U.S. patent application number 09/014522 was filed with the patent office on 2001-07-19 for large-scale production of polyphenols or polyaromatic amines using enzyme-mediated reactions.
Invention is credited to AKKARA, JOSEPH A., AYYAGARI, MADHU S. R., KAPLAN, DAVID L..
Application Number | 20010008768 09/014522 |
Document ID | / |
Family ID | 21765969 |
Filed Date | 2001-07-19 |
United States Patent
Application |
20010008768 |
Kind Code |
A1 |
AKKARA, JOSEPH A. ; et
al. |
July 19, 2001 |
LARGE-SCALE PRODUCTION OF POLYPHENOLS OR POLYAROMATIC AMINES USING
ENZYME-MEDIATED REACTIONS
Abstract
A process for large-scale, low cost, batch or continuous
production of polyphenols using enzyme-mediated reactions and
methods for recycling non-consumed reactants.
Inventors: |
AKKARA, JOSEPH A.;
(HOLLISTON, MA) ; AYYAGARI, MADHU S. R.;
(BRIGHTON, MA) ; KAPLAN, DAVID L.; (STOW,
MA) |
Correspondence
Address: |
U S ARMY SOLDIER SYSTEMS COMMAND
OFFICE OF CHIEF COUNSEL
PATENT COUNSEL
KANSAS STREET
NATICK
MA
017605035
|
Family ID: |
21765969 |
Appl. No.: |
09/014522 |
Filed: |
January 28, 1998 |
Current U.S.
Class: |
435/122 ;
435/156 |
Current CPC
Class: |
C12P 17/12 20130101;
C12P 7/22 20130101 |
Class at
Publication: |
435/122 ;
435/156 |
International
Class: |
C12P 017/12; C12P
007/22 |
Claims
What is claimed is:
1. A low cost method for producing polyphenols or polyaromatic
amines in large scale comprising the steps of polymerizing a
monomer in pre-reaction components in a reactor, wherein
polymerization is catalyzed by an enzyme, yielding phenolic or
aromatic amine polymers, non-consumed reactants, and non-consumed
reaction medium, wherein the non-consumed reactants and
non-consumed reaction medium together comprise post-reaction
components; isolating the polymers from the post-reaction
components; and recycling at least a portion of the post-reaction
components.
2. The method of claim 1, wherein said non-consumed reactants
include hydrogen peroxide, enzyme and monomers, and wherein said
non-consumed reaction medium includes a reversed micellar solution
comprising water, a solvent and a surfactant.
3. The method of claim 1, wherein said non-consumed reactants
include hydrogen peroxide, enzyme and monomers, and wherein said
non-consumed reaction medium includes a buffer and a water-miscible
solvent.
4. The method of claim 1, wherein said non-consumed reactants
include hydrogen peroxide, enzyme and monomers, and wherein said
non-consumed reaction medium includes water.
5. The method of claim 1, wherein said non-consumed reactants
include hydrogen peroxide, enzyme and monomers and the non-consumed
reaction medium includes two phases, wherein said first phase
comprises water that dissolves hydrogen peroxide, enzyme and some
monomer, and said second phase comprises a water-immiscible solvent
that dissolves the remaining monomer; and said reactor comprising a
stirred tank to create a dynamic emulsion.
6. The method of claim 1, wherein said polymer is isolated from
said post-reaction components by means of a filtration unit
yielding isolated polymers and a filtrate, said filtrate comprising
said post-reaction components.
7. The method of claim 1, wherein said polymer is isolated from
said post-reaction components by means of a centrifuge yielding
isolated polymers and a filtrate, said filtrate comprising said
post-reaction components.
8. The method of claim 1, wherein the recycling step includes
recycling a solvent.
9. The method of claim 1, wherein the recycling step includes
isolating a sample of the post-reaction components by means of a
sampling port; analyzing said sample by an analyzing means to
determine concentration and volume levels of the post-reaction
components; recycling said post-reaction components into a mixing
unit; communicating said concentration and said volume levels for
said sample by a first communication means from said analyzing
means to a feed back controller which determines calculated amounts
of monomer and pre-reaction components, said amounts of monomer and
pre-reaction components based on predetermined optimal ratios, to
be added to said post-reaction components in said mixing unit,
wherein said monomer, pre-reaction components and post-reaction
components comprise a reaction solution; and introducing said
reaction solution into said reactor.
10. The method of claim 3, wherein said enzyme is immobilized on an
inert support, said support provided in said reactor.
11. A low cost method for producing polyphenols or polyaromatic
amines in large scale comprising the steps of mixing pre-reaction
components comprising reversed micelles; an enzyme, horseradish
peroxidase; and hydrogen peroxide in a mixing port; introducing
said pre-reaction components and a monomer into a reactor, said
monomer selected from the group consisting of
2,6-Dihydroxy-naphthalene p-Ethylphenol p-Naphthol p-butylphenol
1-Pyrenol Aniline Benzidine p-Hydroxythiophenol; polymerizing said
monomer in said pre-reaction components in said reactor yielding a
phenolic or aromatic amine polymer, non-consumed reactants, and
non-consumed reaction medium, wherein said non-consumed reactants
and non-consumed reaction medium comprise post-reaction components;
isolating said polymer from said post-reaction components by an
isolating means; dehumidifying said post-reaction components by
means of molecular sieves; isolating a sample of said post-reaction
components by means of a sampling port; analyzing said sample by an
analyzing means to determine concentration and volume levels of the
post-reaction components; recycling said post-reaction components
into a mixing unit; communicating said concentration and said
volume levels for said sample by a first communication means from
said analyzing means to a feed back controller which determines
calculated amounts of monomer and reaction components, said amounts
of monomer and reaction components based on predetermined optimal
ratios, to be added to said post-reaction components in said mixing
unit, wherein said monomer, pre-reaction components and
post-reaction components comprose a reaction solution; and
introducing said reaction solution into said reactor.
12. The method of claim 1, wherein said enzyme is immobilized on an
inert support, said support provided in said reactor.
13. The method of claim 1 wherein the reactor comprises a packed
bed reactor.
14. The method of claim 1, wherein said reactor comprises a plug
flow reactor.
15. A low cost method for producing polyphenols or polyaromatic
amines comprising the steps of mixing pre-reaction components: said
pre-reaction components comprising a bulk organic solvent mixture;
horseradish peroxidase; water; a surfactant; and hydrogen peroxide
in a mixing port; introducing said reaction components and a
monomer into a reactor, said monomer selected from the group
consisting of Anisole Aniline p-Amino m-cresol 1,2-Benzenediol
Benzidine p-n-Butylphenol p-n-Butylaniline 8-Hydroxyquinoline
Cresols (p, m, & p) p-sec-Butylaniline p-Hydroxythiophenol
1,3-Dihydroxynaphthalene 2,6-Diethylaniline Isoquinoline
1,5-Dihydroxynaphthalene 3,5-Diethylaniline 2-Methyl 8-quinolinol
3,4-Dimethylphenol 2,6-Dimethylaniline p-Phenylazophenol
p-Ethylphenol 3,5-Dimethylaniline Tyrosine Methoxyphenol (p &
m) p-Ethylaniline Naphthol (.alpha. & .beta.)
3-Phenylenediamine p-n-Octylphenol p-n-Propylaniline Phenol
p-Phenoxyphenol Phenylphenol (m & p)
3-(3-Phenoxyphenoxy)phenol; polymerizing said monomer in said
pre-reaction components in said reactor yielding a phenolic or
aromatic amine polymer, non-consumed reactants, and non-consumed
reaction medium, wherein said non-consumed reactants and
non-consumed reaction medium together are post-reaction components;
introducing said polymer and said post-reaction components into a
filtration unit; isolating said polymer from said post-reaction
components by an isolating means; isolating a sample of said
post-reaction components by means of a sampling port; analyzing
said sample by an analyzing means to determine concentration and
volume levels of said post-reaction components; recycling at least
a portion of said post-reaction components; communicating said
concentration and said volume levels for said sample by a first
communication means from said analyzing means to a feed back
controller which determines calculated amounts of monomer and
reaction components, said amounts of monomers and reaction
components based on predetermined optimal ratios, to be added to
said post-reaction components in said mixing unit, wherein said
monomer, pre-reaction components and post-reaction components
comprise a reaction solution; and introducing said reaction
solution into said reactor.
16. The method of claim 15, wherein said enzyme is immobilized on
an inert support, said support provided in said reactor.
17. The method of claim 15, wherein the reactor comprises a packed
bed reactor.
18. The method of claim 15, wherein said reactor comprises a plug
flow reactor.
19. A low cost method for producing polyphenols and polyaromatic
amines in large scale comprising the steps of mixing a monomer and
pre-reaction components in a reactor; wherein said pre-reaction
components comprising water, an enzyme, hydrogen peroxide, and a
water-immiscible organic; stirring the monomer and pre-reaction
components in the reactor with a stirring means to create a dynamic
macroemulsion; polymerizing said monomer in said pre-reaction
components in said reactor yielding a phenolic or aromatic amine
polymer, non-consumed reactants, and non-consumed reaction medium,
wherein said non-consumed reactants and said non-consumed reaction
medium together are post-reaction components; introducing said
polymer and said post-reaction components into a filtration unit;
isolating said polymer from said post-reaction components by an
isolating means provided in said filtration unit; isolating a
sample of said post-reaction components by a means of a sampling
port; analyzing each said sample by an analyzing means to determine
concentration and volume levels for said post-reaction components;
recycling at least a portion of said post-reaction components into
a mixing unit; communicating said concentration and said volume
levels for said sample by a first communication means from said
analyzing means to a feed back controller which determines
calculated amounts of monomer and reaction components, said amounts
of monomer and reaction components based on predetermined optimal
ratios, to be added to said post-reaction components in said mixing
unit, wherein said monomer, pre-reaction components and
post-reaction components comprise a reaction solution; and
introducing said reaction solution back into the reactor.
Description
BACKGROUND OF INVENTION
[0001] Phenolic resins such as novolacs and resoles are
commercially produced by condensing phenol and formaldehyde at
various molar ratios in the presence of acid or base catalysts.
However, the confirmed carcinogenic nature of formaldehyde poses a
major threat to personnel involved in polyphenol production in
industry and to the end user. Residual amount of formaldehyde in
the finished product is unavoidable and undesirable.
[0002] Alternatively, inorganic catalysts or biocatalysts can be
used to produce polyphenols without the need for formaldehyde.
Enzymatically synthesized polyphenols may find applications in
coatings, laminates, wood composites, color developers, and
recording materials. Such materials could be cast into thin films
or fabric coatings. The polymers can also be tailored for
applications in the detergent industry. In addition, polyphenols
may be used in photolithography, rechargeable lightweight
batteries, and electromagnetic shielding. Enzymatic polymerization
of phenols and aromatic amines in mixtures of water-miscible
solvents and water was first reported by Klibanov and co-workers,
(J. S. Dordick, M. A. Marlett, A. M. Klibanov, Biotechnol. Bioeng.
1987, 30, 31-36.) and Pokora and Cyrus, (A. R. Pokora, W. L. Cyrus,
U.S. Pat. No. 4,647,952, 1987, Mead Corporation., U.S.A.).
[0003] There are numerous advantages to using enzymes to catalyze
phenol polymerization including mild reaction conditions, fast
reaction rates, high substrate specificity and minimal by-product
formation. Polymers produced by enzymatic reactions have the
additional advantage of having extensive backbone conjugation
leading to electronic and electro optic applications. Horseradish
peroxidase (HRP) is the most commonly used enzyme for these
polymerization reactions carried out in solvent/water mixtures and
microemulsions.
[0004] Dordick et al., Vol. # 30,1987 Biotechnol. Bioeng. 31-36,
used HRP in a dioxane/water system to prepare a number of polymers
and copolymers from various phenolic monomers. Akkara et al., 29 J.
Polym. Sci. A 1561 (1991), prepared polymers and copolymers of
various phenols and aromatic amines using these same reactions and
carried out detailed characterization of the polymer products.
Para-alkylphenols were also polymerized at oil-water (reversed
micelles) and air-water (Langmuir-Blodgett trough) interfaces.
Because of their amphophilic nature, the alkylphenols are
partitioned at the interface, and in the presence of HRP and
hydrogen peroxide the monomers are oxidatively coupled to form
polymers. The poly(para-alkylphenols) prepared in reverse micelles
were shown to exhibit relatively more uniform molecular weight
distribution than those prepared in bulk organic solvents.
[0005] However, earlier attempts to control the polymer molecular
weight and molecular weight distribution by varying the time of
reaction or hydrogen peroxide concentration were unsuccessful in
both reversed micelles and bulk solvents. Initial hydrogen peroxide
concentration was found to be stoichiometrically proportional to
the monomer conversion, a hallmark of stepwise polymerization and a
phenomenon observed previously, and there was no effect on the
polymer molecular weight and polydispersity.
[0006] The polymers can be modified by adding functional groups to
the polymeric backbone, significantly enhancing the utility of
these polymers. "Functionalization" enables the polymers to be used
to treat fabrics, to form selectively permeable membranes, and to
improve the performance of integrated circuit chips, among other
applications.
[0007] Palmitoyl chloride may be added to the polymer to make the
polymer easily processable, e.g., as coatings, films, or finishes.
Cinnamoyl chloride may be added to create controlled pore size
membranes (e.g., "molecular sieves") or to enhance the polymers'
ability to absorb UV radiation (e.g. photolithography, sunglasses,
etc.). In their latter use, the modified polymers are applied to a
silicon substrate as an undercoating (under non-functionalized
polyphenols or polyaromatic amines that are then applied as a spin
coating) in an IC chip to control the precision of UV etching, by
inhibiting UV scattering, of circuitry into the spin-coated polymer
layer. In addition, these cinnamoyl chloride-modified polymers are
very thermostable, which allows their use in a variety of
applications where heat is ordinarily a problem. In addition,
photosensitive functional groups may be added to enhance the
utility of the polymers in other applications.
[0008] The polymers also may be modified to create active matrices
and systems allowing the controlled-release of materials, such as
drugs, insecticides, and fertilizers. If biotin or other ligands
are added to the polymer chain, the polymer can be used as
chromatography packing, which may be used to separate and purify
proteins.
[0009] Despite the study of how the functionality of the polymers
varies depending upon whether, and with what, the molecules are
modified, it has not been shown that the molecular weight and the
molecular weight distribution (i.e. "polydispersity") of
polyphenols and polyaromatic amines also can significantly
influence the functional properties of the polymers.
[0010] Although the reactions employed by the invention are known
in the industry, processes have not been developed for large-scale
production of phenolic or aromatic amine polymers in
enzyme-mediated reactions from which non-consumed reactants are
recycled This invention relates to such a process for large-scale
production of phenolic or aromatic amine polymers in
enzyme-mediated reactions from which non-consumed reactants are
recycled. The process specifically relates to recycling the solvent
to minimize waste and lower processing costs.
SUMMARY OF INVENTION
[0011] It is therefore an object of this invention to provide a low
cost process for large-scale production of polyphenols or
polyaromatic amines using enzyme-mediated reactions from which
non-consumed reactants are recycled back into the reaction
system.
[0012] More particularly it is an object of this process to provide
a closed system to recycle the solvent back into the reaction
system to minimize waste and to lower processing costs.
[0013] It is a further object of this invention to provide a method
for producing polyphenols or polyaromatic amines which incorporates
methods for recycling non-consumed reactants and non-consumed
reaction medium back into the system and for controlling reaction
conditions such that high product yields, low molecular weight, and
low polydispersity are obtained.
[0014] The process generally includes polymerizing a monomer in a
reactor with various pre-reaction components, wherein
polymerization is catalyzed by an enzyme, yielding phenolic or
aromatic amine polymers, non-consumed reactants and reaction
medium, wherein the non-consumed reactants and reaction medium form
post-reaction components; isolating the polymers from the
post-reaction components and recycling at least a portion of the
post-reaction components back into a mixing unit.
[0015] The reactants comprise monomers, enzyme, and hydrogen
peroxide. The reaction medium may be a reversed micellar solution
comprising water, a solvent and a surfactant. The reaction medium
may alternatively include water and a water-miscible solvent in a
monophasic system. Additionally, in a biphasic system the reaction
medium may comprise a first phase, where the first phase comprises
water, and a second phase, where the second phase comprises a
water-immiscible solvent. In this latter embodiment the reactor may
comprise a stirred tank to create a dynamic emulsion.
[0016] After polymerization, the polymer may be isolated from the
reaction components by means of a filtration unit yielding isolated
polymers and a filtrate, where the filtrate includes the
non-consumed reactants and the reaction medium.
[0017] After isolating the polymer, the recycling step may include
the following steps.
[0018] (A) In a monophasic solvent system by: 1) isolating a sample
of the filtrate; 2) analyzing the sample by means of a monitoring
device to determine concentration of the nonconsumed reactants and
volume of the reaction medium, together comprising post-reaction
components; 3) recycling the non-consumed reactants and reaction
medium back into the system; 4) communicating the concentration and
the volume levels for the post-reaction components to all flow
controllers; and 5) introducing the calculated amounts of each
reactant and reaction medium into the reactor by means of
controlling the respective flow rate.
[0019] (B) In a reversed micellar solution by: 1) isolating a
sample of the filtrate by means of a sampling port; 2) analyzing
the sample by an analyzing means to determine the concentrations of
nonconsumed reactants, surfactant and water and volume of the
reaction medium, together the post-reaction components; 3)
recycling the post-reaction components to a mixing unit via a
feedback controller; 4) communicating the calculated concentrations
and volume information from the feed back controller to the
respective flow controllers; 5) introducing the calculated amounts
of the reactants, surfactant, water and solvent into their
respective mixing ports/units; 6) introducing the solution into the
reactor; and 7) initiating the polymerization reaction by adding
the calculated amount of hydrogen peroxide at a predetermined
rate.
[0020] (C) In an immobilized system by: 1) analyzing the filtrate
for nonconsumed reactants; and 2) returning the filtrate to the top
of the bed after adding the calculated amounts of reactants and
solvent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Other objects, features and advantages will occur to those
skilled in the art from the following description of a preferred
embodiment and the accompanying drawings in which:
[0022] FIG. 1 is a schematic of meta- and para-substituted phenol
polymerization catalyzed by horseradish peroxidase;
[0023] FIG. 2 is the .sup.13C-NMR spectra for (a) p-ethylphenol and
(b) poly(p-ethylphenol);
[0024] FIG. 3 is a graph of the effect of LiBr concentration in DMF
on poly(p-ethylphenol) molecular weight;
[0025] FIG. 4a is a differential scanning calorimetry (DSC)
thermogram of poly(p-ethylphenol) prepared in reversed
micelles;
[0026] FIG. 4b is a thermogravimetric analysis (TGA) of
p-ethylphenol and poly(p-ethylphenol) prepared in reversed
micelles;
[0027] FIG. 5 shows the molecular weight distribution of poly
(p-ethylphenol) before and after heating;
[0028] FIG. 6a shows FTIR spectra of poly(p-ethylphenol) (i) before
and (ii) after esterification with palmitoyl chloride;
[0029] FIG. 6b shows FTIR spectra of poly(p-ethylphenol) (i) before
and (ii) after esterification with cinnamoyl chloride; and
[0030] FIG. 7 shows the UV absorbance at 259 nm of poly
(p-ethylphenol) before and after cinnamoylation.
[0031] FIG. 8 is a schematic drawing of a preferred embodiment of
the invention.
[0032] FIG. 9 is a schematic drawing of a preferred embodiment of
the invention.
[0033] FIG. 10 is a cross-sectional view of the reactor of a
preferred embodiment of the invention.
[0034] FIG. 11 is a schematic and perspective view of a preferred
embodiment of the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0035] This invention relates to a process for large-scale
production of phenolic or aromatic amine polymers where the
non-consumed reaction components are recycled back into the system.
The reactions utilize enzymes to catalyze polymerization and may be
carried out in 1) bulk solvents, 2) reversed micelles, or 3) a
biphasic system.
[0036] Free radical polymerization of p-ethylphenol and m-cresol,
catalyzed by horseradish peroxidase, was carried out at ambient
conditions in a number of organic solvent systems. While the
dioctyl sodium sulfosuccinate/isooctane reversed micellar system
afforded complete monomer conversion into polymer with an average
molecular weight of 2,500, the addition of chloroform yielded lower
molecular weights, with narrower distributions. Reactions carried
out in dimethyl formamide produced mostly oligomers with uniform
molecular weights. Poly(m-cresol) molecular weight could be
controlled between 1,400 and 25,000 by appropriate design of the
reaction medium comprised of ethanol-water mixture. Analysis of the
polymers by GPC demonstrated the effect of LiBr on the molecular
weights of poly(p-ethylphenol) and poly(p-phenylphenol). The
polymers showed apparently high molecular weights with DMF as GPC
solvent due to significant inter/intra-molecular associations. At
0.35% LiBr in DMF and above, these associations were eliminated to
permit the estimation of true molecular weights. .sup.13C-NMR and
FTIR studies revealed that the repeat units in poly(p-ethylphenol)
are primarily linked at ortho positions. The hydroxyl groups,
which-are not involved in bond formation, could be derivatized with
palmitoyl and cinnamoyl chlorides.
EXAMPLE 1
[0037] A typical polymerization reaction was carried out in
reversed micelles as follows. A 10 ml solution of 0.15 M dioctyl
sodium sulfosuccinate (AOT) in isooctane was prepared, and 0.4 ml
of an aqueous preparation of horseradish peroxidase (Type II)
(12.5.multidot.M) was added to form a clear reversed micellar
solution having a W.sub.o (molar ratio of water to surfactant) of
about 15. Para-ethylphenol was added to the reversed micellar
solution, and the polymerization reaction was initiated by adding
drops of 30% hydrogen peroxide (w/w) (up to about 30%
stoichiometric excess) while stirring the reaction mixture.
[0038] The reaction was exothermic with rapid formation of a
yellowish precipitate. After continuing the stirring for several
hours, the precipitate was centrifuged and washed repeatedly with
pure isooctane to remove the surfactant and any unreacted monomer.
The final precipitate was dried overnight under a reduced pressure
at 50.degree. C.
EXAMPLE 2
[0039] In cases where a mixture of chloroform and isooctane was
used to form reversed micelles, the same procedure was followed
except that the corresponding solvent mixture was used in place of
isooctane. Dry isooctane and chloroform are used for these
examples. These solvents are stored with molecular sieves to the
solvents dry. However, stable (i.e., transparent and single-phase)
reversed micellar solutions were found to be difficult to form with
a mixture of chloroform and isooctane with 25% or less isooctane. A
stable microemulsion could be obtained only up to a W.sub.o of 9
with 50% chloroform at room temperature, and phase separation
occurred at higher values.
EXAMPLE 3
[0040] In the absence of reversed micelles, reaction mixtures were
prepared by first dissolving the monomer and the enzyme in a
mixture of HEPES (N-[2-Hydroxyethyl]piperazine-N'[2-ethanesulfonic
acid]) buffer and solvent such as N,N-dimethylformamide (DMF). The
reaction was initiated, as before, by the dropwise addition of
hydrogen peroxide. The enzyme was completely soluble at 0.5 mg/ml
concentration in DMF/water mixtures at all solvent compositions
studied.
[0041] A. Structural Characterization
[0042] FIG. 1 illustrates the reaction scheme and the structures of
monomers used. Crosslinking in polymer structure is expected in
those cases where the ortho and para positions in the corresponding
monomer structure are unsubstituted, as is the case with m-cresol.
As shown in FIGS. 2a & 2b, .sup.13C-NMR studies on
poly(p-ethylphenol) indicate that the linkage between any two
adjacent phenyl rings is largely at the ortho positions. However,
this type of linkage may strain the polymer backbone in such a
manner that the phenyl rings are out of plane with respect to the
adjacent rings. As a result, the polymer backbone may be forced
into a coiled structure.
[0043] .sup.13C-NMR spectra on the monomer and polymer were
recorded on a 200 MHz Varian instrument (C broad band probe, Model
XL-200, Palo Alto, Calif.). Deuterated acetone and
tetramethylsilane (TMS) were used as the solvent and the internal
standard, respectively. Infrared spectra were recorded on a
Perkin-Elmer 1760 FTIR-FTRaman spectrophotometer at 4 cm.sup.-1
resolution. The samples were cast as thin films on a KBr window
from chloroform solutions. UV spectroscopy studies were carried out
on a Beckman DU 7500 spectrophotometer.
[0044] FIGS. 2a & 2b illustrate .sup.13C-NMR spectra and peak
assignments for the monomer and the polymer, respectively. The peak
position at 131 ppm is in agreement with the theoretically
calculated peak position for ortho linkages on the ring. On the
other hand, if the monomer were linked at meta positions on the
ring, the peaks for C3 and C5 should shift downfield from 129.4 ppm
in the monomer to 144 ppm in the polymer. However, the polymer
spectrum in FIG. 2b shows no such peak, therefore ruling out
linkages at meta positions. There was no significant change in the
peak position for C4, therefore ruling out ether linkages. Although
the hydroxyl groups are involved in the formation of free radicals
leading to polymer formation, they do not appear to be involved in
bond formation. In addition, previous infrared studies revealed no
ether linkage in the polymer structure. Thus the phenyl rings in
the polymer appear to be linked primarily at ortho positions. The
presence of free hydroxyl groups is also indicated by FTIR [see
FIG. 6a(i) and 6b(i)].
[0045] B. Molecular Weight Determination
[0046] Molecular weights were determined on a Waters LC Module I
instrument with an on-line GPC column (GBR mixed bed linear column
with a molecular weight range of 100 to over 20 million). A UV
detector at 270 nm was used to detect the polymer. The GPC data
were collected and processed with Millennium GPC software supplied
with the instrument. An eluent flow rate of 1 ml/min was maintained
under isocratic conditions. Narrow molecular weight polystyrene
standards were used for calibration. All samples were filtered
through 0.2 micron PTFE filters, (Millipore, Bedford, Mass.), prior
to injection. It was ascertained that the filters did not retain
any polymer during filtration. The effects of LiBr in DMF on
aggregation phenomena, as reflected by the weight average molecular
weight of poly(p-ethylphenol), were also determined. LiBr is used
to get true chromatographic separation based upon M.sub.w (LiBr
breaks apart aggregated polymer molecules). A precise measure of
M.sub.w is necessary to determine the functional utility of
polymer.
[0047] For GPC analysis, poly(p-ethylphenol) was completely
dissolved at a concentration of 1 mg/ml in a series of DMF-based
solutions with varying LiBr concentrations in the range of 0 to 1%
(w/v). A given composition (between 0 and 1% LiBr/DMF) of the GPC
solvent was prepared by mixing pure DMF and 1% LiBr/DMF in
appropriate proportions. For all injections, the composition of the
GPC solvent and the solvent used to prepare the sample for
injection were identical. A mixture of polystyrene standards
(M.sub.w 122 to 2.7 million, narrow distribution with
polydispersity in the range of 1.02 to 1.2) was prepared in all
compositions of LiBr and DMF, and always injected before analyzing
the polyphenol sample in the corresponding solvent.
[0048] Dimethylformamide is a good solvent for solution studies of
polyphenols. Earlier reports used a mixture of DMF and methanol, at
a ratio of 4 to 1, as a GPC solvent in the determination of
molecular weights of polyphenols. DMF is an interesting solvent,
especially for polyhydroxy compounds such as polysaccharides and
polyphenols. For example, amylose is not soluble is DMF, but the
polysaccharide swells as DMF penetrates into and `wets` the
polymer. However, it is well known that in the presence of about 3%
(w/v) LiBr, amylose could be dissolved at a concentration of about
1% (w/v) in DMF. Polyphenol, like a polysaccharide, is also a
polyhydroxy compound. Although DMF easily dissolves poly
(p-ethylphenol), there still may be inter/intramolecular
associations in the polymer. These interactions may result in an
apparently high molecular weight in GPC analysis.
[0049] The potential aggregation of polyphenol molecules, and the
use of a mixture of DMF and methanol to break the association, is
known. Molecular weights in the range of a few hundreds to a few
thousands have been reported for a number of different polyphenols,
with poly(p-phenylphenol) exhibiting a molecular weight of 26,000.
Using an identical GPC solvent composition, molecular weights of
over 400,000 for poly(p-phenylphenol) prepared in a dioxane/water
system have been reported. Similarly, an average molecular weight
of about 20,000 with DMF/methanol solvent mixture as GPC eluent for
poly(p-ethylphenol) prepared in AOT reversed micelles has been
observed. However, it is not clear if the solvent mixture of DMF
and methanol at 4:1 ratio is optimal to deaggregate the polymer
chains and give a true molecular weight. To address this problem,
the molecular weights of poly(p-ethylphenol) prepared in reversed
micelles and dioxane/water system and poly(p-phenylphenol) prepared
in dioxane/water system were analyzed as a function of LiBr
concentration in DMF and DMF/methanol mixture as GPC eluents.
[0050] FIG. 3 illustrates the effect of LiBr concentration in DMF
as GPC eluent on the weight average molecular weight of
poly(p-ethylphenol) prepared in reversed micelles. There is a
dramatic decrease in the molecular weight by over three orders of
magnitude when the LiBr concentration was increased from 0 to
0.35%. The molecular weight and dispersity of the polymer
stabilized at about 2500 and 1.36, respectively, above 0.35% LiBr
in DMF. Above this critical concentration of LiBr in DMF, there is
no additional effect on the polymer a molecular weight.
[0051] An analogous phenomenon was observed with the solubility
studies of amylose in DMF. Although DMF is capable of forming its
own hydrogen bonds with the polysaccharide (as noted earlier, the
polysaccharide swells in DMF, but is insoluble), it may not be able
to completely disrupt the inter- and intramolecular forces.
However, LiBr appears to be very effective in overcoming these
inter- and intramolecular interactions. The polysaccharide becomes
soluble at a concentration of 3% LiBr in DMF. It is possible that
the solubility of the polyhydroxy compound is dictated by a fixed
ratio between amylose and LiBr concentrations in DMF. The same
argument applies to the molecular dissociation of
poly(p-ethylphenol) in the presence of LiBr in DMF. Not
unexpectedly, there was no effect of LiBr on the retention times of
the polystyrene standards due to the lack of strong interchain
interactions.
[0052] A mixture of DMF/methanol at 4:1 ratio was also used as the
GPC solvent to determine the molecular weight of poly
(p-ethylphenol) synthesized in AOT/isooctane reversed micelles. The
result was a bimodal distribution with an average molecular weight
of 90,000 and 300,000 for the two distributions. A similar bimodal
molecular weight distribution was described by Akkara et al. for
poly(p-phenylphenol). Molecular aggregation is still significant in
this solvent system since the molecular weight of the sample
dropped to about 2700 in the presence of 1% LiBr in DMF/methanol
mixture at 4:1 ratio. Identical observations were made with a
sample of poly(p-ethylphenol) synthesized in 85% dioxane/water
system. Subsequently, a sample of poly(p-phenylphenol), synthesized
in 85% dioxane/water, was analyzed for molecular weight both in DMF
and DMF/methanol mixtures at different LiBr concentrations. As
before, the polymer molecular weight dropped from well over 6
million to about 3400 on increasing LiBr concentration from 0 to 1%
(w/v) in DMF. Similarly, poly(p-phenylphenol) showed a significant
shift to lower molecular weight as the LiBr concentration in
DMF/methanol mixture at 4:1 ratio was varied in the same
concentration range as in DMF. In this case, the molecular weight
dropped from about 500,000 to 3200. Table 1 lists the molecular
weight and dispersity profiles of poly(p-ethylphenol) and
poly(p-phenylphenol) synthesized under different conditions as a
function of GPC solvent composition. Poly(p-ethylphenol)
synthesized in reversed micelles exhibited a polydispersity of less
than 1.4, and that prepared in bulk solvent, dioxane/water, >2.
The average molecular weight of the polymer increased slightly as
the surfactant concentration was increased, a phenomenon noted
earlier.
1TABLE 1 Molecular weight and dispersity profiles of
poly(p-ethylphenol) and poly(p-phenylphenol) synthesized under
different conditions as a function of GPC solvent composition
Synthesis M.sub.w(M.sub.w/M.sub.n).sup.1 Sample medium (a) (b) (c)
(d) Poly(p- AOT/ >4.5M 2500 300,000 2700 ethylphenol) isoootane
(<2.5) (1.4) (>2.0) (1.4) reserved micelles Poly(p- 85/15
>6.0M 3400 500,000 3200 ethylphenol) dioxane/ (>2.5)
(>2.0) (>2.0) (>2.0) water Poly(p- 85/15 >6.0M 3000
300,000 3200 phenylphenol) dioxane/ (>2.5) (>2.0) (>2.0)
(>2.0) water .sup.1Molecular weights (M.sub.w) and dispersity
(M.sub.w/M.sub.n) were determined with the following GPC solvents:
(a) DMF; (b) 1% LiBr in DMF; (c) 4:1 DMF/methanol; and (d) 1% LiBr
in 4:1 DMF/methanol.
[0053] C. Thermal Characterization
[0054] Thermal characterization of polymers was carried out on Du
Pont thermal analyzers. For differential scanning calorimetry (DSC)
analysis, the polymers were hermetically sealed, and heated under a
nitrogen atmosphere at a temperature gradient of 10.degree. C. per
minute from room temperature to 300.degree. C. Thermogravimetric
analysis (TGA) was carried out at the same temperature gradient and
under nitrogen atmosphere, but heated to 600.degree. C. The thermal
properties of p-ethylphenol and poly(p-ethylphenol) prepared in
reversed micelles are illustrated as DSC and TGA thermograms in
FIGS. 4a & 4b, respectively. The polymer was reasonably stable
until a temperature of about 250.degree. C., with a loss of less
than 10% of the material (6% loss occurred before 200.multidot.c
presumably in part due to loss of water). The exotherm at about
110.degree. C. in the polymer DSC thermogram may be due to cross
linking in the polymer or due to loss of heat of crystallization.
Once heated over 200.degree. C., the exotherm was irreversibly
lost.
[0055] GPL analysis indicated a 170% increase in molecular weight,
presumably due to cross-linking, when a sample of
poly(p-ethylphenol) was heated to 150.degree. C., and the polymer
became significantly more polydisperse than the corresponding
untreated polymer. FIG. 5 shows portions of the GPC profiles of
poly(p-ethylphenol) before and after heating the polymer to
150.degree. C. Both samples were easily soluble in 1% LiBr/DMF
solution and this solution was also used as eluent. X-ray
diffraction studies on the samples revealed a partial
crystallization of the heat-treated polymer.
EXAMPLE 4
[0056] Phenol polymerization was carried out in a mixture of DMF
and water at various ratios to investigate the solvent effect on
enzyme activity and on the polymer molecular weight. The objective
was to investigate if the molecular weight of polyphenols could be
controlled, while maintaining a reasonably narrow molecular weight
distribution, by varying reaction system parameters such as time of
reaction, hydrogen peroxide concentration, and solvent composition.
Table 2 sets forth the ranges of solubility parameters and
dielectric constants covered by the solvent systems used for
polymerization reactions.
2TABLE 2 Ranges of solubility parameters and dielectric constants
covered by the solvent systems used for polymerization reactions
Solubility parameter Dielectric Solvent systems (MPa.sup.1/2)
constant Isooctane/ 14 2 chloroform 19 5 DMF/ 25 37 water 48 78
1,4-Dioxane/ 20 30 water 48 78 Ethanol/ 26 24 water 48 78
[0057] The solvent mixtures, listed in Table 2, were selected on
the basis of the range of solubility parameters that they cover.
The wide variation in solubility parameters and dielectric
constants for each system was similar to that found in certain
supercritical fluids as a function of pressure. These properties
not only influence the solubility of the growing polymer chain in
the corresponding reaction medium, but significantly affect enzyme
activity (solvents with high dielectric constants are known to
denature the enzyme). However, unlike in supercritical fluids, the
solvent properties can be varied at ambient conditions of pressure
and temperature.
[0058] The reaction medium composition was varied from 100% DMF to
100% water. As before, the reaction was initiated with the addition
of hydrogen peroxide at room temperature and with stirring.
Interestingly, and analogous to the dioxane/water system, there was
no sign of reaction (i.e., no heat or color generation) in the
reaction mixtures containing 85% or more organic solvent, and the
solutions remained clear throughout the addition of hydrogen
peroxide. On the other hand, heat evolution (due to exothermic
reaction) followed the reaction in solvents with 60% or less DMF,
and the solutions became colored and opaque instantaneously.
[0059] It is clear that DMF sustained enzyme activity up to a
concentration of 60%, although the presence of water was necessary.
The monomer solubility in 20% DMF solution was poor, and solution
turned into a stable emulsion prior to initiating the reaction. The
reactions were continued for a few more hours before the solvent
was evaporated under reduced pressure. The precipitates were washed
with water and isooctane to remove buffer salt, the enzyme and any
unreacted monomer. The dried precipitates were dissolved in 1%
LiBr/DMF and their molecular weights were analyzed as described
earlier. Table 3 shows the effect of solvent composition on the
polymer molecular weight and dispersity (reactions in bulk and in
the absence of reversed micelles).
3TABLE 3 Effect of solvent composition on the polymer molecular
weight and dispersity (reactions in the absence of reversed
micelles) Synthesis Monomer Polymer medium Conversion.sup.1 Yield
M.sub.w M.sub.w/M.sub.n Comments 100/0 0% 0% no reaction DMF/water
85/15 20% 10% 281 1.23 dimers soluble DMF/water in 85% DMF 60/40
80% 75% 612 1.20 oligomers DMF/water soluble in 60% DMF 40/60 80%
80% 675 1.05 oligomers DMF/water soluble in 40% DMF 20/80 75% 75%
658 1.02 oligomers DMF/water soluble in 20% DMF 0/100 50% 35% 400
1.90 oligomers DMF/water soluble in 100% DMF 85/15 80% 15% 3000
2.10 Insoluble Dioxane/ polymer water soluble oligomers
.sup.1Monomer converted/monomer added initially
[0060] The polymer yield, defined as a ratio of the amount of
polymer recovered as an insoluble fraction in isooctane to the
amount of monomer converted, was about 75% for cases where the DMF
content in the reaction mixture was between 20% and 60%. Molecular
weight analyses revealed that the polymers were in fact oligomers
with an average molecular weight of about 650, (significantly lower
than that obtained with the AOT/isooctane reversed micellar
system), and a polydispersity of 1.03-1.20. The molecular weight
was not variable with DMF content, indicating that either the
solubility of growing chains during the reaction was not sustained
by DMF/water (up to 60% DMF) or the enzyme became inactive.
[0061] Ethanol-HEPES buffer mixtures were also used to polymerize
m- and p-cresol or p-ethylphenol in the study of molecular weight
control. m-Cresol was studied in greater detail since it allows the
study of a much broader molecular weight range than p-cresol or
p-ethylphenol. Ethanol is a solvent of choice in view of its
environmental compatibility and ease of regeneration. In addition,
the enzyme is active in ethanol/buffer mixtures at levels up to at
least 60% ethanol. Enzyme activity was studied as a function of
ethanol content, and it was found that 20 to 40% ethanol was
optimal for the conversion of about 50% monomer into polymer
(20,000 molecular weight, degree of polymerization of 200). At an
enzyme concentration of 2 mM, the conversion was essentially
complete in about 10 minutes. Although there was no significant
improvement in the monomer conversion when the reaction was
continued for 24 hours, the molecular weight of poly(m-cresol)
increased by about 75%. Table 4 shows the effect of ethanol content
on monomer conversion and the molecular weight of poly(m-cresol) in
ethanol/water systems. Ethanol appears to be a useful solvent when
higher molecular weight polymer is desired. Replacing HEPES buffer
with deionized water resulted in no noticeable change in reaction
rates or in polymer properties.
4TABLE 4 Effect of ethanol content on monomer conversion and the
molecular weight of poly(m-cresol) in ethanol/water systems
Polydispersity in all cases was >2.5 Unless noted otherwise, HRP
concentration was 0.1 mg/ml Solvent Monomer Polymer Composition
conversion M.sub.w Comments 100% buffer 40% 2200 polymer removed
once at the end; 60% conversion possible when HRP and
H.sub.2O.sub.2 added in pulses 100% buffer with 1% 40% 1400 polymer
removed as it KCl formed; 60% conversion possible when HRP and
H.sub.2O.sub.2 added in pulses 80/20 90% 22000 M.sub.w of 6000 to
22000 water/EtOH possible at intermediate stages of reaction 60/40
47% 10000 Lower molecular water/EtOH weights possible at
intermediate stages of reaction 40/60 11% 3000 90% conversion &
water/EtOH 24000 M.sub.w at 5X enzyme concentration 20/80 3% -- 20%
conversion & 2000 water/EtOH M.sub.w at 5X enzyme concentration
100% EtOH 0% Insoluble enzyme
[0062] Although high molecular weight polymers were produced with
poly(m-cresol) due to crosslinking and with ethanol, in some
applications, such as photoresists and detergent formulations,
oligomers are desirable. Hence, m-cresol was polymerized in 100%
buffer/water, and as a result, the polymer molecular weight
decreased to 2,500. During the reaction the polymer molecular
weight gradually increased without significant improvement in
monomer conversion. It was therefore attempted to isolate the
polymer as the polymerization process continued. This was achieved
by carrying out the reaction in the presence of 0.5 to 1 (w/v)% KC1
in the medium. While the salt, up to around 3% had no effect on the
enzyme activity, it caused precipitation of the polymer as it
formed. The polymer precipitate was isolated by filtration and the
filtrate was returned to the reaction vessel. The enzyme in the
filtrate was no longer active, therefore, enzyme and peroxide were
added in pulses such that fresh enzyme was always available after
each filtration to assure continued polymer formation. However, the
rate of polymerization dropped as the reaction continued after each
filtration in spite of the presence of significant amounts of the
residual monomer, fresh enzyme, and hydrogen peroxide. The polymer
yield was about 40% after completely adding all the enzyme and
hydrogen peroxide to a final concentration of 2.multidot.M enzyme
and 30% stoichiometric excess of the peroxide. The polymer
molecular weight was about 1,400. Further polymerization was still
possible in the remaining reaction mixture if fresh enzyme and
peroxide were further added. Therefore, the polymerization process
could be continued, in theory, until all monomer was consumed. When
the polymer was not removed by filtration and the reaction was run
with the identical additions of enzyme and hydrogen peroxide, the
final polymer molecular weight was over 2,000. The polydispersity
was about 2.5 in both cases of whether the polymer was removed
intermittently or not.
[0063] It is thus possible to control the polymer molecular weight
by manipulating the reaction and/or process conditions. In the case
of poly(m-cresol), it is possible to obtain control of molecular
weight from 1,400 to 25,000, with a polydispersity of about 2.5, by
the selection of the reaction medium. Enzyme activity is a function
of the reaction medium composition which influences monomer
conversion. However, the polymer molecular weight is also strongly
influenced by the polymer solubility and the length of time it is
in contact with the enzyme and reacting species in the reaction
mixture, even after precipitation.
[0064] In order to minimize the enzyme inactivation, a less polar
solvent than DMF or ethanol was sought as the reaction medium.
Accordingly, chloroform was used to carry out the phenol
polymerization reaction. A mixture of chloroform and isooctane was
used for the reactions, and the polarity of the medium was
gradually varied by adding chloroform and thus changing the
composition from 100% isooctane to 100% chloroform. However, the
enzyme powder was poorly dispersed in this solvent system, and it
became necessary to prepare AOT reversed micelles with the
chloroform/isooctane mixture, as described earlier. As the
isooctane content in the reaction mixture was lowered from 100%,
the polymer yield dropped from 100% (in pure isooctane reversed
micelles) to about 10% (in pure chloroform reversed micelles) These
results are shown in Table 5.
5TABLE 5 Effect of solvent composition on the polymer molecular
weight and dispersity (reactions in the AOT reversed micelles)
using isooctane and chloroform Synthesis Monomer Polymer medium
Conversion.sup.1 Yield M.sub.w M.sub.w/M.sub.n Comments 100% 100%
100% 2500 1.36 W.sub.0 = 15 Isooctane 100% 90% 100% 2500 1.38
W.sub.0 = 9 Isooctance 75/25 100% 85% 1681 1.53 W.sub.0 = 9
Isooctane/ CHCL.sub.3 50/50 100% 75% 3461 1.85 W.sub.0 = 9
Isooctane/ CHCl.sub.3 25/75 75% 35% 3601 1.83 W.sub.0 = 9
Isooctane/ phase CHCl.sub.3 separation 100% CKCl.sub.3 20% 10% 1000
1.07 W.sub.0 = 9 phase separation
[0065] Polymer molecular weight was maximum in 50-75% chloroform in
isooctane with a polydispersity of 1.5 to 1.9. However, the polymer
exhibited a low polydispersity of 1.07 in 100% chloroform. The poor
polymer yields at high chloroform contents are perhaps due to the
formation of unstable microemulsion systems leading to phase
separation. As a result, the contact between the enzyme and the
monomer is inefficient and the polymer yield is poor. Smaller
W.sub.o values also contribute to poor monomer conversion. One
approach is to eliminate the surfactant altogether by polymerizing
phenolic monomers in a biphasic system where large amount of water
containing enzyme is mechanically dispersed in a hydrophobic
organic solvent containing the monomer. Preliminary results
indicate that a number of polymers including poly(p-ethylphenol) of
molecular weight 2500 can be prepared in chloroform/buffer (50:50
v/v) or an isooctane/water (50/50) biphasic system.
[0066] The hydroxyl groups in enzymatically prepared polyphenols do
not a participate in bond formation, as noted earlier from
.sup.13C-NMR studies. The FTIR spectrum of the polymer, shown in
FIGS. 6a(i) and 6b(i), also illustrates the point with a broad peak
at 3400 cm.sup.-1 due to 0-H stretch. Thus the hydroxyls on the
polymer are available for chemical modifications such as
esterification. Esterification was carried out in chloroform with
palmitoyl and cinnamoyl chlorides in the presence of stoichiometric
amount of pyridine to scavenge HCI produced in the reaction.
[0067] FIGS. 6a and 6b illustrate FTIR spectra of
poly(p-ethylphenol) before and after functionalization with
palmitoyl and cinnamoyl moieties, respectively, at the hydroxyl
groups of the polymer. FIG. 6a shows the presence of alkyl chains
in the polymer due to palmitoyl groups, confirmed by the presence
of strong peaks between 2800 and 3000 cm.sup.-1 due to asymmetric
and symmetric C--H stretch in methyl and methylene groups of the
alkyl chains.
[0068] In addition, the peak for 0-H stretch at 3400 cm.sup.-1
disappeared in the esterified polymer indicating the participation
of the hydroxyl groups in the reaction. The ester formation was
also confirmed by the presence of C.dbd.O stretch at 1750 cm.sup.-1
in the modified polymer. Similarly, cinnamoylation of the polymer
was confirmed by the disappearance of 0-H stretch as well as from
the strong presence of C.dbd.C ring stretch at 1600 cm.sup.-1,
shown in FIG. 6b. UV spectroscopic studies, carried out with
acetonitrile solutions of the polymer, showed an increased
absorbance for the cinnamoylated polymer at 259 nm due to the
presence of additional phenyl ring, as shown in FIG. 7.
[0069] A preferred embodiment, shown in FIG. 8, comprises
polymerizing a monomer by horseradish peroxidase (HRP) at the
oil-water interface of water-in-oil microemulsions (reversed
micelles). FIG. 1 is a schematic of meta- and para-substituted
phenol polymerization catalyzed by HRP. The side chains do not
participate in the reaction, but they have an effect on polymer
properties, i.e. the position of the polymer link depends on the
position of the side chain.
[0070] FIG. 8 is a schematic diagram of enzymatic polyphenol
synthesis in reversed micelles. For example, p-ethylphenol could be
polymerized in AOT/isooctane reversed micelles with complete
monomer conversion into the polymer. The resultant polymer exhibits
relatively narrow polydispersity, as shown in Table 6.
6TABLE 6 Monomer conversion and polymer molecular weight and
dispersity in different reaction media SAMPLE SYNTHESIS MEDIUM
M.sub.w (p.d.).sup.1 COMMENTS Poly AOT/isooctane 2,500 (1.4) 100%
monomer conversion (p-ethylphenol) reversed micelles to polymer
Poly 85/15 dioxane/water 3,400 (>2.0) Good monomer conversion;
(p-ethylphenol) poor polymer yield Poly Isooctane/buffer biphasic
1,700 (>2.0) Good monomer conversion; (p-ethylphenol) system
fair polymer yield Poly 85/15 dioxane/water 3,000 (>2.0) Good
monomer conversion; (p-phenylphenol) fair polymer yield
.sup.1Molecular weights were determined with 1% LiBr in DMF as GPC
solvent. p.d. = polydispersity
[0071] In this embodiment, the reversed micelles act as
three-dimensional templates for the organization of the monomer
prior to polymerization.
[0072] The reversed micelles are prepared from isooctane and the
anionic surfactant AOT. The resultant reversed micelles and the
Isooctane are then introduced from reservoirs 10 and 12,
respectively, into mixing port 28 through flow controllers 20 and
22, respectively. The contents of mixing port 28 are then
introduced into mixing unit 30 and mixed with an enzymatic solution
of HRP introduced into mixing unit 30 from reservoir 14 through
flow controller 24. The contents of mixing unit 30 are then
introduced into mixing unit 32 and mixed with a suitable monomer
such as para-ethylphenol introduced into mixing unit 32 from
reservoir 16 through flow controller 26. After mixing, the contents
of mixing unit 32 are introduced into reactor 34, which is cooled
by cooling jacket 44, where polymerization is initiated by the
introduction of H.sub.2O.sub.2 through flow controller 18 into
reactor 14. As the monomers are polymerized, the dense polymers
settle to the bottom of the reactor.
[0073] After polymerization is complete, the contents of reactor 34
are introduced into centrifuge 36 where the polymer is isolated.
The polymer may alternatively be isolated using filters.
[0074] After the polymer is isolated, the water in the recycled
reaction components is removed by contacting with molecular sieves
in dehumidifier 38. Concentrations of water, surfactant, enzyme and
monomers are anlyzed in the sampling port 40 and the information is
relayed to the feedback controller 42. Based upon this information,
the feed back controller communicates with different flow
controllers (18, 20, 22, 24 and 26) to adjust the concentrations in
the feed to the reactor 34.
[0075] Monomers which can be polymerized in reversed micelles
include the following phenols, aromatic amines, and mixed
functional groups:
7 Phenols Aromatic Amines Mixed Groups 2,6-Dihydroxy- Aniline
p-Hydroxythiophenol naphthalene Benzidine p-Ethylphenol Naphthol
p-butylphenol 1-Pyrenol
[0076] Polymer yields as high as 95% are possible for reactions in
reversed micelles and molecular weight can be controlled by
controlling the polarity of the oil. Uniformity in size of the
reverse micelles leads to narrow polydispersity in the resultant
polymer.
[0077] A second preferred embodiment, shown in FIG. 9, includes a
monophasic system where the monomer is polymerized in a polar
solvent/water mixture such as a dioxane/water mixture where the
monomer conversion may be controlled by hydrogen peroxide and HRP.
The molecular weight of the polymer may be controlled by adjusting
the residence time of the reacting species in the reactor, by
molecular weight cutoff filtration, and by the amount of reactants.
It is preferable that the ratio of enzyme and hydrogen peroxide is
maintained throughout the reaction which can require more than 24
hours for completion. The amount of enzyme and hydrogen peroxide
are determined by the amount of monomer.
[0078] FIG. 9 is a schematic diagram of enzymatic polyphenol
synthesis in an ethanol/water mixture. The polar solvent, monomer,
water, hydrogen peroxide, and enzymatic solution are introduced
into reactor 64 from their respective reservoirs (46, 48, 50, and
52) through their respective flow controllers (54, 56, 58, and 60),
and mixed. After polymerization, the polymer and any post-reaction
components (non-consumed reaction components) are run through
filtration unit 68, as shown in FIG. 9., to isolate the polymer
from the reactants. A centrifuge and/or filters can be used to
isolate the polymer. After filtration, the filtrate is introduced
into analyzer/controller 66 through flow controller 62 to determine
concentration and volume, and then recycled back into reactor 64.
Unwanted filtrate is purged through flow controller 62. Fresh
reaction components and monomer may then be added to the reactor
for continuous production of the polymer.
[0079] Suitable polar solvents for use in the monophasic system may
include organic solvents such as methanol, ethanol, acetone,
tetrahydrofuran, dimethyformamide, isopropyl alcohol, dioxane and
dimethylsulfoxide.
[0080] Suitable monomers for polymerization in the polar
solvent/water mixture include the following phenols, aromatic
amines, and mixed functional groups:
8 Phenols Aromatic Amines Mixed Groups Anisole Aniline p-Amino
m-cresol 1,2-Benzenediol Benzidine 2- p-n-Butylphenol
p-n-Butylaniline Hydroxybenzylalcohol Cresols (o, m, & p)
p-sec-Butylaniline 8-Hydroxyquinoline 1,3-Dihydroxynaphthalene
2,6-Diethylaniline p-Hydroxythiophenol 1,5-Dihydroxynaphthalene
3,5-Diethylaniline Isoquinoline 3,4-Dimethylphenol
2,6-Dimethylaniline 2-Methyl 8-quinolinol p-Ethylphenol
3,5-Dimethylaniline p-Phenylazophenol Methoxyphenol (o & m)
p-Ethylaniline Tyrosine Naphthol (alpha & beta)
3-Phenylenediamine p-n-Octylphenol p-n-Propylaniline Phenol
p-Phenoxyphenol Phenylphenol (m & p) 3-(3-Phenoxyphenoxy)
phenol
[0081] A third preferred embodiment of the method, partially shown
in FIG. 11, comprises a biphasic system in which water and a
water-immiscible organic solvent, such as chloroform or hexane, and
a monomer are introduced into stirred tank reactor 80. Upon
addition of hydrogen peroxide and enzyme, the reaction takes place
at the oil-water interface. The enzyme remains soluble in the
aqueous phase while the hydrophobic monomer remains largely in the
organic phase. A dynamic macroemulsion is created by vigorous
stirring the two phases with stirring device 82. Since no
surfactant is involved in creating the emulsion, the product is
free from possible surfactant contamination. Following
polymerization, the contents of reactor 80 are analyzed and removed
for phase separation, product recovery, and solvent recycling as in
the above described embodiments.
[0082] In contrast to the homogenous dispersion of the enzyme in
the above described embodiments, the enzyme can also reused by
immobilizing the enzyme on an inert support such as silica gel,
e.g: retractable enzyme bed 74, as shown in FIG. 10, or on the
inside walls of a tube reactor to construct a packed bed reactor or
a plug flow reactor. FIG. 10 illustrates an embodiment of packed
bed reactor 76 with distribution cap 70 and cooling jacket 72. In
this embodiment, the monomer conversion and polymer molecular
weight can be controlled by varying the residence time of the
monomer. Residence time can be regulated by controlling the flow
rate of monomer solution through the enzyme bed. Over time the
immobilized enzyme will become contaminated and will require
removal for cleaning purposes.
[0083] Although specific features of the invention are shown in
some drawings and not others, this is for convenience only as some
feature may be combined with any or all of the other features in
accordance with the invention.
[0084] Other embodiments will occur to those skilled in the art and
are within the following claims.
* * * * *