low molecular weight lignin suppresses activation of NF-κB and HIV-1 promoter

Article abstract of DOI:10.1016/j.bmc.2007.11.041

Human immunodeficiency virus type 1 (HIV-1) is a cytopathic retrovirus and the primary etiological agent of acquired immunodeficiency syndrome (AIDS) and related disorders. In cells chronically infected with HIV-1, nuclear factor-κB (NF-κB) activation by external stimuli such as tumor necrosis factor α (TNFα) augments replication of HIV-1. NF-κB involves in many diseases such as inflammation, cancer, and Crohn's disease. In this paper, we exhibit that (i) HIV-1gene expression was inhibited by lignin, (ii) fraction of small molecular mass in HBS lignin (less than 0.5 kDa) had stronger inhibitory effects than large molecular mass (more than 1.3 kDa), (iii) lignin also inhibited activation of NF-κB induced by TNFα, (iv) among six lignin dimer-like compounds, compound 6 containing β-5 bond has more potent inhibitory activity than compounds 1, 2, 3, 4 and 5, which contain β-1, β-O-4, 5-5, or β-β structural units. These results suggested that small molecules of lignin inhibit HIV-1 replication through suppression of HIV-1 transcription from LTR including activation via NF-κB. Low molecular lignin may be a beneficial material or drug leads as a new class for AIDS and NF-κB-related diseases.

Full text of DOI:10.1016/j.bmc.2007.11.041

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2645–2650
Low molecular weight lignin suppresses activation
of NF-jB and HIV-1 promoter
Shinya Mitsuhashi,a Takao Kishimoto,a Yasumitsu Uraki,a
Takashi Okamotob and Makoto Ubukataa,*
aDivision of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
bDepartment of Molecular and Cellular Biology, Nagoya City University Graduate School of Medical Sciences,
Nagoya 467-8601, Japan
Received 25 July 2007; revised 13 November 2007; accepted 15 November 2007
Available online 21 November 2007
Abstract—Human immunodeficiency virus type 1 (HIV-1) is a cytopathic retrovirus and the primary etiological agent of acquired
immunodeficiency syndrome (AIDS) and related disorders. In cells chronically infected with HIV-1, nuclear factor-jB (NF-jB) acti-
vation by external stimuli such as tumor necrosis factor a (TNFa) augments replication of HIV-1. NF-jB involves in many diseases
such as inflammation, cancer, and Crohn’s disease. In this paper, we exhibit that (i) HIV-1gene expression was inhibited by lignin,
(ii) fraction of small molecular mass in HBS lignin (less than 0.5 kDa) had stronger inhibitory effects than large molecular mass
(more than 1.3 kDa), (iii) lignin also inhibited activation of NF-jB induced by TNFa, (iv) among six lignin dimer-like compounds,
compound 6 containing b-5 bond has more potent inhibitory activity than compounds 1, 2, 3, 4 and 5, which contain b-1, b-O-4, 5-5,
or b–b structural units. These results suggested that small molecules of lignin inhibit HIV-1 replication through suppression of HIV-
1 transcription from LTR including activation via NF-jB. Low molecular lignin may be a beneficial material or drug leads as a new
class for AIDS and NF-jB-related diseases.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
transcription from HIV-1 provirus is conceived to be
crucial for viral replication since amplification of the vir-
al genetic information is attainable only through tran-
scription. HIV-1 transcription is directed by the
promoter located in the 50-long terminal repeat (LTR)
of the integrated provirus. In cells chronically infected
with HIV-1, activation of nuclear factor-jB (NF-jB)
by external stimuli such as tumor necrosis factor a
(TNF-a) and its binding to LTR triggers the initiation
of transcription of viral genes, which results in explosive
HIV-1 replication.4–6 It is expected that inhibitor of
HIV-1 transcription could be used for chemotherapy.
Lignin is a polyphenolic material arising from oxidative
polymerization of three phenylpropanoid monomers,
p-coumaryl, coniferyl, and sinapyl alcohols (Fig. 1),
and are amorphous cell wall polymer is covalently
bound to cellulose and hemicelluloses.
Human immunodeficiency virus type 1 (HIV-1) is a
cytopathic retrovirus and the primary etiological agent
of acquired immunodeficiency syndrome (AIDS) and re-
lated disorders. The recent progress in combination
therapy against viral reverse transcriptase and protease
has achieved considerable reduction of the viral load
in HIV-1-infected individuals and significant improve-
ment in survival, however, chemotherapy could not ter-
minate latent infection, and HIV-1 replicates
continuously, even during the prolonged asymptomatic
period between primary infection and the development
of AIDS.1–3 To control HIV-1 replication from latently
infected cells, it is important to search for new drugs.
Among the various steps of viral life cycle, the step of
Abbreviations: AIDS, acquired immunodeficiency syndrome; DHP,
synthetic lignin-like polymer; HBS lignin, High-boiling solvent lignin;
HIV-1, human immunodeficiency virus type 1; LTR, 50-long terminal
repeat; NF-jB, nuclear factor-jB; TNF-a, tumor necrosis factor-a.
Keywords: HBS; HIV-1; Lignin; LTR; NF-jB; TNF-a.
The structural elements comprising lignin are linked by
various species of carbon-carbon and ether bonds.7 Lig-
nin extracted from natural products has anti-HIV activ-
ities toward cultured cell.8–11 The synthetic lignin-like
polymer (DHP) also has anti-HIV effect in vivo. DHP
suppresses the absorption of HIV-1 onto the cultured
*
Corresponding author. Tel./fax: +81 11 706 3638; e-mail: m-ub@
for.agr.hokudai.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.11.041

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S. Mitsuhashi et al. / Bioorg. Med. Chem. 16 (2008) 2645–2650
γCH2OH
CH2OH
1.25
CH2OH
β
α
1
1
0.75
5
OMe
MeO
OMe
4
0.5
0.25
0
OH
-Coumaryl alcohol
Figure 1. Structures of lignin monomer.
OH
OH
p
Coniferyl alcohol
Sinapyl alcohol
1
2
3
4
5
cell surface, and lignin and DHP inhibit activity of HIV-
1 protease in vitro. However, mechanism of inhibition of
lignin on HIV-1 replication is still obscure. Moreover,
there is no information about relationship between
structure of lignin and its inhibitory activity.
Figure 2. Suppression of HIV-1 gene expression by lignin.
2.2. Distribution of molecular weights of HBS lignin
It is thought that the average molecular weight of lignin
considerably differs among extraction methods. To
investigate relationship between molecular weight and
anti-HIV effect, HBS lignins were fractionated between
ether-based Soxhlet extracts and insoluble residue, and
their average molecular weights were determined by size
exclusion chromatography (Table 1). The average
molecular weights of A- and B-HBS lignins were smaller
than those of lignin alkali, and those of ether soluble
fraction were smaller than those of ether insoluble frac-
tion. The number-average molecular weights of ether
soluble fractions of A- and B-HBS lignins were 390
and 470, respectively. Thus, these results suggested that
main components of ether soluble fraction are dimer
and trimer lignins.
In this paper, we exhibit that (i) HIV-1gene expression
was inhibited by lignin, (ii) fraction of small molecular
mass in HBS lignin12 (less than 0.5 kDa) had stronger
inhibitory effects than large molecular mass (more than
1.3 kDa), (iii) lignin also inhibited activation of NF-jB
induced by TNFa, (iv) among six lignin dimer-like com-
pounds, compound 6 containing b-5 structure has more
strong inhibitory activity than the other compounds,
which contain b-1, b-O-4, 5-5, or b–b structures. These
results suggested that small molecules of lignin inhibit
HIV-1 replication through suppression of HIV-1 tran-
scription from LTR including activation of NF-jB. This
paper is the first report about the mechanism of inhibi-
tory effect of lignin on HIV-1 replication, and demon-
strating for the first time about relationship between
structure of lignin and its inhibitory activity.
2.3. Relationship between molecular weight and effect on
HIV-1 gene expression
As shown in Table 2, the 50% inhibitory concentrations
(IC50) of ether soluble fraction of A- and B-HBS lignins
for HIV-1gene expression were 25 and 19 lg/ml, respec-
tively. The insoluble fractions of A- and B-HBS lignins
showed IC50 values of 308 and >400 lg/ml, respectively.
From these results, it is thought that low molecular
weight lignin (less than 0.7 kDa) has inhibitory effect
on transcription of HIV-1 gene rather than high molec-
ular weight lignin (over 1.3 kDa).
2. Results
2.1. Suppression of HIV-1 gene expression by lignin
There are several methods for extraction of lignin from
wood, and properties of those lignins are different,
respectively. Therefore, three kinds of lignin, lignin alka-
li, lignin organosolve, and HBS lignin,12 were used. At
first, to assess whether lignin inhibits HIV-1gene expres-
sion, we examined transient luciferase assay using repor-
ter plasmid, CD-12-luc is widely used in studying
transcription of HIV-1 gene.13,14 Transfected cells were
cultured in the absence or presence of lignin for 20 h.
As shown in Figure 2, lignin alkali did not have effect
on HIV-1gene expression. However, 71%, 60%, and
70% decrease in expression were observed with 100 lg/
ml lignin organosolve, A-HBS lignin, and B-HBS lignin,
respectively. This result suggested that anti-HIV effect of
lignin differs with kinds of lignin.
2.4. Suppression of NF-jB activation by lignin
NF-jB is an inducible cellular transcription factor that
regulates viral gene expression via the two NF-jB sites
Table 1. Distribution of molecular weights of HBS lignin
Mn
Mw
Mw/Mn
Lignin alkali
A-HBS lignin
Ether soluble
Ether insoluble
B-HBS lignin
Ether soluble
Ether insoluble
5000a
900
28,000a
3020
530
5.6a
3.6
1.3
2.9
2.6
1.3
2.1
390
The cells were co-transfected with pCD-12-luc and
pCMV-b-galactosidase. Transfected 293-T cells were
cultured for 4 h and then incubated for 20 h with vehicle
(column 1), 100 lg/ml lignin alkali (column 2), 100 lg/
ml lignin organosolve (column 3), 100 lg/ml A-HBS lig-
nin (column 4), and 100 lg/ml B-HBS lignin (column 5).
Data are means from three independent experiments.
1320
890
3860
2310
630
470
1470
3050
Mn and Mw indicate number–average molecular weight and weight–
average molecular weight, respectively.
a Data from Sigma–Aldrich catalogue.

S. Mitsuhashi et al. / Bioorg. Med. Chem. 16 (2008) 2645–2650
Table 2. Effects of lignin on LTR (HIV-1) promoter
2647
OH
O
MeO
MeO
HO
Lignin
Yield (%)
IC50 (lg/ml)
LTR (HIV-1)
OH
OH
O
O
HO
Lignin alkali
>400
78
91
Lignin organosolve
A-HBS lignin
Ether soluble
Ether insoluble
B-HBS lignin
Ether soluble
Ether insoluble
MeO
MeO
100
14.1
85.9
100
32
25
1
2
308
82
19
>400
Me
68
OH
Transfected cells were cultured for 4 h and then treated with or without
lignin for 20 h.
MeO
HO
Me
OH
MeO
Table 3. Effects of lignin on NF-jB activity
HO
HO
OMe
Lignin
IC50 (lg/ml)
NF-jB + TNF
OH
OMe
NF-jB
3
4
Lignin alkali
>400
68
>400
84.5
100
37
Lignin organosolve
A-HBS lignin
Ether soluble
Ether insoluble
B-HBS lignin
Ether soluble
Ether insoluble
OMe
200
37
HO
OH
>400
71
33
271
67.5
21
OH
O
O
H
H
>400
>400
‘NF-jB’ column: Transfected cells were cultured for 4 h and then
treated with or without lignin for 20 h. ‘NF-jB + TNF’ column:
Transfected cells were cultured for 19 h and then treated with or
without lignin for 1 h. Treated cells were stimulated with 3 ng/ml
TNFa for 5 h.
O
OMe
HO
HO
OMe
OMe
5
6
Figure 3. Structures of lignin model compounds.
located in the HIV-1 LTR enhancer region.4–6 Treat-
ment with compounds that block NF-jB activation
inhibits HIV-1 gene expression and viral replication.
Hence, we examined whether lignin suppresses NF-jB
activation.
Table 4. Effects of lignin model compounds on LTR (HIV-1)
promoter and NF-jB activity
Compound
IC50 (lg/ml)
LTR (HIV-1)
NF-jB + TNF
As shown in Table 3, lignin organosolve, A-HBS lignin,
and B-HBS lignin also inhibited NF-jB activity in the
absence and presence of TNFa, whereas lignin alkali
did not show significant inhibitory effects. The ether sol-
uble fractions have more inhibitory effect than ether
insoluble fraction and whole HBS lignin. These results
suggested that lignin supresses HIV-1 gene expression
via inhibition of NF-jB activity, and inhibitory effect
is attributed to low molecular weight lignin.
1
2
3
4
5
6
>150
107
126
43
>150
>150
>150
34
>150
>150
49
19
‘LTR (HIV-1)’ column: Transfected cells were cultured for 4 h and
then treated with or without lignin model compound for 21 h.
‘NF-jB + TNF’ column: Transfected cells were cultured for 20 h and
then treated with or without lignin model compound for 1 h. Treated
cells were stimulated with 3 ng/ml TNFa for 5 h.
2.5. Inhibitory effects on HIV-1 gene expression and NF-
jB activation of lignin model compounds
There are various structures in the lignins from plant
and DHP. It is important to explore what is the most
effective structural unit. Therefore, we synthesized six
lignin dimer-like compounds (Fig. 3) and measured their
inhibitory activities (Table 4). Compounds 1 and 2 were
used as lignin dimer model of b-O-4 substructure, and
compounds 3–6 were dimer models of b-1, 5–5, b–b,
and b-5 substructures, respectively. These substructures
are representative structural units in lignin. Among
them, compound 6 showed most potent inhibitory activ-
ity on LTR (HIV-1) and NF-jB, and compound 2 also
had significant activities. Values of IC50 on LTR and
NF-jB activities of compound 6 were 34 and 19 lg/ml,
respectively.
3. Discussion
In addition to transcription factor, several targets such
as reverse transcriptase, protease, and integrase have
been well studied for chemotherapy of AIDS.15–20 Many
designed drugs were modified from nucleic acid and pep-

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S. Mitsuhashi et al. / Bioorg. Med. Chem. 16 (2008) 2645–2650
tide. However, there are also severe issues accompany-
ing strong side effects and high cost of production. Thus,
it is important to explore a new class of anti-HIV agents.
In this study, we have addressed the three questions;
(i) whether lignin can inhibit HIV-1 gene expression,
(ii) what is the mechanism of inhibitory action of lignin,
(iii) what is the most important structural unit in decom-
posed lignin for anti-HIV-1 activity. We observed that
lignin olganosolve and HBS lignin inhibit transcription
from HIV-1 LTR (Fig. 2 and Table 2). NF-jB activa-
tion, which is a critical element for HIV-1 replication,
was also inhibited by lignin (Table 3). Moreover, frac-
tion of low molecular weight in lignin and a lignin di-
mer-like compound containing b-5 structural unit
among six model compounds show potent inhibitory
activities (Tables 2–4). The most abundant substructure
in lignin is a b-O-4 structure. However, our previous
observations suggested that phenolic ether linkages in
lignin were cleaved homolytically via quinone methide
intermediates and main recombination structures from
the b-O-4 structure were phenylcoumaran (b-5) struc-
tures under HBS pulping conditions. Pinoresinol (b–b)
was identified as a radical coupling product from a phe-
nolic b-aryl ether model compound and b–b substruc-
tures were detected in lignin samples by 1H–13C
correlation 2D NMR measurements.12 Compound 6
having representative b-5 structure showed significant
effects on both of LTR (HIV-1) promoter and NF-jB
activity as shown in Table 4. Compound 5 having repre-
sentative b–b structure showed selective inhibitory activ-
ity against NF-jB mediated transcription of HIV-1.
Interestingly, compound 2, a-keto b-O-4 model com-
pound, had moderate inhibitory activities on both
LTR (HIV-1) and NF-jB mediated transcription.
to solubility of the components in ether by the Soxhlet
method.
4.2. Lignin model compounds
Lignin model compounds were obtained by reported
methods with slight modifications.22–25
4.2.1. Compounds 1 and 2. To a solution of 10 g of x-(2-
methoxyphenoxy)-acetoguaiacone in 100 ml of ethanol
were added 5 g of potassium carbonate and 100 ml of
formalin. The reaction mixture was stirred for 2 h at
50 ꢁC. Standard workup and recrystallization from a
mixed solution of ethyl ether and benzene gave 6.3 g
of a desired crystalline compound 2 in 56.7% yield, mp
104 ꢁC (lit.22 98 ꢁC).
To a solution of 15 g of compound 2 in 1000 ml of 0.1 N
NaOH was added 2 g of sodium borohydride. The resul-
tant mixture was stirred for 12 hours at room tempera-
ture. The reaction mixture was adjusted to pH 3 with
diluted HCl and extracted with CHCl3. The organic
solution was dried over Na2SO4 and concentrated in va-
cuo to give 9.0 g of solid material. Recrystallization of
above solidified product from ethyl ether gave 6.1 g of
pure compound 1 in 40.9% yield, mp 114.5 ꢁC (lit.22
119–120 ꢁC).
1
4.2.1.1. Compound 1. H NMR (CDCl3): d 2.81 (1H,
br t, Cc-OH), 3.48 (1H, br m, Cc-Ha), 3.62 (br d, Cc-
Hb), 3.73 (1H, s, Ca-OH), 3.86 (3H, s, OCH3), 3.90
(3H, s, OCH3), 4.02 (1H, ddd, Cb-H), 4.95 (1H, d,
Ca-H), 5.77 (1H, s, Ph-OH), 6.00–6.60 (6H, m,
aromatics).
Lignin may be a beneficial material or drug leads as a
new class of anti-HIV agents possibly effective in the
treatment of AIDS. Inhibition of HIV-1 replication
by lignin was previously demonstrated. The effective
dose, based on 100% inhibition of HIV-1-induced cyto-
pathogenicity in MT-4 cells, of wheat bran lignin was
125 lg/ml.11 The DHP fractions having a 0.5–1 kDa
molecular mass showed stronger anti-HIV-1 activity
rather than wheat bran lignin, whereas monomers of
p-coumaric acid and ferulic acid had no effect on
HIV-1 replication.11 These observations and our results
suggest that small decomposed-lignins or lignin model
compounds are profitable for suppression of HIV-1.
Such small molecules having lignin like structure would
have higher cell permeability than high molecular
weight lignins.
13C NMR (CDCl3): d 55.9 (OCH3), 55.9 (OCH3), 61.1
(Cc), 74.0 (Ca), 89.5 (Cb), 109.5, 112.2, 114.4, 120.3,
121.0, 121.7, 124.2, 131.5, 145.6, 146.7, 147.7, 151.3
(aromatics).
1
4.2.1.2. Compound 2. H NMR (CDCl3): d 3.04 (1H,
br t, Ca-OH), 3.86 (3H, OCH3), 3.94 (3H, OCH3), 4.07
(2H, br t, Cc-H), 5.40 (1H, t, Cb-H), 6.15 (1H, s, Ph-
OH), 6.40–7.00 (5H, m, aromatics), 7.60–7.70 (2H, m,
aromatics).
4.2.2. Compound 3. To a solution of 10 ml of LDA
(2.0 M) in THF were added dropwise a solution of
methyl benzylhomovanillate (4.29 g, 0.015 mol) in
20 ml over 20 min at ꢀ70 ꢁC and stirred for 30 min.
Benzylvanillin (3.63 g, 0.015 mol) in 20 ml of THF was
added dropwise to the resultant greenish yellow solution
at the same temperature. The reaction temperature was
gradually raised to ꢀ58 ꢁC over 1 h and quenched with
dry ice. The standard workup followed by recrystalliza-
tion with EtOH gave 2.74 g of erythro-b-hydroxy ester
as first crystals and 2.91 g of threo-b-hydroxy ester as
second crystals. Erythro isomer was recrystallized
repeatedly from 1% MeOH in CHCl3 and 20% MeOH
in CHCl3 to yield a pure erythro isomer.
4. Experimental
4.1. Lignin
Lignin Alkali and Lignin Organosolve were purchased
from Sigma Chemical Co (St. Louis, MO, USA).
High-boiling solvent lignin (HBS lignin) was previously
described.12 A-HBS lignin and B-HBS lignin were pre-
pared from Acacia mangium and Betula platyphylla,
respectively.21 HBS lignins were fractionated according
To a suspension of the erythro isomer in 30 ml of THF
were added 3.19 ml (22.7 mmol) of triethylamine and

S. Mitsuhashi et al. / Bioorg. Med. Chem. 16 (2008) 2645–2650
2649
1.4 ml (11.4 mmol) of TMSCl at 0 ꢁC. The reaction tem-
perature was raised to 70 ꢁC and stirred for 2.5 hours.
The above mixture was transferred to a dropping funnel
and LiAlH4 (0.863 g, 22.7 mmol) in 25 ml of anhydrous
THF to give 1.78 g (94% yield) of pure 1,2-bis-(4-benzyl-
oxy-3-methoxyphenyl)-propane-1,3-diole after a stan-
dard workup and recrystallization from EtOAc-hexane
(1:2). Hydrogenolysis of the resultant compound
(1.66 g, 3.32 mmol) with 1 g of Pd–C in 200 ml of
MeOH under hydrogen atmosphere gave 0.9 g (yield
18.7%) of crystalline compound (3) after standard work-
up followed by recrystallization from EtOAc-hexane,
mp 158 ꢁC (lit.26 151–154 ꢁC; lit.27 158–159 ꢁC).
56.0 (OCH3), 71.7 (Cc), 85.8 (Ca), 108.5 (C2), 114.2
(C5), 118.9 (C6), 132.9 (C1), 145.1 (C4), 146.6 (C3).
1
4.2.4.2. Compound 6. H NMR (acetone-d6): d 3.53
(1H, br q, J = 6.4, Cb-H), 3.78–3.86 (3H, m, c-OH,
Cc-H), 3.82 (3H, s, OCH3), 3.86 (3H, s, OCH3), 4.15–
4.22 (3H, m, c0-OH, Cc-H), 5.56 (1H, d, J = 6.4, Ca-
H), 6.25 (1H, dt, J = 15.8, 5.4, Cb-H), 6.53 (1H, d,
J = 15.8, Ca-H), 6.79–7.04 (5H, m, aromatics), 7.65
(1H, s, C4-OH); 13C NMR (acetone-d6) d 54.7 (Cb),
56.2 (OCH3), 56.3 (OCH3), 63.3 (Cc0), 64.5 (Cc), 88.4
0
0
(Ca), 110.3 (C2), 111.5 (C2 ), 115.5 (C5), 115.9 (C6 ),
0
119.4 (C6), 128.1 (Cb’), 130.2 (C5 ), 130.3 (Ca’), 131.7
0
(C1 ), 134.2 (C1), 144.9 (C3 ), 147.1 (C4), 148.1 (C3),
0
4.2.2.1. Compound 3. 1H NMR(DMSO-d6): d 2.72 (br
q, 1H, Cb-H), 3.43–3.49 (m, 1H, Cc-Ha), 3.60, 3.64 (s,
6H, OCH3 · 2), 3.62–3.68 (m, 1H, Cc-Hb), 4.38 (br t,
1H, Cc-OH), 4.80 (br t, 1H, Ca-H), 4.88 (br d, 1H,
Ca-OH), 6.46–6.62 (m, 6H, aromatics), 8.54, 8.64 (s,
2H, Ph-OH · 2); 13C NMR (DMSO-d6): d 55.4 (Cb),
55.6, 55.7 (OCH3 · 2), 62.9 (Cc), 72.6 (Ca), 110.9,
114.0, 114.6, 118.7, 121.9, 131.5, 136.0. 144.7, 144.9,
146.6, 146.8 (aromatics).
148.7 (C4 ).
0
4.3. Plasmids
Construction of HIV-1 LTR luciferase expression plas-
mid: CD-12-luc (containing the HIV-1 LTR U3 and
R) was previously described.6 The pNF-jB-luc reporter
plasmid was purchased from Stratagene (Garden Grove,
CA, USA). The expression vector, pCMV-b-galactosi-
dase, was previously described.30
4.2.3. Compound 4. To a solution of above 4-propyl-
guaiacol (1.0 g) in EtOH (20 ml) was added a solution
of horseradish peroxidase (3 mg, 100 U/mg) in 20 ml
of water. To the reaction mixture was added dropwise
3% H2O2 (3.5 ml) over 30 min with vigorous stirring.
The resultant crystals were collected and washed with
50% aqueous EtOH to give crude compound. Recrystal-
lization from EtOH gave 0.81 g of 4,40-dipropyl-6,60-
biguaiacol (4) (yield 43.5%), mp 149.5 ꢁC (lit.28 150–
152 ꢁC; lit.29 152 ꢁC ).
4.4. Luciferase assay
293-T cells were maintained in RPMI-1640 medium
(WAKO, Osaka, Japan) containing 10% fetal bovine
serum, 100 lg/ml streptomycin, and 20 U/ml penicillin
G at 37 ꢁC under 5% CO2. For transient transfections
cells were transfected using Fugene-6 (Roche Diagnos-
tics Inc., Mannheim, Germany) according to the manu-
facturer’s recommendation.
1H NMR (CDCl3): d 0.96 (6H, t, J = 7.3, Cc-H), 1.65
(4H, sextet, Cb-H), 2.56 (4H. t. J = 7.7, Ca-H), 3.92
(6H, s, OCH3), 6.02 (2H, Ph-OH), 6.72 (2H, s, C6-H),
6.74 (2H, s, C2-H).
The cells in 10-cm dishes were co-transfected with 5 lg
reporter plasmid and 0.008 lg pCMV-b-galactosidase.
Two hours later, cells were harvested into new 12-well
plates and cultured for 3–19 h, treated with or without
lignin, and then stimulated with or without 5 ng/ml
TNFa (PEPROTECH EC Ltd, London, UK). Lucifer-
ase activity was measured with the Luciferase Assay Sys-
tem (Promega, Madison, WI, USA). b-Galactosidase
activity was measured with the b-GloTM Assay System
(Promega). Chemiluminescence was determined by a
microplate luminometer, VeritasTM (Promega). b-Galac-
tosidase activities were used to normalize transfection
efficiency and cell number.
4.2.4. Compounds 5 and 6. To distilled water was added
a solution of coniferyl alcohol (18.0 g) in acetone
(33 ml). To the resultant solution was added a solution
of horseradish peroxidase (6.8 mg) in distilled water
(150 ml), and then dropwise 0.5% H2O2 (1000 ml) over
60 min with stirring. The reaction mixture was stirred
for 1 h and added NaCl (250 g). The resultant solution
was extracted with EtOAc and organic layer was
washed with brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was chromatographed
on silica gel (MeOH: CHCl3, 5:95 to 10:90) to give
716 mg (yield 4.0%) of pinoresinol (5), mp 157.1 ꢁC
(lit.28 156.5–158 ꢁC) and 3.87 g (yield 21.6%) of dehyd-
rodiconiferylalcohol (6), mp 118.2 ꢁC (lit.29 120–
121 ꢁC) after recrystallization from acetone-hexane
(1:1) and acetone, respectively.
4.5. Size exclusion chromatography
Acetylation of lignin samples was done with acetic anhy-
dride and pyridine at room temperature. The obtained
acetylated lignin samples were purified according to
the method of G. Gellerstedt,31 and then samples were
dissolved in tetrahydrofuran and were analyzed for
average molecular weights by Size exclusion chromatog-
raphy. A HITACHI Liquid Chromatograph L-6200
with a UV detector, L-4000 (280 nm) was used. Shodex
GPC packed column KF-802 and KF-803 L were con-
nected in a series and molecular weight was calibrated
with standard polystyrene (tetrahydrofuran, flow-rate:
0.5 ml/min, 40 ꢁC).
1
4.2.4.1. Compound 5. H NMR (CDCl3): d 3.10 (2H,
m, Cb-H), 3.86 (2H, dd, J = 3.5, Cc-H1), 3.91 (6H, s,
OCH3), 4.25 (2H, dd, J = 6.8, 8.9, Cc-H2), 4.74 (2H,
d, J = 4.1, Ca-H), 5.59 (2H, s, C4-OH), 6.82 (2H, d,
J = 8.1, C5-H), 6.87 (2H, s, C2-H), 6.89 (2H, dd,
J = 1.4, 8.1, C6-H); 13C NMR (CDCl3): d 54.2 (Cb),

2650
S. Mitsuhashi et al. / Bioorg. Med. Chem. 16 (2008) 2645–2650
10. Nakashima, H.; Murakami, T.; Yamamoto, N.; Naoe, T.;
Acknowledgments
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We thank Dr. J. Magae (Institute of Research and Inno-
vation, Kashiwa, Japan) and N. H. Heintz (University
of Vermont, Burlington, VT) for providing us with
pCMV-b-galactosidase. This work was supported in
part by Grants-in-Aid for Scientific Research (A) (No.
19208011), and for Scientific Research on Priority Area
‘Creation of Biologically Functional Molecules’ by the
Ministry of Education, Culture, Sports, Science and
Technology of Japan (MU). We are grateful to Northan
Advancement Center for Science & Technology (Advi-
ser: Dr. Y. Sano), the Hokkaido Development Agency.
Shinya Mitsuhashi was supported by a postdoctoral fel-
lowship from the Japan Society for the Promotion of
Science.
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