Enzymatic oxidative polymerization of p-t-butylphenol and characterization of the product polymer

Article abstract of DOI:10.1246/bcsj.76.375

For structural control and analysis of an enzymatically synthesized polyphenol, peroxidase-catalyzed polymerization of p-t-butylphenol in a mixture of polar organic solvent and phosphate buffer has been examined in detail. The resulting products were subjected to preparative HPLC. Two dimers and one trimer were isolated and their structures were determined by ¹H and ¹³C NMR. ESI-TOF mass analysis showed the formation of two kinds of dimer and of at least four kinds of trimer. These data clearly showed that the polymer was composed of a mixture of phenylene and oxyphenylene units. The ratio of phenylene and oxyphenylene units in the product could be controlled by changing the solvent composition. The phenylene unit linearly increased as a function of the water content in the mixed solvent.

Full text of DOI:10.1246/bcsj.76.375

c. Jpn.
© 2003 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 76, 375–379 (2003)
375
e Chemical
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Enzymatic Oxidative Polymerization of p-t-Butylphenol and
Characterization of the Product Polymer
Enzymatic Polymerization of p-t-Butylphenol
N. Mita et al.
Naruyoshi Mita,# Naoyuki Maruichi,† Hiroyuki Tonami,† Ritsuko Nagahata,†† Shin-ichiro Tawaki,†††
Hiroshi Uyama,† and Shiro Kobayashi*,†
03
5
9
Joint Research Center for Precision Polymerization, Japan Chemical Innovation Institute, NIMC,
1-1 Higashi, Tsukuba, Ibaraki 305-8565
SJA8
09-2673
229
†Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501
††Research Center of Macromolecular Technology, National Institute of Advanced Industrial Science and Technology,
AIST Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565
02
02
†††Catalysis Science Laboratory, Mitsui Chemicals, Inc., 580-32 Nagaura, Sodegaura, Chiba 299-0265
(Received August 15, 2002)
3.6
.1
For structural control and analysis of an enzymatically synthesized polyphenol, peroxidase-catalyzed polymeriza-
tion of p-t-butylphenol in a mixture of polar organic solvent and phosphate buffer has been examined in detail. The re-
sulting products were subjected to preparative HPLC. Two dimers and one trimer were isolated and their structures were
determined by 1H and 13C NMR. ESI-TOF mass analysis showed the formation of two kinds of dimer and of at least four
kinds of trimer. These data clearly showed that the polymer was composed of a mixture of phenylene and oxyphenylene
units. The ratio of phenylene and oxyphenylene units in the product could be controlled by changing the solvent compo-
sition. The phenylene unit linearly increased as a function of the water content in the mixed solvent.
Enzymatic synthesis of polyphenols is considered as a
promising alternative to preparation of conventional phenolic
resins (novolak and resol resins), owing to no use of toxic
formaldehyde, mild reaction conditions (in neutral solvents at
room temperature) and facile procedures.1 Peroxidases were
reported to catalyze an oxidative polymerization of various
phenol derivatives to produce a new class of useful and func-
tional polyphenols efficiently.2 Chemoselective polymeriza-
tion of phenols bearing an unsaturated group took place via the
peroxidase catalysis, yielding reactive polyphenols.3
Scheme 1.
structural control and analysis of an enzymatically synthesized
polyphenol (Scheme 1). In order to obtain more data on the
structure of the polyphenols, some oligomers were isolated
and their structures were determined by NMR. The solvent
composition was systematically investigated for precise con-
trol of the polymer structure.
Structures of the polyphenols are often very complicated; in
the case of p-substituted phenols, we described the polymer as
mainly consisting of phenylene and oxyphenylene units;4 how-
ever, other groups reported exclusive formation of the polymer
having an ortho–ortho linkage.5 Very recently, we briefly re-
ported the control of the coupling selectivity (ratio of phe-
nylene and oxyphenylene units) in the peroxidase-catalyzed
polymerization of p-substituted phenols by selection of the
monomer substituent and nature of solvent.6 The hydrophobic
parameters of the monomer substituent and organic solvent
strongly affected the polymer structure.
In an oxidative coupling of p-substituted phenols, three re-
action positions are conceivable, whereas unsubstituted and m-
substituted phenols have four sites. Formation of Pummerer’s
ketone is involved as a side-reaction in the oxidative coupling
of p-substituted phenols with small substituents.7 In this study,
therefore, we used p-t-butylphenol (1) as a model monomer for
Results and Discussion
Isolation and Identification of Oligophenols.
In this
study, horseradish and soybean peroxidases (HRP and SBP, re-
spectively) were used as catalysts. The HRP-catalyzed oxida-
tive polymerization of 1 was first performed using hydrogen
peroxide as oxidizing agent in an equivolume mixture of meth-
anol and phosphate buffer (pH 7) at room temperature under
air. Hydrogen peroxide (5%) was added dropwise to the reac-
tion mixture for 2 h. Powdery polymeric precipitates were
quickly formed by the addition of hydrogen peroxide. Then,
after 1 h, the polymer was isolated by filtration (yield 83%).
HPLC analysis of the reaction mixture showed that 90% of 1
was consumed. The molecular weight of the polymer was
measured by size exclusion chromatography (SEC). The num-
# Catalysis Science Laboratory, Mitsui Chemicals, Inc.

376 Bull. Chem. Soc. Jpn., 76, No. 2 (2003)
Enzymatic Polymerization of p-t-Butylphenol
Fig. 1. HPLC traces of the product obtained from 1 in an
equivolume mixture of methanol and phosphate buffer us-
ing (A) HRP and (B) SBP catalyst.
ber-average molecular weight (Mn) was 570 and its index was
1.9.
HPLC analysis of the product was performed using an in-
verse-phase silica-gel column with methanol/water (98:2
vol%) eluent. On the HPLC chart of the reaction mixture,
many peaks were observed (Fig. 1(A)). The main fractions A–
C were separated by using preparative HPLC column and their
structures were analyzed by NMR spectroscopy. Two dimers
2, 3 and one trimer 4 were identified by 1H and 13C NMR spec-
troscopies (Fig. 2). In the SBP-catalyzed polymerization of 1
under similar reaction conditions, the peak pattern of the prod-
uct was very similar to that obtained for HRP (Fig. 1(B)).
These data suggest little structural difference between the
polymers obtained by using HRP and SBP catalysts.
Fig. 2. Structure of isolated dimers (2 and 3) and trimer (4)
and their assignment of 1H and 13C NMR peaks.
ESI-TOF Mass Analysis of Poly(1). In order to confirm
the polymer structure consisting of a mixture of phenylene and
oxyphenylene units, a liquid chromatography/electrospray ion-
ization-time of flight mass (LC/ESI-TOF MS) measurement
was carried out. There were many peaks observed in a total
ion chromatogram of poly(1) obtained by HRP catalyst in an
equivolume mixture of methanol and the buffer (Fig. 3(A)), al-
though the peak separation was not good. The peaks’ pattern
was somewhat different from that by UV detector (Fig. 1),
probably owing to the difference of the detection methods. In
the detection of the mass of a dimer plus a sodium ion (m/z =
321.1), two sharp peaks were detected (Fig. 3(B)), supporting
the formation of two dimers 2 and 3. If the oxidative coupling
of 1 proceeds via the formation of C–C and C–O bonds, five
trimers are conceivable (Chart 1). In case of the molecular
mass of a trimer (m/z = 469.2), four or five peaks were detect-
ed (Fig. 3(C)), which may be due to these trimers. As for a tet-
ramer (m/z = 617. 3) and a pentamer (m/z = 765.4), many
peaks were observed in their spectra (Figs. 3(D) and 3(E), re-
spectively). These data support the conclusion that the poly-
mer structure was of a mixture of phenylene and oxyphenylene
units.
Fig. 3. LC/ESI-TOF mass chromatograms of poly(1) with
detector of (A) total ion, (B) m/z 321.1, (C) m/z 469.2, (D)
m/z 617.3, and (E) m/z 765.4.
In the HPR-catalyzed polymerization of phenol in a mixture of
methanol and buffer, the polymer structure could be controlled
by changing the methanol content of the mixed solvent.2b,2c In
the HRP-catalyzed oxidative polymerization of p-substituted
Solvent Engineering for Control of Polymer Structure.

N. Mita et al.
Bull. Chem. Soc. Jpn., 76, No. 2 (2003) 377
Table 1. Peroxidase-Catalyzed Polymerization of 1 in a Mixture of 2-Propanol and Phosphate Buffera)
Polymer
Mw/Mnc)
Entry
Catalyst
Content of 2-propanol/%
Conv.b)/%
Yield/%
95
Mnc)
800
1000
1400
1400
1500
920
1200
1300
1400
1500
Ph/Oxd)
71/29
60/40
56/44
50/50
45/55
68/32
60/40
57/43
53/47
45/55
1
2
3
4
5
6
7
8
9
HRP
HRP
HRP
HRP
HRP
SBP
SBP
SBP
SBP
SBP
30
40
50
60
70
30
40
50
60
70
97
94
92
63
33
100
98
98
93
68
1.7
1.7
1.5
1.5
1.6
1.6
1.4
1.5
1.5
1.6
92
87
51
9
91
89
91
86
10
44
a) Polymerization of 1 (0.75 g, 5.0 mmol) using HRP (2.0 mg, 440 units) or SBP (8.0 mg, 420 units) catalyst in
a mixture of 2-propanol and pH 7 phosphate buffer (25 mL) at room temperature for 3 h under air. b) Deter-
mined by HPLC. c) Determined by SEC using DMF as eluent with polystyrene standards. d) Determined by
titration.
Chart 1.
phenols, the coupling selectivity could be controlled by chang-
ing the hydrophobic parameters of the solvent and monomer
substituent, yielding soluble polyphenols with a different ratio
of phenylene/oxyphenylene units.6 Here, such solvent effects
on the structural control in the peroxidase-catalyzed oxidative
Fig. 4. FT-IR spectra of poly(1) (entry 3 in Table 1) (A) be-
fore acetylation and (B) after acetylation.
polymerization of 1 in an aqueous organic solvent were inves-
tigated in detail.
The ratio of phenylene and oxyphenylene units (Ph/Ox) was
determined by conventional titration methods;8 the polymer
was reacted with an excess of acetic anhydride in pyridine and
the acetylated amount was determined by the titration. Figure
4 shows FT-IR spectra of the polymer before and after the
acetylation at 95–100 °C for 1 h. A characteristic broad peak
due to phenolic O–H bond was observed at 3400 cm−1 in
poly(1) (Fig. 4(A)). After the acetylation, this peak completely
disappeared (Fig. 4(B)), indicating that the acetylation pro-
ceeded quantitatively under the present reaction conditions;
thus, the residual phenolic group in poly(1) can be correctly
determined by the titration. By 1H NMR analysis of the acety-
lated poly(1), the quantitative acetylation of the phenolic group
was confirmed (data not shown).
HRP-catalyzed polymerization of 1, the polymer was formed
in high yields when the 2-propanol content was in the range
from 30 to 50% and the molecular weight increased with in-
creasing the 2-propanol content (entries 1–3). When the 2-pro-
panol content was higher than 60%, the monomer conversion
and polymer yield became smaller (entries 4 and 5). This is
probably due to the HRP denaturation in the solvent with the
high content of 2-propanol.9 In using SBP as catalyst (entries
6–10), a similar reaction pattern was observed except for the
higher conversion and yield in the 2-propanol content of 60 or
70% (entries 9 and 10). This may be because the activity loss
of SBP in such a solvent is smaller than that of HRP.
The unit ratio of the polymer was determined by the titra-
tion. For both enzymes, the phenylene content linearly de-
creased as a function of the 2-propanol content (Fig. 5); corre-
lation coefficients for HRP and SBP catalysts were 0.984 and
0.986, respectively. These data suggest that the precise control
In a mixed solvent of 2-propanol and phosphate buffer (pH
7), the effects of the mixed ratio have been systematically ex-
amined (Table 1). When the 2-propanol content was less than
20%, the monomer was partly insoluble in the medium. In the

378 Bull. Chem. Soc. Jpn., 76, No. 2 (2003)
Enzymatic Polymerization of p-t-Butylphenol
Table 2. Peroxidase-Catalyzed Polymerization of 1 in an Equivolume Mixture of Organic Solvent and Phosphate Buffera)
Polymer
Entry
Catalyst
Organic solvent
Ethylene Glycol
N,N-Dimethylformamide
Methanol
1,4-Dioxane
Acetone
2-Propanol
1-Propanol
t-Butyl Alcohol
Methanol
1,4-Dioxane
Acetone
2-Propanol
log P
Conv.b)/%
Yield/%
38
Mnc)
390
760
570
930
1500
1400
1600
1600
670
1100
1500
1300
Mw/Mnc)
2.0
Ph/Oxd)
85/15
68/32
72/28
64/36
56/44
56/44
47/53
51/49
73/27
64/36
56/44
57/43
1e)
2
HRP
HRP
HRP
HRP
HRP
HRP
HRP
HRP
SBP
SBP
SBP
SBP
−1.36
−1.01
−0.77
−0.42
−0.24
0.05
0.25
0.35
−0.77
−0.42
−0.24
0.05
92
95
90
95
93
92
78
96
98
100
94
98
70
83
98
67
87
64
93
96
1.8
1.9
1.8
1.6
1.5
1.5
1.5
1.6
3e)
4e)
5
6e)
7e)
8
9
10
11
12
96
88
91
1.8
1.7
1.5
a) Polymerization of 1 (0.75 g, 5.0 mmol) using HRP (2.0 mg, 440 units) or SBP (8.0 mg, 420 units) catalyst in an equiv-
olume mixture of organic solvent and pH 7 phosphate buffer (25 mL) at room temperature for 3 h under air. b) Deter-
mined by HPLC. c) Determined by SEC using DMF as eluent with polystyrene standards. d) Determined by titration.
e) Data from Ref. 6.
Fig. 5. Relationships between 2-propanol content and poly-
mer structure in the peroxidase-catalyzed polymerization
of 1 in a mixture of 2-propanol and phosphate buffer.
Fig. 6. Relationships between log P of organic solvents and
polymer structure in the peroxidase-catalyzed polymeriza-
tion of 1 in an equivolume mixture of organic solvent and
phosphate buffer.
of the polyphenol structure can be achieved by changing the
mixing ratio. A similar tendency was observed in the HRP-
catalyzed polymerization of unsubstituted phenol in aqueous
methanol.2b,2c
phobicity increased. The structure of the polymer obtained by
using HRP catalyst was almost the same as that obtained by
SBP.
Table 2 shows polymerization results in equivolume mix-
tures of various polar organic solvents and the phosphate buff-
er. Here, log P was used as a measure of hydrophilicity of the
solvent.6,10 In most cases, the polymer was obtained in good
yields. When the log P value of the cosolvent was less than
−0.4, the molecular weight of the polymer was relatively low
(< 1000). The peroxidase origin affected the polymerization
behaviors only slightly; the polymerization results obtained by
using HRP catalyst were very similar to those by SBP.
Figure 6 shows relationships between log P of the organic
cosolvent and the phenylene unit of poly(1). A relatively good
first-order correlation was observed and the phenylene unit de-
creased as log P increased. Data of Figs. 5 and 6 clearly
showed that the phenylene unit decreased as the solvent hydro-
Conclusion
For structural analysis of enzymatically synthesized poly(1),
two dimers and one trimer were isolated from the resulting
polymer by using preparative HPLC and their structure was
determined by NMR analysis. LC/ESI-TOF MS analyses sug-
gest the formation of at least four kinds of trimers. These data
clearly indicate that the enzymatically synthesized polyphenol
consisted of phenylene and oxyphenylene units. The solvent
composition affected the control of the polyphenol structure.
In the polymerization of 1 in a mixture of 2-propanol and
phosphate buffer, the phenylene unit content linearly decreased
as a function of 2-propanol content. A relatively good first-or-
der correlation between log P of organic solvent and phenylene

N. Mita et al.
Bull. Chem. Soc. Jpn., 76, No. 2 (2003) 379
References
a) S. Kobayashi, S. Shoda, and H. Uyama, Adv. Polym. Sci.,
unit was observed in a mixed solvent of polar organic solvent
and phosphate buffer.
1
121, 1 (1995). b) S. Kobayashi, S. Shoda, and H. Uyama, “Catal-
ysis in Precision Polymerization,” ed by S. Kobayashi, John Wiley
& Sons, Chichester (1997), Chap. 8. c) S. Kobayashi, J. Polym.
Sci., Polym. Chem. Ed., 37, 3041 (1999). d) S. Kobayashi, H.
Uyama, and M. Ohmae, Bull. Chem. Soc. Jpn., 74, 613 (2001). e)
R. A. Gross, A. Kumar, and B. Kalra, Chem. Rev., 101, 2097
(2001). f) S. Kobayashi, H. Uyama, and S. Kimura, Chem. Rev.,
101, 3793 (2001).
Experimental
Materials. HRP and SBP were purchased from Wako Pure
Chemical Industries, Ltd. and Sigma Chemical Co., respectively,
and were used without further purification. Other reagents and
solvents were commercially available and were used as received.
Enzymatic Polymerization. A typical run was as follows
(entry 3 in Table 1). p-t-Butylphenol (0.75 g, 5.0 mmol) and HRP
(2.0 mg, 440 unit) were dissolved in an equivolume mixture of 2-
propanol and 0.1 M phosphate buffer (pH 7) (25 mL). To this so-
lution, 3.2 mL of 5% hydrogen peroxide (5.3 mmol) was added
dropwise for 2 h. The mixture was stirred at room temperature
under air. After 1 h, the precipitated materials were collected by
centrifugation and washed with a mixture of methanol and water
(1:1 vol) repeatedly, followed by drying in vacuo to give 0.66 g of
the polymer (yield 87%). 1H NMR (DMSO-d6) δ 0.8–1.5 (m,
CH3), 6.5–7.5 (m, Ar). IR (KBr) 3400 (ν O-H), 2963, 2906, 2869
(ν C-H), 1586, 1508 (ν CwC of Ar), 1217 (ν C(Ar)–O–C(Ar) and
C(Ar)–OH), 1120 cm−1 (ν C(Ar)–O–C(Ar)).
2
a) H. Uyama and S. Kobayashi, CHEMTECH, 29(10), 22
(1999). b) T. Oguchi, S. Tawaki, H. Uyama, and S. Kobayashi,
Macromol. Rapid Commun., 20, 401 (1999). c) T. Oguchi, S.
Tawaki, H. Uyama, and S. Kobayashi, Bull. Chem. Soc. Jpn., 73,
1389 (2000). d) B. K. Mandal, C. J. Walsh, T. Sooksimuang, and
S. J. Behroozi, Chem. Mater., 12, 6 (2000). e) M. H. Reihmann
and H. Ritter, Macromol. Chem. Phys., 201, 1593 (2000). f) T.
Fukuoka, H. Tonami, N. Maruichi, H. Uyama, S. Kobayashi, and
H. Higashimura, Macromolecules, 33, 9152 (2000). g) H. Uyama,
N. Maruichi, H. Tonami, and S. Kobayashi, Biomacromolecules,
3, 187 (2002).
Titration. Poly(1) (0.1 g) was dissolved in pyridine contain-
ing 2.5% acetic anhydride (5 mL). The solution was kept at 95–
100 °C for 1 h under gentle stirring. After cooling to room tem-
perature, water (0.5 mL) was added to the reaction mixture, then
the mixture was again heated at 95–100 °C for 10 min. The solu-
tion was titrated with 0.2 M (1 M = 1 mol dm−3) potassium hy-
droxide in ethanol in the presence of phenolphthalein as indicator.
Measurements. For SEC and HPLC measurements, a Tosoh
SC8020 apparatus was used. SEC analysis was carried out by us-
ing a refractive index (RI) detector at 60 °C under the following
conditions: two TSKgel α-M columns and DMF containing 0.09
M LiCl eluent at a flow rate of 1.0 mL min−1. The calibration
curves were obtained using polystyrene standards. HPLC analysis
was performed using a UV monitor (278 nm) at 40 °C under the
following conditions: two YMC-Pack ODS AM-312 columns and
methanol/17 mM phosphoric acid eluent at a flow rate of 1.8 mL
min−1 or a TSKgel ODS-80Ts column and methanol/water (98:2
vol%) eluent at a flow rate of 0.5 mL min−1. NMR spectra was re-
corded on JEOL JNM-LA 600 and Bruker DPX400 spectrome-
ters. LC/ESI-TOF MS analyses were carried out using a PE Bio-
systems Mariner ESI/TOF equipped with a Hewlett Packard HP
1100 under the following LC conditions: an HP-ODS Hypersil
column and methanol/water (90:10 vol%) eluent at a flow rate of
1 mL/min. FT-IR measurements were carried out with a Perkin-
Elmer Paragon 1000 spectrometer.
3
a) H. Uyama, C. Lohavisavapanich, R. Ikeda, and S.
Kobayashi, Macromolecules, 31, 554 (1998). b) H. Tonami, H.
Uyama, S. Kobayashi, T. Fujita, Y. Taguchi, and K. Osada, Bio-
macromolecules, 1, 149 (2000). c) M. H. Reihmann and H. Ritter,
Macromol. Chem. Phys., 201, 798 (2000).
4
a) H. Kurioka, I. Komatsu, H. Uyama, and S. Kobayashi,
Macromol. Rapid Commun., 15, 507 (1994). b) H. Uyama, H.
Kurioka, J. Sugihara, I. Komatsu, and S. Kobayashi, J. Polym.
Sci., Polym. Chem. Ed., 35, 1453 (1997). c) H. Tonami, H.
Uyama, S. Kobayashi, K. Rettig, and H. Ritter, Macromol. Chem.
Phys., 200, 1998 (1999).
5
a) M. S. Ayyagari, K. A. Marx, S. K. Tripathy, J. A.
Akkara, and D. L. Kaplan, Macromolecules, 28, 5192 (1995). b)
P. Wang, D. Martin, S. Parida, D. G. Rethwisch, and J. S. Dordick,
J. Am. Chem. Soc., 117, 12885 (1995). c) M. Ayyagari, J. A.
Akkara, and D. L. Kaplan, Acta Polymerica, 47, 193 (1996). d) J.
A. Akkara, M. S. R. Ayyagari, and F. F. Bruno, Trends Biotechnol.,
17, 67 (1999).
6
N. Mita, S. Tawaki, H. Uyama, and S. Kobayashi, Chem.
Lett., 2002, 402.
7
H. Uyama, H. Kurioka, J. Sugihara, I. Komatsu, and S.
Kobayashi, Bull. Chem. Soc. Jpn., 68, 3209 (1995).
8
9
JIS K 0070-1992.
a) H. Tonami, H. Uyama, S. Kobayashi, and M. Kubota,
Macromol. Chem. Phys., 200, 2365 (1999). b) T. Oguchi, A.
Wakisaka, S. Tawaki, H. Tonami, H. Uyama, and S. Kobayashi, J.
Chem. Phys. B, 106, 1421 (2002).
10 C. Laane, S. Boeren, K. Vos, and C. Veeger, Biotechnol.
Bioeng., 30, 81 (1987).
This work was partly supported by NEDO for the project on
Technology for Novel High-Functional Materials in Industrial
Science and Technology Frontier Program, AIST.