U.S. patent application number 09/244005 was filed with the patent office on 2001-06-14 for process to control the molecular weight and poldispersity of substituted polyphenols and polyaromatic amines by enzymatic synthesis inorganic solvents microemulsions and biphasic systems.
Invention is credited to AKKARA, JOSEPH A., AYYAGARI, MADHO, KAPLAN, DAVID L..
Application Number | 20010003774 09/244005 |
Document ID | / |
Family ID | 24396715 |
Filed Date | 2001-06-14 |
United States Patent
Application |
20010003774 |
Kind Code |
A1 |
AKKARA, JOSEPH A. ; et
al. |
June 14, 2001 |
PROCESS TO CONTROL THE MOLECULAR WEIGHT AND POLDISPERSITY OF
SUBSTITUTED POLYPHENOLS AND POLYAROMATIC AMINES BY ENZYMATIC
SYNTHESIS INORGANIC SOLVENTS MICROEMULSIONS AND BIPHASIC
SYSTEMS
Abstract
A process of controlling the molecular weight and dispersity of
poly(p-ethylphenol) and poly(m-cresol) synthesized enzymatically by
varying the composition of the reaction medium. Polymers with low
dispersities and molecular weights from 1000 to 3000 are
synthesized in reversed micelles and biphasic systems. In
comparison, reactions in bulk solvents resulted in a narrow range
of molecular weights (281 to 675 with poly(p-ethylphenol) in a
DMF/water system and 1,400 to 25,000 with poly(m-cresol) in an
ethanol/water system). Poly(p-ethylphenol) was functionalized at
hydroxyl positions with palmitoyl, cinnamoyl, and biotin
groups.
Inventors: |
AKKARA, JOSEPH A.;
(HOLLISTON, MA) ; KAPLAN, DAVID L.; (STOW, MA)
; AYYAGARI, MADHO; (BRIGHTON, MA) |
Correspondence
Address: |
VINCENT J RANUCCI
USA SOLDIER & BIOLOGICAL CHEMICAL CMD
SOLDIER SYSTEMS CENTER
15 KANSAS STREET
NATICK
MA
017605035
|
Family ID: |
24396715 |
Appl. No.: |
09/244005 |
Filed: |
February 4, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09244005 |
Feb 4, 1999 |
|
|
|
08598737 |
Jan 16, 1996 |
|
|
|
6096859 |
|
|
|
|
Current U.S.
Class: |
528/86 ; 435/132;
435/156; 435/190; 435/192; 528/210; 528/211; 528/212; 528/214;
528/215; 528/422; 528/501; 528/503 |
Current CPC
Class: |
C08G 61/10 20130101 |
Class at
Publication: |
528/86 ; 528/501;
528/503; 528/210; 528/212; 528/214; 528/215; 528/211; 528/422;
435/132; 435/156; 435/190; 435/192 |
International
Class: |
C08G 065/38; C08G
002/00; C08J 003/00; C08F 006/00 |
Goverment Interests
[0001] The invention described herein may be manufactured and used
by the Government for governmental purposes without the payment of
any royalty thereon.
Claims
What is claimed is:
1. A method of preparing polyphenols and polyaromatic amines, the
method comprising the following steps: (a) preparing a synthesis
reaction medium allowing for the control of molecular weight and
polydispersity of the polymerization product, the synthesis
reaction medium being selected from the group consisting of
reversed micelles, bulk organic solvent mixtures, and biphasic
reaction media; (b) adding to the synthesis reaction medium an
aqueous preparation of an enzyme; (c) adding to the synthesis
reaction medium and enzyme preparation a monomer selected from the
group consisting of phenols, aromatic amines, and their derivatives
to form a reaction mixture; (d) initiating a polymerization
reaction by adding dropwise 30% hydrogen peroxide (w/w) (up to
about 30% stoichiometric excess) while stirring the reaction
mixture; (e) continuing stirring for several hours; (f)
centrifuging the precipitate formed; (g) repeated washing of the
precipitate with pure isooctane to remove substances selected from
the group consisting of the surfactant and any unreacted monomer;
and (h) drying the final precipitate overnight under a reduced
pressure at 50.degree. C.
2. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the reversed micelles are formed from
dioctyl sodium sulfosuccinate and isooctane.
3. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the reversed micelles are formed from
chloroform and/or isooctane.
4. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the bulk organic solvent mixtures are
formed from dimethylformamide and water.
5. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the bulk organic solvent mixtures are
formed from dioxane and water.
6. The method of producing poly phenols and polyaromatic amines, as
claimed in claim 1, wherein the bulk organic solvent mixtures are
formed from ethanol and water.
7. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the bulk organic solvent mixtures are
formed from isooctane and methylchloride.
8. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the biphasic systems are formed from
organic solvents and water.
9. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the enzyme is selected from the group
consisting of peroxidases, tyrosinases, laccases, phenoloxidases,
aromatic aminoxidases.
10. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the monomer is p-ethylphenol.
11. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the monomer is m-cresol.
12. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the molecular weight of the polymer
molecules is between 600 and 3,600.
13. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the molecular weight of the polymer
molecules is between 1,400 and 25,000.
14. The method of producing polyphenols and polyaromatic amines, as
claimed in claim 1, wherein the polydispersity of the polymer
molecules ranges from 1.02 to greater than 2.
15. A method of producing derivatives of polyphenols and
polyaromatic amines, the method comprising the following steps: (a)
preparing a synthesis reaction medium allowing for the control of
molecular weight and polydispersity of the polymerization product,
the synthesis reaction medium being selected from the group
consisting of reversed micelles, bulk organic solvent mixtures, and
biphasic reaction media; (b) adding to the synthesis reaction
medium an aqueous preparation of an enzyme; (c) adding to the
synthesis reaction medium and enzyme preparation a monomer selected
from the group consisting of phenols, aromatic amines, and their
derivatives, and to form a reaction mixture; (d) initiating a
polymerization reaction by adding dropwise 30% hydrogen peroxide
(w/w) (up to about 30% stoichiometric excess) while stirring the
reaction mixture; (e) continuing stirring for several hours; (f)
centrifuging the precipitate formed; (g) repeated washing of the
precipitate with pure isooctane to remove the surfactant and/or any
unreacted monomer; and (h) drying the final precipitate overnight
under a reduced pressure at 50.degree. C.
16. The method of producing derivatives of polyphenols and
polyaromatic amines, as claimed in claim 15, wherein the derivative
comprises palmitoyl chloride.
17. The method of producing derivatives of polyphenols and
polyaromatic amines, as claimed in claim 15, wherein the derivative
comprises cinnamoyl chloride.
18. The method of producing derivatives of polyphenols and
polyaromatic amines, as claimed in claim 15, wherein the derivative
comprises biotin compounds.
19. A polymerization product produced by: (a) preparing a synthesis
reaction medium allowing for the control of molecular weight and
polydispersity of the polymerization product, the synthesis
reaction medium being selected from the group consisting of
reversed micelles, bulk organic solvent mixtures, and biphasic
systems; (b) adding to the synthesis reaction medium an aqueous
preparation of an enzyme; (c) adding to the synthesis reaction
medium and enzyme preparation a monomer selected from the group
consisting of phenols, aromatic amines, and their derivatives to
form a reaction mixture; (d) initiating a polymerization reaction
by adding dropwise 30% hydrogen peroxide (w/w) (up to about 30%
stoichiometric excess) while stirring the reaction mixture; (e)
continuing stirring for several hours; (f) centrifuging the
precipitate formed; (g) repeated washing of the precipitate with
pure isooctane to remove the surfactant and any unreacted monomer;
and (h) drying the final precipitate overnight under a reduced
pressure at 50.degree. C.
20. The polymerization product, as claimed in claim 19, wherein the
reversed micelles are formed from dioctyl sodium sulfosuccinate and
isooctane.
21. The polymerization product, as claimed in claim 19, wherein the
reversed micelles are formed from AOT, chloroform, and
isooctane.
22. The polymerization product, as claimed in claim 19, wherein the
bulk organic solvent mixtures are formed from dimethylformamide and
water.
23. The polymerization product, as claimed in claim 19, wherein the
bulk organic solvent mixtures are formed from dioxane and
water.
24. The polymerization product, as claimed in claim 19, wherein the
bulk organic solvent mixtures are formed from ethanol and
water.
25. The polymerization product, as claimed in claim 19, wherein the
bulk organic solvent mixtures are formed from isooctane and
methylchloride.
26. The polymerization product, as claimed in claim 19, wherein the
biphasic systems are formed from organic solvents and water.
27. The polymerization product, as claimed in claim 19, wherein the
enzyme is selected from the group consisting of peroxidases,
tyrosinases, laccases, phenoloxidases, aromatic aminoxidases.
28. The polymerization product, as claimed in claim 19, wherein the
monomer is p-ethylphenol.
29. The polymerization product, as claimed in claim 19, wherein the
monomer is m-cresol.
30. The polymerization product, as claimed in claim 19, wherein the
molecular weight of the polymer molecules is between 600 and
3,600.
31. The polymerization product, as claimed in claim 19, wherein the
molecular weight of the polymer molecules is between 1,400 and
25,000.
32. The polymerization product, as claimed in claim 19, wherein the
polydispersity of the polymer molecules ranges from 1.02 to greater
than 2.
33. A film comprising a polymerization product produced by: (a)
preparing a synthesis reaction medium allowing for the control of
molecular weight and polydispersity of the polymerization product,
the synthesis reaction medium being selected from the group
consisting of reversed micelles, bulk organic solvent mixtures, and
biphasic systems; (b) adding to the synthesis reaction medium an
aqueous preparation of an enzyme; (c) adding to the synthesis
reaction medium and enzyme preparation a monomer selected from the
group consisting of phenols, aromatic amines, and their derivatives
to form a reaction mixture; (d) initiating a polymerization
reaction by adding dropwise 30% hydrogen peroxide (w/w) (up to
about 30% stoichiometric excess) while stirring the reaction
mixture; (e) continuing stirring for several hours; (f)
centrifuging the precipitate formed; (g) repeated washing of the
precipitate with pure isooctane to remove the surfactant and any
unreacted monomer; and (h) drying the final precipitate overnight
under a reduced pressure at 50.degree. C.
34. A photoresist material comprising a polymerization product
produced by: (a) preparing a synthesis reaction medium allowing for
the control of molecular weight and polydispersity of the
polymerization product, the synthesis reaction medium being
selected from the group consisting of reversed micelles, bulk
organic solvent mixtures, and biphasic systems; (b) adding to the
synthesis reaction medium an aqueous preparation of an enzyme; (c)
adding to the synthesis reaction medium and enzyme preparation a
monomer selected from the group consisting of phenols, aromatic
amines, and their derivatives to form a reaction mixture; (d)
initiating a polymerization reaction by adding dropwise 30%
hydrogen peroxide (w/w) (up to about 30% stoichiometric excess)
while stirring the reaction mixture; (e) continuing stirring for
several hours; (f) centrifuging the precipitate formed; (g)
repeated washing of the precipitate with pure isooctane to remove
the surfactant and any unreacted monomer; and (h) drying the final
precipitate overnight under a reduced pressure at 50.degree. C.
35. UV absorbing materials comprising a polymerization product
produced by: (a) preparing a synthesis reaction medium allowing for
the control of molecular weight and polydispersity of the,
polymerization product, the synthesis reaction medium being
selected from the group consisting of reversed micelles and bulk
organic solvent mixtures; (b) adding to the synthesis reaction
medium an aqueous preparation of an enzyme; (c) adding to the
synthesis reaction medium and enzyme preparation a monomer selected
from the group consisting of phenols, aromatic amines, and their
derivatives to form a reaction mixture; (d) initiating a
polymerization reaction by adding dropwise 30% hydrogen peroxide
(w/w) (up to about 30% stoichiometric excess) while stirring the
reaction mixture; (e) continuing stirring for several hours; (f)
centrifuging the precipitate formed; (g) repeated washing of the
precipitate with pure isooctane to remove the surfactant and any
unreacted monomer; and (h) drying the final precipitate overnight
under a reduced pressure at 50.degree. C.
Description
FIELD OF THE INVENTION
[0002] The present invention relates generally to the preparation
of phenolic and aromatic amine polymers, wherein the reaction
conditions are controlled such that high product yields, molecular
weight, and a uniform molecular weight distribution are
obtained.
BACKGROUND OF THE INVENTION
[0003] Phenolic and aromatic amine polymer resins constitute a very
important and useful class of chemical compounds. They have a
number of uses, e.g., as coatings and laminates that provide a
number of functional advantages. Besides possessing good thermal
properties, these polymers can be doped to make them electrically
conductive, making them a key component of integrated circuit (IC)
chips.
[0004] At present, these polymers are prepared by chemical
synthesis, e.g., as from phenol and formaldehyde. The polymers's
linearity/network structure (and, by extension, their functional
properties) varies depending on the monomer and type of reaction
conditions used. However, the use of certain constituent chemicals,
such as formaldehyde, is being restricted in the chemical industry
because of their toxicity. Accordingly, the enzyme-mediated
synthesis of polyphenols and polyaromatic amines offers a viable
alternative to the currently used chemical synthesis of such
commercial phenolic resins.
[0005] Peroxidase-catalyzed free radical polymerization of phenol,
aromatic amines, and their derivatives is well known. Horseradish
peroxidase (HRP) is the most widely used biocatalyst in the
polymerization of phenol, aniline, or their derivatives. HRP has
been shown to be active in a number of organic solvents or solvent
mixtures and the reaction is typically initiated by the addition of
hydrogen peroxide as an oxidant.
[0006] 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.
Poylm. 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.
p-Alkylphenols were also polymerized at oil-water (reversed
micelles) and air-water (Langmuir-Blodgett trough) interfaces.
Because of their amphiphilic nature, the alkylphenols are
positioned at the interface, and in the presence of HRP and
hydrogen peroxide the monomers are oxidatively coupled to form
polymers. The poly(p-alkylphenols) prepared in reversed micelles
were shown to exhibit relatively more uniform molecular weight
distribution than those prepared in bulk organic solvents.
[0007] 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.
[0008] 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 IC chips, among other applications.
[0009] Palmitoyl chlorides may be added to the polymer to make the
polymer easily processable, e.g., as coatings, films, or finishes.
Cinnamoyl chlorides may be added to create controlled pore size
membranes (e.g., "molecular sieves") or to enhance the polymers's
ability to absorb UV radiation (e.g., for sunglasses), thereby
enabling their use as anti-reflective coatings in photoresists. 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 contrast, photosensitive
functional groups may be added to enhance the utility of the
polymers in other applications.
[0010] The polymers also may be modified by the addition of
proteins (e.g., enzymes, antibodies, etc.) to create active
matrices and systems allowing the controlled-release of materials,
such as drugs, insecticides, and fertilizers If biotin groups are
added to the polymer chain, the polymer can be used as
chromatography packing, which may be used to separate and purify
proteins.
[0011] 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., the "polydispersity") of
polyphenols and polyaromatic amines also can significantly
influence the functional properties of the polymers.
[0012] Accordingly, it is an object of this invention to overcome
the above illustrated inadequacies and problems of extant
polyphenols and polyaromatic amines by providing an improved method
of their manufacture suitable for use in applications that would
benefit from uniform polymer size.
[0013] It is another object of this invention to provide a method
of producing polyphenols and polyaromatic amines wherein it is
possible to control the molecular weight distribution of the
polymer molecules.
[0014] Yet another object of the present invention is to provide a
method of producing polyphenols and polyaromatic amines wherein the
molecular weight distribution of the polymer molecules is between
600 and 3,600.
[0015] It is a further object of the present invention to provide a
method of producing polyphenols and polyaromatic amines wherein the
molecular weight distribution of the polymer molecules is between
1,400 and 25,000.
[0016] A still further object is to provide a method of producing
polyphenols and polyaromatic amines wherein it is possible to
control the polydispersity of the polymer molecules.
[0017] It is another object of this invention to provide a method
of producing polyphenols and polyaromatic amines wherein the
polydispersity of the polymer molecules ranges from 1.02 to greater
than 2.
[0018] It is yet another object of the present invention to provide
a method to modify the polymer prepared by adding functional groups
to the polymer using palmitoyl chloride, cinnamoyl chloride, and
biotin compounds.
SUMMARY OF THE INVENTION
[0019] The objects of the present invention are met by a method of
enzymatically synthesizing polyphenols and polyaromatic amines
under controlled reaction conditions. More particularly, the
invention relates to the control of molecular weight and
polydispersity in enzymatically synthesized polyphenols and
polyaromatic amines by manipulating the several reaction
parameters.
[0020] The present invention defines reaction conditions for any
given phenol/aromatic amine monomer necessary to control M.sub.W
and polydispersity within a defined range. Such control of M.sub.W
and polydispersity has been found to increase the utility of these
polymers.
[0021] In particular, the ability to control the molecular weight
and dispersity of poly(p-ethylphenol) and poly(m-cresol) has been
achieved. The polymers were synthesized enzymatically in different
organic solvents and a water-in-oil microemulsion. Using solubility
parameters, the composition of the reaction medium was varied to
study the effects on polymer yield, molecular weight, and
dispersity. It has been discovered that polymers with low
dispersities and with molecular weights ranging from 1000 to 3000
can be synthesized in reversed micelles. In addition, it has been
discovered that reactions conducted in bulk solvents resulted in a
narrow range of molecular weights (281 to 675 with
poly(p-ethylphenol) in a dimethylformamide (DMF)/water system and
1,400 to 25,000 with poly(m-cresol) in an ethanol/water
system).
[0022] With DMF as the chromatography eluent, the effect of LiBr in
DMF on the molecular aggregation of poly(p-ethylphenol) was
determined using gel permeation chromatography (GPC). The presence
of LiBr (at 0.35 w/v %) in DMF resulted in complete dissociation of
the aggregates in solution. Further, poly(p-ethylphenol) was
functionalized at hydroxyl positions with palmitoyl and cinnamoyl
groups. Structural characterization of the polymers was carried out
by .sup.13C-NMR, UV, and FTIR spectroscopies.
[0023] Other objects, features and advantages will be apparent from
the following detailed description of preferred embodiments thereof
taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a schematic of ortho- and para-substituted phenol
polymerization catalyzed by horseradish peroxidase (HRP);
[0025] FIG. 2 is the .sup.13C-NMR spectra for (a) p-ethylphenol and
(b) poly(p-ethylphenol);
[0026] FIG. 3 is a graph of the effect of LiBr concentration in DMF
on poly(p-ethylphenol) molecular weight;
[0027] FIG. 4a is a differential scanning calorimetry (DSC)
thermogram of poly(p-ethylphenol) prepared in reversed
micelles;
[0028] FIG. 4b is a thermogravimetric analysis (TGA) of
p-ethylphenol and poly(p-ethylphenol) prepared in reversed
micelles;
[0029] FIG. 5 shows the molecular weight distribution of
poly(p-ethylphenol) before and after heating;
[0030] FIG. 6a shows FTIR spectra of poly(p-ethylphenol) (i) before
and (ii) after esterification with palmitoyl chloride;
[0031] FIG. 6b shows FTIR spectra of poly(p-ethylphenol) (i) before
and (ii) after esterification with cinnamoyl chloride; and
[0032] FIG. 7 shows the UV absorbance at 259 nm of
poly(p-ethylphenol) before and after cinnamoylation;
DETAILED DESCRIPTION OF EMBODIMENTS
[0033] 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
AOT/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 DMF 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 poly(p-ethylphenol) 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 in 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
[0034] 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
.mu.M) was added to form a clear reversed micellar solution having
a W.sub.O (molar ratio of water to surfactant) of about 15.
p-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.
[0035] 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
[0036] 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. Isooctane and chloroform were stored with molecular
sieves to remove water from the solvents. 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
[0037] 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.
[0038] A. Structural Characterization
[0039] 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
p-phenylphenol and 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.
[0040] .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.
[0041] FIGS. 2a & 2b illustrate .sup.13C-NMR spectra and peak
assignments for the monomer and the polymer, respectively. The peak
for C2 & C6 (at 115.9 ppm in the monomer and 117 ppm in the
polymer) diminished while an additional peak appeared at 131 ppm in
the polymer. 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)].
[0042] B. Molecular Weight Determination
[0043] 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 prior to injection. It was
ascertained that the filters did not retain any polymer during
filtration.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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 intermolecular forces. However, LiBr
appears to be very effective in overcoming these intermolecular
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.
[0050] 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/isooctane >4.5M 2500 300,000 2700 ethylphenol)
reversed micelles (>2.5) (1.4) (>2.0) (1.4) Poly(p- 85/15
>6.0M 3400 500,000 3200 ethylphenol) dioxane/water (>2.5)
(>2.0) (>2.0) (>2.0) Poly(p- 85/15 >6.0M 3000 300,000
3200 phenyl- dioxane/water (>2.5) (>2.0) (>2.0) (>2.0)
phenol) .sup.1Molecular weights 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.
[0051] C. Thermal Characterization
[0052] 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.
[0053] 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 at 200.degree. 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.
[0054] A 170% increase in molecular weight was observed, 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 that was used as eluent.
X-ray diffraction studies on the samples revealed a partial
crystallization of the heat-treated polymer.
EXAMPLE 4
[0055] 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 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 system (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
[0056] 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.
[0057] 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.
[0058] 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 ot reversed
micelles). Synthesis Monomer Polymer Mw/ medium Conversion.sup.1
Yield Mw Mn 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/water polymer soluble oligomers .sup.1Monomer
converted/monomer added initially
[0059] 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.
Analogous to the dioxane/water system, the monomer conversion was
either absent or poor at higher DMF contents, presumably due to
significant enzyme inactivity.
[0060] 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, DP of 200). At an enzyme concentration of
2 .mu.M, 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 40% 1400 polymer
removed as it with 1% 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 weights water/EtOH possible at
intermediate stages of reaction 40/60 11% 3000 90% conversion &
24000 M.sub.w water/EtOH at 5.times. enzyme concentration 20/80 3%
-- 20% conversion & 2000 M.sub.w at water/EtOH 5.times. enzyme
concentration 100% EtOH 0% -- Insoluble enzyme
[0061] Although high molecular weight polymers were produced with
poly(m-cresol) due to cross-linking and with ethanol, in some
applications, such as photoresists, 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 .mu.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.
[0062] 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.
[0063] 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 (reaction in AOT reversed micelles) using
isooctane and chloroform. Synthesis Monomer Polymer medium
Conversion Yield Mw Mw/Mn Comments 100% 100% 100% 2500 1.36 W.sub.O
= 15 Isooctane 100% 90% 100% 2500 1.38 W.sub.O = 9 Isooctane 75/25
100% 85% 1681 1.53 W.sub.O = 9 Isooctane/CHCl.sub.3 50/50 100% 75%
3461 1.85 W.sub.O = 9 Isooctane/CHCl.sub.3 25/75 75% 35% 3601 1.83
W.sub.O = 9 Isooctane/CHCl.sub.3 phase separation 100% 20% 10% 1000
1.07 W.sub.O = 9 CHCl.sub.3 phase separation
[0064] Polymer molecular weight was maximum at 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.
[0065] 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 O-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 HCl produced in the reaction.
[0066] 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.
[0067] In addition, the peak for O-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=O stretch at 1750 cm.sup.-1 in
the modified polymer. Similarly, cinnamoylation of the polymer was
confirmed by the disappearance of O-H stretch as well as from the
strong presence of C=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.
[0068] It will now be apparent to those skilled in the art that
other embodiments, improvements, details and uses can be made
consistent with the letter and spirit of the foregoing disclosure
and within the scope of this patent, which is limited only by the
following claims, construed in accordance with the patent law,
including the doctrine of equivalents.
* * * * *