Polyaromatic compounds and method for their production

Polyaromatic compound made by polymerizing one or more aromatic monomers, least one of the one or more aromatic monomers having the following structure: ##STR1## wherein X is selected from the group consisting of OH and NH.sub.2, Y is selected from the group consisting of a halogen, a halogen-containing group, a boron-containing group, a phosphorus-containing group, a carboxyl group, a hydroxyacid group, a carboxyacid group, an amino group, an amino acid group, an amide group, an ester group, a hydroxyl group and a sulfonic acid group, R is an alkyl, phenyl, naphthyl, hydroxyphenyl, hydroxy naphthyl or hydroxy (.alpha., .beta., .gamma.) alkyl group, and n is an integer from 0 to 12. The (R).sub.n --Y group is preferably either ortho or para to X and more preferably is para to X. The polyaromatic compound may be a homopolymer consisting of one species of aromatic monomer of formula (I), a copolymer consisting of two or more species of aromatic monomers of formula (I), or a copolymer comprising one or more species of aromatic monomer of formula (I) and one or more species of an aromatic monomer not of formula (I). Preferably, the one or more aromatic monomers are reacted with a stoichiometric amount of hydrogen peroxide in the presence of horseradish peroxidase, the reaction taking place in water or in a mixture of water and one or more polar solvents. To obtain a substantial yield, the pH of the reaction medium is preferably around or below the pK.sub.a of the functional group of the monomer(s) being polymerized.

BACKGROUND OF THE INVENTION 
The present invention relates generally to polyaromatic compounds and 
methods for their production and more particularly to the synthesis of 
polyaromatic compounds using biocatalysts. 
Traditionally, phenolic resins, such as novolacs and resoles, have been 
commercially prepared by condensing phenol and formaldehyde at various 
desired molar ratios in the presence of a particular acid or base 
catalyst. However, due to the carcinogenic nature of formaldehyde, the use 
of formaldehyde in phenolic resin production poses a major threat to the 
health and safety of personnel involved therein. In addition to the 
aforementioned health and safety risks associated with the use of 
formaldehyde in phenolic resin production, residual amounts of 
formaldehyde are typically present in the finished product--a result that 
is both undesirable and largely unavoidable. 
One alternative to using formaldehyde to prepare phenolic resins has been 
to use inorganic catalysts (e.g., copper halide+an aliphatic amine) or 
biocatalysts (e.g., peroxidase enzymes). In addition to avoiding the 
problems associated with the use of formaldehyde, phenolic polymers 
produced by enzymatic reactions typically have the additional advantage of 
having extensive backbone conjugation, thereby leading to conductivity 
under doped conditions. Other advantages of using enzymes to catalyze 
phenol polymerization include mild reaction conditions, fast reaction 
rates, high substrate specificity and minimal by-product formation. 
Horseradish peroxidase is the most commonly used enzyme for these 
polymerization reactions, which are typically carried out in solvent/water 
mixtures and microemulsions. Polymer molecular weight and polydispersity 
are controllable using the aforementioned enzymatic method. 
One shortcoming that has been noted in connection with the aforementioned 
phenolic resins is that they have limited solubility in water. As a 
result, such resins are limited in their utility in certain applications, 
such as in use in rechargeable batteries, photolithography, fire 
retardants and detergents. For example, polypyrrole is the most widely 
used conjugated polymer in enzyme-mediated glucose detection. 
Polythiophene, polydiacetyline and polyaniline are a few other polymers 
used in this context. However, these polymers lack the flexibility to 
incorporate recognition sites into their backbone and/or an ease of 
synthesis and/or modification. Polypyrroles, for example, are usually 
obtained as water-insoluble films which are used to physically trap or to 
sandwich biomolecules in the preparation of biosensing elements. 
Incorporation by covalently linking the biomolecules to the polymer 
backbone requires the use of functionalized monomers with carboxyl or 
amine groups. Such functionalized thiophene monomers have been used to 
develop a generic molecular assembly for pesticide sensor applications. 
In U.S. Pat. No. 5,508,180, inventors Johnson et al., which issued Apr. 16, 
1996, and which is incorporated herein by reference, there is disclosed a 
biocatalytic oxidative process for preparing phenolic resins using soybean 
peroxidase. According to said patent, phenols which are preferred for 
polymerization are represented by the following formula: 
##STR2## 
wherein Y and Z are selected from the group consisting of a hydrogen atom, 
a halogen atom, an alkyl group, an alkoxy group, an aryl group, an allyl 
group, a phenylalkyl group, a --COOR group, a --NR.sup.1 R.sup.2, where R 
represents a hydrogen atom or a lower alkyl group, and R.sup.1 and R.sup.2 
represent a hydrogen atom, an alkyl group, or a phenylalkyl group or Z in 
conjunction with the adjacent meta position forms a condensed benzene 
ring. Since polymerization proceeds via the ortho or para positions, when 
Y is at the ortho or para position, at least one of Y and Z must be a 
hydrogen atom or Z must form said condensed benzene ring. Y is preferably 
para to the phenolic hydroxyl group. Otherwise, the phenol adds as a 
terminal group as discussed below. At the para position, long chain alkyl 
groups have a tendency to slow the reaction. The reaction appears to 
proceed best when Y is p-phenyl, p-methoxy or p-halogen. A single phenol 
or a mixture of phenols may be used in the polymerization reaction. In 
certain applications, it may be desirable to produce phenolic resins 
having certain terminal groups. 
In U.S. Pat. No. 5,420,237, inventors Zemel et al., which issued May 30, 
1995, and which is incorporated herein by reference, there is disclosed a 
method for enzymatically synthesizing electrically conductive and 
substituted and unsubstituted polyanilines. According to said patent, 
aniline monomer(s), an oxidizing agent, which comprises an enzyme and an 
electron acceptor, and an acidifying agent are reacted together to form 
polyanilines. The patent identifies the following as illustrative of such 
aniline monomers: 
##STR3## 
wherein n is an integer from 0 to 4; m is an integer from 1 to 5 with the 
proviso that the sum of n and m is equal to 5 and that at least one 
position on the aniline ring is a moiety which allows oxidative coupling 
at that position; R.sub.1 is a hydrogen or a permissible R.sub.2 
substituent; and R.sub.2 is the same or different at each occurrence and 
is selected from the group consisting of alkyl, alkenyl, alkoxy, 
cycloaklyl, cycloalkenyl, alkanoyl, alkylthio, aryloxy, alkylthioalkyl, 
alkylaryl, arylalkyl, amino, alkylamino, dialkylamino, aryl, 
alkylsulfinyl, aryloxyalkyl, alkylsulfinylalkyl, alkoxyalkyl, 
alkylsulfonyl, aryl, arylthio, alkylsulfonyl, carboxylic acid, hydroxy, 
halogen, cyano, sulfonic acid, nitro, mercapto, alkylsilane or alkyl 
substituted with one or more sulfonic acid, carboxylic acid, halo, nitro, 
mercapto, cyano, or epoxy moieties; or any two R.sub.2 groups together may 
form an alkylene or alkenylene chain completing a 3, 4, 5, 6 or 7 membered 
aromatic or alicyclic ring, which ring may optionally include one or more 
divalent nitrogen, sulfur, sulfonyl, ester, carbonyl, sulfonyl, or oxygen 
atoms, or R.sub.2 is an aliphatic moiety having repeat units of the 
formula: 
EQU --(OCH.sub.2 CH.sub.2)qO-- or --(OCH.sub.2 CH(CH.sub.3))qO-- 
in which q is a positive whole number. A moiety which allows oxidative 
coupling is any moiety that does not hinder the head-to-tail coupling of 
the monomers in forming polyaniline. An example of such a moiety is 
hydrogen or deuterium. 
In Ayyagari and Akkara, "Enzymatic synthesis of multifunctional polyphenols 
for biosensor applications," Polymeric Materials Science & Engineering, 
76:610-611 (1997), which is incorporated herein by reference, there is 
disclosed the copolymerization of p-ethylphenol (EP) and 
3-(4-hydroxyphenyl)propionic acid (HPPA) at different molar ratios. 
According to the publication, the copolymerization reaction involves 
dissolving the monomers in ethanol and adding HEPES buffer (1 mM, pH 7.5) 
to obtain a final ethanol content of about 25% (v/v). The final monomer 
concentration is 0.15M. An aliquot of enzyme solution, prepared by 
dissolving horseradish peroxidase (Type II) in 
N-[2-hydroxyethyl]piperazine-N'[2-ethanesulfonic acid] (HEPES) buffer at a 
known concentration, is added to the monomer solution such that the final 
enzyme concentration is 0.1 mg/ml. The reaction is initiated by dropwise 
addition of H.sub.2 O.sub.2. The total amount of H.sub.2 O.sub.2 added is 
30% in excess of stoichiometric amount. The reactions are continued for 24 
hours. At the end, solvent is evaporated under the flow of nitrogen at 
room temperature. The dry material is washed with isooctane and water 
separately to remove unreacted monomers, salts and the enzyme. The 
material is then dried at 40.degree. C. under vacuum. 
Additional patents of interest include the following, all of which are 
incorporated herein by reference: U.S. Pat. No. 5,639,806, inventors 
Johnson et al., issued Jun. 17, 1997; U.S. Pat. No. 5,606,010, inventors 
Erhan et al., issued Feb. 25, 1997; U.S. Pat. No. 5,367,043, inventors 
Butler et al., issued Nov. 22, 1994; U.S. Pat. No. 5,278,055, inventors 
Cyrus, Jr. et al., issued Jan. 11, 1994; U.S. Pat. No. 4,900,671, 
inventors Pokora et al., issued Feb. 13, 1990; U.S. Pat. No. 4,882,413, 
inventor Erhan, issued Nov. 21, 1989; and U.S. Pat. No. Re. 35,247, 
inventors Cyrus, Jr. et al., reissued May 21, 1996. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide novel polyaromatic 
compounds and a method for their production. 
According to one aspect of the present invention, there is disclosed a 
polyaromatic compound made by polymerizing one or more aromatic monomers, 
at least one of said one or more aromatic monomers having the following 
structure: 
##STR4## 
wherein X is selected from the group consisting of OH and NH.sub.2, Y is 
selected from the group consisting of a halogen, a halogen-containing 
group, a boron-containing group, a phosphorus-containing group, a carboxyl 
group, a hydroxyacid group, a ketoacid group, an amino group, an amino 
acid group, an amide group, an ester group, a hydroxyl group and a 
sulfonic acid group, R is an alkyl, phenyl, naphthyl, hydroxyphenyl, 
hydroxy naphthyl or hydroxy (.alpha., .beta., .gamma.) alkyl group, and n 
is an integer from 0 to 12. Preferably, the (R).sub.n --Y group is either 
ortho or para to X; more preferably the (R).sub.n --Y group is para to X. 
One benefit associated with the polyaromatic compounds of the present 
invention is that said polyaromatic compounds can be prepared with a 
controlled functional group density and type of desired functional groups. 
For example, using a particular density of ionizable functional groups, 
the resultant polymer can be imbued with a desired solubility for a given 
solvent or mixtures of solvents. Accordingly, such a polymer may be 
well-suited for use in the fields of photoresists, rechargeable batteries 
and detergency. In a like manner, a polymer made containing halogen 
functional groups of a particular density (or number of functional groups 
per unit mole of polymer) should exhibit fire resistant properties. It is 
expected that such a polymer can also be blended with nylon 6 to enhance 
the fire retardant and fire resistant properties of the nylon. 
Another benefit associated with the polyaromatic compounds of the present 
invention is that said polyaromatic compounds can be prepared so as to be 
amenable for derivatization (e.g., for attachment of a particular type of 
ligand thereto) based upon specific end-use applications. 
Still another benefit associated with the polyaromatic compounds of the 
present invention is that said polyaromatic compounds can be generated on 
a large scale in either a batch mode or a continuous mode of operation, 
with high polymer yields (e.g., 95% yield); at the same time, monomer 
conversion and polymer molecular weight can also be controlled where 
desired. 
Additional objects, features, aspects and advantages of the present 
invention will be set forth, in part, in the description which follows 
and, in part, will be obvious from the description or may be learned by 
practice of the invention. Certain embodiments of the invention will be 
described hereafter in sufficient detail to enable those skilled in the 
art to practice the invention, and it is to be understood that other 
embodiments may be utilized and that structural or other changes may be 
made without departing from the scope of the invention. The following 
detailed description is, therefore, not to be taken in a limiting sense, 
and the scope of the present invention is best defined by the appended 
claims.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
As noted above, the present invention is directed to a class of 
polyaromatic compounds and to a method for their production. The 
polyaromatic compounds of the present invention are made by polymerizing 
one or more aromatic monomers, at least one of said one or more aromatic 
monomers having the following structure: 
##STR5## 
wherein X is selected from the group consisting of OH and NH.sub.2, Y is 
selected from the group consisting of a halogen (e.g., bromine), a 
halogen-containing group, a boron-containing group, a 
phosphorus-containing group, a carboxyl group, a hydroxyacid group, a 
ketoacid group, an amino group, an amino acid group, an amide group, an 
ester group, a hydroxyl group and a sulfonic acid group, R is an alkyl, 
phenyl, naphthyl, hydroxyphenyl, hydroxy naphthyl or hydroxy (.alpha., 
.beta., .gamma.) alkyl group, and n is an integer from 0 to 12. 
Preferably, the (R).sub.n --Y group is either ortho or para to X; more 
preferably the (R).sub.n --Y group is para to X. 
Examples of aromatic monomers of formula (I) in which the (R).sub.n --Y 
group includes a carboxyl group are 4-hydroxybenzoic acid, 4-hydroxyphenyl 
acetic acid, 3-(4-hydroxyphenyl)propionic acid, 4-hydroxycinnamic acid and 
4-(4-hydroxyphenyl)benzoic acid. 
An example of an aromatic monomer of formula (I) in which the (R).sub.n --Y 
group includes a hydroxyacid group is 4-hydroxyphenyl lactic acid. An 
example of an aromatic monomer of formula (I) in which the (R).sub.n --Y 
group includes a ketoacid group is 4-hydroxyphenyl pyruvic acid. 
Examples of aromatic monomers of formula (I) in which the (R).sub.n --Y 
group includes an amino group are 4-hydroxyphenyl methyl amine, 
4-hydroxyphenyl ethyl amine and N-(4-hydroxyphenyl)glycine. Examples of 
aromatic monomers of formula (I) in which the (R).sub.n --Y group includes 
an amino acid group are 4-hydroxyphenylglycine and 4-hydroxyphenylalanine. 
Examples of aromatic monomers of formula (I) in which the (R).sub.n --Y 
group includes an amide group are 4-hydroxyphenylacetamide, sulfanilamide, 
4-hydroxybenzamide and N-(4-hydroxyphenyl)stearamide. 
Examples of aromatic monomers of formula (I) in which the (R).sub.n --Y 
group includes an ester group are tyrosine methyl ester, tyrosine ethyl 
ester, tyrosine propyl ester, methyl 4-hydroxybenzoate, methyl 
4-hydroxyphenyl acetate, propyl 4-hydroxybenzoate and 
3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester. Examples of 
aromatic monomers of formula (I) in which the (R).sub.n --Y group includes 
an alcohol are 4-hydroxyphenylmethyl alcohol, 4-hydroxyphenylethyl 
alcohol, 4-hydroxyphenylpropyl alcohol, and bisphenol A. Examples of 
aromatic monomers of formula (I) in which the (R).sub.n --Y group includes 
a sulfonic acid group are 4-hydroxybenzene sulfonic acid, 
4-hydroxyphenylethane sulfonic acid, 4-aminobenzene sulfonic acid, 
sulfanilamide and diaminobenzene sulfonic acid. 
Examples of aromatic monomers of formula (I) in which the (R).sub.n --Y 
group includes a halogen are 2-bromophenol, 4-bromophenol, 4-chlorophenol, 
4-hydroxyphenyl methyl bromide, 3,5-bis(trifluoromethyl)phenol and 
4-hydroxyphenyl ethyl bromide. 
The polymers of the present invention may be homopolymers consisting of one 
species of aromatic monomer of formula (I), copolymers consisting of two 
or more species of aromatic monomers of formula (I), or copolymers 
comprising one or more species of aromatic monomer of formula (I) and one 
or more species of an aromatic monomer not of formula (I), such as a 
p-alkylphenol (e.g., p-ethylphenol). 
Preferably, the polyaromatic compounds of the present invention are 
prepared by reacting together said one or more aromatic monomers with 
hydrogen peroxide in the presence of horseradish peroxidase, the reaction 
taking place in water or in a mixture of water and another polar solvent 
(e.g., ethanol). The total amount of hydrogen peroxide added to the 
reaction mixture is preferably in excess of that of the monomer; however, 
to prevent denaturation of the horseradish peroxidase, the hydrogen 
peroxide is preferably added to the reaction mixture in small increments 
over an extended period of time (e.g., one-tenth of total amount added 
every thirty minutes). 
Instead of using horseradish peroxidase to catalyze the polymerization 
reaction, other oxidases and peroxidases may be used, such as lignin-, 
Coprinus cinerus-, and soybean peroxidases and isoenzymes of horseradish 
and soybean peroxidases; tyrosinases; laccases; phenoloxidases; aromatic 
amineoxidases; and cytochrome oxidase. Moreover, catalysts other than 
peroxidases may be used, such as heme and heme-containing compounds, 
examples of which include hemoglobin, hematin, cytohemin, myoglobin and 
cytochromes (e.g., c, c1, c2, c3, c551, f, and P450). 
In those instances in which the polymerization reaction is conducted under 
aerobic conditions and tyrosinase is used as the biocatalyst, hydrogen 
peroxide need not be added to the reaction mixture since tyrosinase will 
produce a sufficient amount of hydrogen peroxide from air for the 
polymerization reaction. Hydrogen peroxide can also be generated in situ 
and, therefore, need not be added to the reaction mixture (regardless of 
the particular biocatalyst used) by adding, under aerobic conditions, 
glucose oxidase and glucose to the reaction mixture. 
It is to be understood that peroxides other than hydrogen peroxide, 
examples of which include perbenzoic acid and peracetic acid, may be used 
instead of hydrogen peroxide in the polymerization reaction. 
The pH of the reaction mixture is preferably in the range of about 2-8, 
more preferably 3-7, even more preferably 3-5. Moreover, to obtain a 
substantial yield, the pH of the reaction medium should be around or below 
the pK.sub.a of the functional group of the monomer(s) being polymerized 
(i.e., a pH of approximately 3-4 as in 4-hydroxyphenyl propionic acid). 
One advantage to using some of the non-peroxidase biocatalysts identified 
above is that they are not as sensitive as are the peroxidases to being 
adversely affected by the low pH conditions necessary to polymerize 
monomers whose functional groups have low pK.sub.a values. Therefore, when 
using such non-peroxidase biocatalysts, even lower pH conditions than 
those specified above may be possible. 
The experiments described below are intended merely to be illustrative of 
the principles of the present invention and are in no way intended to be 
limiting. 
Materials and Methods 
All reagents were obtained from Aldrich Chemical Company (Milwaukee, Wis.). 
Horseradish peroxidase (Type II) was supplied by Sigma Chemical Company 
(St. Louis, Mo.). Reaction mixtures were prepared by first dissolving the 
monomer in HEPES buffer (1 mM to 100 mM, pH 7.5) or a mixture of ethanol 
and buffer. Enzyme dissolved in buffer was added, and the reaction was 
initiated by adding hydrogen peroxide in small aliquots to a 30% 
stoichiometric excess. At the end of the reaction, polymers were 
precipitated by adding KCl, when necessary, and washed repeatedly with 
water and/or mixture of ethanol and water to remove monomer, salts and 
enzyme. The polymer was then dried under vacuum at not more than 
50.degree. C. Polymer yield was calculated as the weight ratio of the dry 
polymer product obtained finally to the weight of the monomer added at the 
beginning of the reaction. Polymer molecular weight was obtained on a 
Waters instrument (LC Module 1, Milford, Mass.) fitted with a gel 
permeation column (GBR mixed bed linear column, 100 to 20+million, Jordi 
Associates, Bellingham, Mass.). A UV detector calibrated at 270 nm was 
used with the instrument and the data was processed with Millenium 2010 
Chromatography Manager software (Waters, Milford, Mass.). Polystyrene was 
used as a relative standard and N,N-dimethylformamide containing 1% LiBr 
was used as the eluent. The polymer samples were coated on Zn-Se slides to 
record infrared spectra on a Perkin-Elmer 1760 FTIR-FT Raman 
spectrophotometer (Norwalk, Conn.) at a 4 cm.sup.-1 resolution. 
Results and Discussion 
The conventional reaction scheme for the synthesis of polyaromatic 
compounds using horseradish peroxidase (HRP) as a catalyst is as follows: 
##STR6## 
A detailed discussion of HRP-catalyzed reactions is available elsewhere 
(e.g., Ayyagari et al., Acta Polymerica, 47:193-203 (1996), which is 
incorporated herein by reference). Briefly, stoichiometric amount of 
hydrogen peroxide is necessary for the oxidative coupling among the 
monomer units and growing oligomers. Table I summarizes the results for 
reactions involving 4-hydroxybenzoic acid (4-HBA), 4-hydroxyphenyl acetic 
acid (4-HPAA) and 3-(4-hydroxyphenyl)propionic acid (4-HPPA), 
4-hydroxybenzene sulfonic acid (HBSA), sulfanilinic acid (SAA) and 
2,5-diaminobenzene sulfonic acid (2,5-DABSA). 
TABLE I 
______________________________________ 
pH adjusted by 
addition of 
Monomer HCl/NaOH Buffer Comments 
______________________________________ 
4-HPPA (i) yes (pH 
(i) 0.1M (i,ii) reaction mixture 
adjusted from HEPES, pH 7.5 transparent and turned yel- 
4.3 to 7.5 with (ii) 0.1M low; no polymer precipitate 
NaOH) HEPES, pH 7.5 formed; identical results 
(ii) no (pH 4.3) (iii) 0.1M with 0% to 20% ethanol in 
(iii) yes (pH HEPES, pH 7.5 the medium 
adjusted from (iv) 0.01M (iii) reaction mixture turned 
4.3 to 3.0 with HEPES, pH 7.5 yellow and opaque; poly- 
HCl) (v) 0.01M mer yield was 70% 
(iv) yes (pH HEPES, pH 7.5 (iv) reaction mixture trans- 
adjusted from (vi) no buffer parent and turned reddish 
4.3 to 7.4 with brown; no polymer precipi- 
NaOH) tate 
(v) no (pH 3.3) (v) reaction mixture turned 
(vi) no (2.4) yellow & opaque; polymer 
yield 70% with 0% ethanol 
(100% buffer) and 85% 
with 20% ethanol (80% 
buffer) in the reaction 
medium 
(vi) reaction mixture turned 
yellow and opaque; poly- 
mer yield 50% medium 
4-HPAA (i) yes (pH (i) 0.1M (i) reaction mixture trans- 
adjusted from HEPES, pH 7.5 parent and turned yellow; 
3.7 to 7.5 with (ii) 0.1M no polymer precipitate 
NaOH) HEPES, pH 7.5 with 0% to 20% ethanol 
(ii) no (pH 3.1) (iii) no buffer media 
(iii) no (ii) reaction mixture turned 
(pH 2.3) yellow & opaque. Polymer 
precipitate formed after 
long time with about 15% 
yield in 20% ethanol 
(iii) reaction mixture turned 
yellow and slightly opaque; 
no polymer precipitate 
formed 
4-HBA (i) yes (pH (i) 0.1M (i) reaction mixture dark 
adjusted from HEPES, pH 7.5 brown instantly; no poly- 
3.5 to 7.5 with (ii) 0.01M mer precipitate formed 
NaOH) HEPES, pH 7.5 (ii) reaction mixture turned 
(ii) no (pH 3.0) brown and opaque; polymer 
precipitate formed after 
long time with about 10% 
yield in 20% ethanol 
4-HBSA yes (pH 0.01M HEPES, reaction mixture turned 
adjusted from pH 7.5 dark brown at higher pH; 
0.4 to x with very small amount of 
NaOH) polymer precipitate at 
x = 1-7 low pH values 
SAA and no (pH 5.7) 0.1M phos- reaction mixture turned 
2,5- phate, pH 6.0 dark with both monomers; 
DABSA reaction mixture with 
2,5-DABSA was dialyzed 
against water, evaporated 
to dryness, precipitate 
extracted with methanol 
and molecular weight 
determination showed 
mostly monomers and 
dimers 
______________________________________ 
Clearly, only 4-HPPA was polymerizable with high yields while 4-HPAA and 
4-HBA yielded much lower amounts of corresponding polymers under optimized 
conditions. Buffer strength and, consequently, pH of the monomer solution 
were found to have a profound effect on the polymerizability in these 
reactions. Although this observation may not be surprising from the 
viewpoint of pH effects on enzyme activity, it is interesting to note how 
the degree of ionization of the carboxy groups on the monomers affects 
their polymerizability. Table I summarizes the results from these 
experiments conducted in triplicate. Unless noted otherwise, all reactions 
were carried out either in 100% aqueous phase or in 20% ethanol/buffer 
mixtures. In general, reactions at any buffer concentration and at a pH&gt;4 
yielded little or no polymer product. For example, the pH of a 0.1 M 
solution of 4-HPPA in 0.1 M HEPES buffer (pH 7.5) was 4.3, which is close 
to the pK.sub.a (about 4.5) of the carboxy group in the monomer. Little or 
no polymer formation was observed in this system. On the other hand, the 
pH of a 0.1 M solution of the same monomer in 10 mM HEPES buffer (pH 7.5) 
was 3.2, and formation of poly(4-HPPA) was significant. Moreover, 
adjusting the pH of any solution to the optimal pH value (about 7) for the 
enzyme clearly was not useful for the reaction. Thus, polymerization of 
4-HPPA was possible at pH values less than the pK.sub.a value, leading to 
the theory that the ionization of the carboxy functionality plays a 
significant role in the polymerizability of a given monomer. 
Polymerization of these monomers was possible even in the absence of 
buffer so long as the pH of the monomer solution was adjusted to about 3, 
and reasonably good polymer yield was obtained. It should be noted, 
however, that such low pH conditions are generally unsuitable for the 
optimal performance of HRP. According to the supplier, HRP used in this 
study has an optimal pH of 6, and the activity drops significantly as the 
pH is varied from 6. That the yields of poly(4-HPAA) and poly(4-HBA) were 
poor at low pH values is perhaps indicative of the necessity of further 
lowering pH values (note that their pK.sub.a values are lower, albeit 
slightly, than that of 4-HPPA) for efficient catalysis and the enzyme 
activity is affected at such low pH conditions. It can be concluded from 
these results that carboxy phenols in their ionized state (occurring 
significantly at pH&gt;4) are not polymerizable under these conditions and 
that HRP is much more active up to and above a pH of about 3. This leads 
to a decrease in polymerization efficiency on the order of 4-HPPA to 
4-HPAA to 4-HBA as the pK.sub.a of monomers decreases in the same order. 
Sulfonic acids being stronger acids than carboxy acids, the operating pH 
required to keep sufficient number of the sulfonate groups protonated was 
too low for HRP to be catalytically active to a substantial degree. No 
polymerization was possible in solutions at pH&gt;3 as the pK.sub.a for the 
monomer was much lower than 3. Indeed, as noted in Table I, under the 
conditions at hand, sulfonated phenol (4-HBSA) and aniline (SAA and 
2,5-DABSA) were not polymerizable in the pH range of 1-7. However, in 
cases where the enzyme deactivation kinetics are slower than the reaction 
kinetics, some degree of polymerization is possible. 
4-ethylphenol (4-EP) was copolymerized with 4-HPPA to demonstrate that the 
number density of functional groups in the copolymer can be controlled 
simply by adjusting the molar ratio between the monomers. FIG. 1 shows the 
FT-IR spectra of the copolymers at different 4-EP to 4-HPPA molar ratios. 
The intensity ratio for the peaks at 1710 cm.sup.-1 (C.dbd.O stretch) to 
2900 cm.sup.-1 (C-H stretch) clearly increased as the molar fraction of 
4-HPPA increased in the copolymer. Molecular weights ranging from 700 
(pentamers) to 2500 could be obtained. Subsequent to the synthesis of the 
EP/4-HPPA copolymers, the carboxy groups were further derivatized to 
attach 4-amino TEMPO (a 2,2,6,6-tetramethyl-1-piperidine-N-oxyl spin label 
for electron paramagnetic resonance (EPR) spectroscopy) and the binding 
was ascertained by EPR spectroscopy. In a separate experiment, the 
copolymer of 4-EP and tyramine was prepared similar to 4-EP/4-HPPA 
copolymer, and derivatized with biotin for subsequent specific binding to 
the conjugates of the protein streptavidin. It is thus possible to 
enzymatically synthesize polyphenols with desired functional groups. 
Ionizable polymers of 4-HPPA and some of its copolymers with 4-EP are 
soluble in water under neutral and slightly alkaline conditions. Coupled 
with the conjugated nature of their backbones, such polymers find 
applications in detergency, batteries and biosensors. 
Poly(4-bromophenol) or P4BP was synthesized for the fire retardant 
properties of the bromopolymer. The reaction was conducted in 100% aqueous 
media and in media containing 20% ethanol. There was a significant 
improvement in both the polymer yield (from 30% to 90%) and molecular 
weight (from 1500 to 2500) in the presence of ethanol, a fact noted 
earlier in the synthesis of polycresol (see Ayyagari et al., Acta 
Polymerica, 47:193-203 (1996)). Interestingly, the pH of the reaction 
medium dropped as the polymerization proceeded in 100% aqueous media. 
These changes are reported below in TABLE II. 
TABLE II 
______________________________________ 
Reaction pH Changes of 
Medium Buffer Reaction Medium Comments 
______________________________________ 
(i) 100% buffer 
1 mM HEPES, 
pH dropped to 
(i,ii,iii) Polymer 
pH 7.5 2.3 on yield and molecular 
polymerization weight, 30% and 
(ii) 10 mM pH dropped to 1500, respectively, 
100% buffer HEPES, pH 7.5 6.8 on low compared to a 
polymerization reaction medium 
(iii) 25 mM pH dropped to with 20% ethanol 
100% buffer HEPES, pH 7.5 7.2 on 
polymerization 
(iv) 80% buffer 1 mM HEPES, pH dropped to (iv,v,vi) Polymer 
20% ethanol pH 7.5 2.3 on yield and molecular 
polymerization weight &gt;90% and 
2500, respectively, 
are higher compared 
to 100% buffer 
reaction medium 
(v) 80% buffer 10 mM pH dropped to (i,ii,iii,iv,v,vi) 
20% ethanol HEPES, pH 7.5 6.8 on Elemental analysis 
polymerization of the polymer 
(vi) 80% buffer 25 mM pH dropped to prepared indicated 
20% ethanol HEPES, pH 7.5 7.2 on following composi- 
polymerization tion (elemental ana- 
lysis of the mono- 
mer is given in the 
bracket): carbon 
47.7% (41.7%), 
hydrogen 2.8% 
(2.9%), oxygen 
11.7% (9.2%), 
bromine 35.4% 
(46.2%). Calculated 
elemental compo- 
sition of 12-mer 
polymer with 66% 
theoretical brom- 
ine: C - 49.7%, 
H - 2.5%, O - 
11.0%, & Br - 
36.7%. 
______________________________________ 
Elemental analysis of the product showed a 33% loss of bromine in the 
polymer. It remains to be studied whether this loss is a result of the 
elimination of bromine from the ring due to bond formation at para 
position and whether the loss of bromine contributes to the drop in pH. 
The embodiments of the present invention recited herein are intended to be 
merely exemplary and those skilled in the art will be able to make 
numerous variations and modifications to it without departing from the 
spirit of the present invention. All such variations and modifications are 
intended to be within the scope of the present invention as defined by the 
claims appended hereto.