Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
31L26~8
This invention relates to crosslinked polymers,
and in particular, to crosslinked polymers which form
water-swellable, water-insoluble gels and water-swellable,
water-insoluble foams.
Linear polyalkylenepolyamines are rendered
water-insoluble through the addition of diisocyanates
at a temperature between 30C and 200C, using techniques
disclosed in U.S. 4,087,413. Unfortunately, the resulting
water-insoluble products are prepared either in neat
form or in the presence of a suitable solvent. Reactions
of amines with diisocyanates are disclosed in
U.S. 4,177,038. However, it is typically necessary to
prepare such products under essentially anhydrous
conditions because isocyanates moieties are hydrolyzed
rapidly by water to yield substituted ureas.
In view of the fact that polymers such as
acylated polyalkylenepolyamines are water~soluble, and
in view of the fact that it would be highly desirable
to render such polymers water-insoluble in an aqueous
medium; it would be highly desirable to provide a
,~, jl
30,785h-F -1-
--2--
~Z6~
process for preparing a crosslinked polyalkenepolyamine
in a non-anhydrous environment.
In view of the fact that known processes
provide gelled materials, it would also be highly
desirable to provide a process for efficiently preparing
stable, water-swellable but water-insoluble foams.
The present invention is a process for cross-
linking a polyalkylenepolyamine wherein a polyisocyanate
is contacted with an aqueous liquid comprising a poly-
alkylenepolyamine which is at a specific pH and whichis subjected to a sufficiently high rate of shear
agitation to yield a water-swellable, essentially
water-insoluble gel. By the term "specific pH" is
meant a pH which can range from slightly acidic to
slightly basic.
The advantage of the process of this inven-
tion is that gelled materials can be prepared quickly
and efficiently under conditions which do not require
an anhydrous environment. The gels are useful in a
wide variety of applications in which water-insoluble,
swellable gels are useful. Of particular interest, are
those uses in which a gel is useful as a biosupport for
the isolation and purification of enzymes and other
p~-oteins.
Thus, in another aspect the present invention
is a process for covalently immobilizing a protein
wherein said protein is contacted with a polyalkylene-
polyamine and a polyisocyanate in an aqueous liquid
which is at a specific pH and which is subjected to a
sufficiently high rate of agitation to yield a water-
swellable, essentially water-insoluble gel.
30,785~-F -2-
--3--
~;~6~
Still another aspect, the present invention
is a pxocess for providing stable, water-swellable,
essentially water-insoluble foam wherein a polyisocyanate
is contacted with an aqueous liquid comprising a gelatin
which is at a specific pH and which is subjected to a
sufficiently high rate of shear agitation. By the term
"specific pH" is meant a pH which carl range from slightly
acidic to slightly basic.
Thus, another advantage of the process of
this invel~tion is that foamed materials can be prepared
quickly and efficiently under conditions which do not
require an anhydrous environment. Foams which are
prepared can be dried in order to provide gels which
are hard, soft, friable, etc. Upon drying the foam
structure is maintained and the original gel can be
regenerated by swelling in water. Typically, foams are
closed cell foams. The foams are useful in a wide
variety of applications in which water-insoluble,
swellable foams are useful. Of particular interest,
are those uses in which a foam is useful as a biosupport
for the isolation and purification of enzymes and other
proteins where a proteinaceous environment is desirable.
Thus, still in another aspect the present
invention is a process for covalently immobilizing a
protein wherein said protein is contacted with a gelatin
and a polyisocyanate in an aqueous liquid which is at a
specific pH and which is subjected to a sufficiently
high rate of agitation to yield a water-swellable,
essentially wa~er-insoluble foam.
The immobilized proteins are useful for a
varie~y of uses. For example, immobilized proteins can
be used as catalysts or in the purification of bioproducts.
30,785A-F -3-
~Z64~9~
As used herein, the term "gelatin" refers to
those types of proteins which are obtained by the
hydrolysis of collagen by boiling skin, ligaments,
tendons, etc. Type A (obtained from acid treated raw
materials) and Type B (obtained from alkali treated raw
- materials) can be employed. Most grades of commercially
available purified gelatins can be employed. Gelatins
having a high bloom number; for example, greater than
about 100; are preferred.
Polyalkylenepolyamines have repeating units
which can be independently represented as follows:
~N-~CHR)
, x (I)
tN-(CHR)X-N-(CHR) t
H c-Rl x (II)
20 or~N-(CHR) - N-C-~CHR) ~
x x (III)
wherein R is independently hydrogen or a C1 to C3 lower
alkyl; Rl is Cl to Cl8 alkyl, aryl, alkylaryl or
substituted alkyl, alkylaryl or aryl; and x is 2 or 3.
Such types of polymers are prepared using techniques
disclosed in U.S. 4,087,413. In general, the polyalky-
lenepolyamines of this invention are any water-soluble
30,785A-F -4-
-5
polyalkylenepolyamines containing secondary amine
groups. The preferred polyalkylenepolyamine is poly-
ethylenepolyimine. In the case of the partially hydro-
lyzed acylated polyamines, the extent of hydrolysis can
range from 10 to 85, preferably 50 weight percent,
based on the weight of the polymerized monomers.
Preferably, the molecular weight of the polyalkylene-
polyamine can range from 10,000 to 1,000,000; most
preferably from 50,000 to 75,000. Poor gels are obtained
or no gelation occurs if the molecular weight of the
polymer is not sufficiently high.
Organic polyisocyanates which can be employed
include aromatic, aliphatic and cycloaliphatic poly-
isocyanat:es and combinations thereof. Representative
of these types are the diisocyanates such as m-phenylene
diisocyanate, tolylene-2,4-diisocyanate, tolulene-2,6-
diisocyanate, hexamethylene-1,6-diisocyanate, tetra-
methylene-1,4-diisocyanate, cyclohexane-1,4-diiso-
cyanate, hexahydrotolylene diisocyanate tand isomers),
naphthylene-1,5-diisocyanate, 1-methoxyphenyl-
-2,4-diisocyanate, diphenylmethane-4,4-diisocyanate,
4,4'-biphenylene diisocyanate, 3,3'-dimethoxy-
~-4,4'-biphenyl diisocyanate, 3,3'-dimethyl-4,4'-biphenyl
diisocyanate, and 3,3'-dimethyldiphenylmethane-
-4,4'-diisocyanate; the triisocyanates such as
4,4',4'-triphenylmethane triisocyanate, polymethylene
polyphenylisocyanate and tolylene-2,4,6-triisocyanate;
and the tetraisocyanates such as 4,4'-dimethyldiphenyl-
methane-2,2',5,5'-tetraisocyanate. Especially useful
due to their availability and properties are tolulene
diisocyanate, diphenylmethane-4,4'-diisocyanate and
pol~methylene polyphenylisocyanate.
30,785A-F -5-
-6-
~12~
Crude polyisocyanate can also be used in the
practice of the present invention, such as crude tolu-
ene diisocyanate obtained by the phosgenation of a
mixture of toluene diamines or crude diphenylmethylene
diisocyanate obtained by the phosgenation of crude
diphenylmethylenediamine. The preferred undistilled or
crude isocyanates are disclosed in U.S. 3,215,652O
The amount of polyisocyanate which is employed
relative to the polyalkylenepolyamine can vary depending
upon factors such as the reactivity of the species, the
concentration of reactants relative to the aqueous
liquid, the amount of crosslinking desired and the
like. Preferably, the amount of polyisocyanate ra~ges
from 5 to 25, preferably 10 to 15 weight percent, based
on the weight of the dry polyalkylenepolyamine. Typically,
poor gels are obtained or no gelation occurs if there
is employed an insufficient amount of polyisocyanate.
The amount of polyisocyanate which is employed
relative to the gelatin can vary depending upon factors
such as the reactivity of the species, the concentration
of reactants relative to the aqueous liquid, the amount
of crosslinking desired and the like. Preerably, the
amount of polyisocyanate ranges from 1 to 20, preferably
2 to 8 weight percent, based on the weight of the dry
gelatin. Typically, poor foams are obtained or no
permanent foaming occurs if there is employed an
insufficient amount of polyisocyanate.
The process of this invention is perfomed ln
an aqueous liquid. Preferably, the aqueous liquid
comprises essentially water. As used herein, the term
"aqueous liquid" means a liquid comprising water which
30,7~5A-F -6-
-7-
~264~
can contain additives soluble therein or immiscible
therewith. For example, the aqueous liquid can contain
an aqueous phase comprising water and additives soluble
therein, and a water immiscible organic solvent. The
amount of the water immiscible organic solvent can
range, for example, from O.5:1 to 5:1 relative to the
volume of the aqueous phase. Examples of water
immiscible organic solvents include benzene, toluene,
methylenechloride, chloroform, and the like.
Typically, depending upon the polyamine, the
pH of the aqueous liquid can range from 4 to 9,
preferably S to 8, more preferably from 5 to ~.5, most
preferably from 6 to 6.5. Also, depending upon gelation,
the pH of the aqueous liquid can range from 4 to lO,
preferably 6 to 8. The pH of the aqueous liquid is
critical as the rate of crosslinking is sensitive to
pH. That is, for example, the crosslinkin~ reaction
can occur too quickly if the p~ is exceedingly low.
Conversely, the crosslinking reaction can occur very
2a slowly if the pH is exceedingly high. In addition,
poor gels and foams are obtained or no gelation or
foaming occurs if the pH of the aqueous liquid is not
within a suitable (i.e., specific) range. The pH
typically decreases by a small amount (i.e., less than
1 unit) during the crosslinking reaction. This change
in pH can be controlle~ by a suitable buffer.
The amount o aqueous liquid which is employed
depends upon the molecular weight and the amount of
polyalkylenepolyamine or gelatin which is employed.
Typically, poor gelation or foaming occurs if the
polym r solution is very dilute or the molecular weight
of the polymer is very low. Conversely, the formation
30,785A-F -7-
~L;Z6~ 8
of desirable gels or foams is a difficult process to
perform if the polymer concentratlon in the aqueous
liquid is very high or the molecular weight of the
polymer is very high. Preferably, the amount of
polyalkylenepolyamine present in the aqueous liquid can
range from 10 to 50, preferably from ~0 to 30 weight
percent, based on the weight of the polymer and aqueous
liquid. Preferably, the amount of gelatin pr~sent in
the aqueous liquid can range from 0.5 to 20, preferably
from 1 to 10 weight percent, based on the weight of the
polymer and aqueous liquid.
The temperature at which the process of this
invention is performed is not particularly critical and
can vary. For example, the process of this invention
can be performed from 0 to 30C. Typically, crosslinking
can occur at room temperature. However, the a~ueous
liquid can also be heated above 30C.
The process of this invention is highly
dependent upon the rate of agitation to which the
aqueous polyalkyleneamine/polyisocyanate or gelatin/-
polyisocyanate mixture is subjected. Little or no
gellation or foaming occurs when a conventional magnetic
stirrer or mechanical ~tirrer is employed as an agitation
device. For this reason, it is necessary to provide a
sufficiently high rate of agitation using a device such
as a homogenizer, ultra-sonic stirring device or blender.
Typically, the agitation rate should exceed 500 rpm,
preferably 3,000 rpm for formation process. Typically,
the agitation rate should exceed 3,000 rpm for the
gelation process. Preferred agitation rates range from
5,000 rpm to 50,000 rpm, most preferably from 10,000
rpm to 20,000 rpm. It is understood that practicall.y
30,785A-F -8-
~26415~3
any device capable of providing such a high rate o
agitation can be effectively employed.
The polyalkyleneamine/polyisocyanate or
gelative/polyisocyanate mixture is subjected to a high
rate of agitation for a period of time which can ~ary.
For example, the period of time over which the mixture
is subjected to agitation can depend upon factors such
as the amount of reactants, the concentration of reactants
in the aqueous liquid, etc. T~pically, the period of
time over which the polyalkyleneamine/polyisocyanate
mixture i~ subjected to agitation can range from 2
seconds to several seconds, most preferably from 3
seconds to 5 seconds. Typically, the period of time
over which the gelatin/polyisocyanate mixture is s~`ojected
to agitation can range from 2 seconds to several minutes,
preferably from 20 seconds to 2 minutes, most preferably
for 30 seconds.
In the situation in which a two phase (i.e.,
a~ueous solvent and water-immiscible solvent) solvent
20 system is employed, hydrolysis of the isocyanate is
believed to be retarded. Thus, conventional stirring
devices such as magnetic stirrers or mechanical stirrers
can be employed to yield good results. However, the
rate of crosslinking is much slower than that observed
25 when high rates of agitation are employed. That is,
reaction times ranging from several minutes to 1 hour
may be necessary for sufficient crosslinking to occur.
In any event, the actual reaction time can depend upon
factors such as the particular polymer, the isocyanate
concentration, the relative amount of solvent, the
proportions of aqueous phase and water im~iscible
phase, etc.
30,785A-F -9-
--10-
~Z6~8
After the mixture is subjected to agitation,
the mixture is allowed to settle for a period of time
sufficient for gellation or foams formulation
(i.e., crosslinking) to be completed. This period of
time ranges from lO minutes to 60 minutes, preferably
from 15 to 20 minutes for gelation. This period of
time ranges from a few minutes to several hours,
preferably from l hour to 8 hours, most preferably for
2 hou.rs for foam formulations. Setting can occur over
a wide temperature range, but preferably occurs at room
temperature.
The process of this invention is most prefer-
ably carried out in a predominately aqueous phase. It
is unders1~ood that most additives common in an aqueous
liquid can be present. For convenience purposes, the
water-soluble polyalkylenepolyamine or gelatin is
dispersed in the aqueous liquid and a solution is
formed, which solution has the appropriate pH. This
solution is contacted with the polyisocyanate, and the
mixture is subjected to a sufficiently high rate of
agitiation for a short period of time. The mixture is
then allowed to gel or set as appropriate. If desired,
the resulting gel or foam can be dispersed in an aqueous
liquid by providing further agitation. If desired, the
foam can be dried. In any case, the gel or foam can
be further treated, as desired, for the par-ticular
application for which it is employed.
Proteins containing free primary amine groups,
such as enzymes and antibodies, can be immobilized
using the process of this invention. For example,
glucose oxidase, esterase, catalase, alkaline phosphatase,
and the like can be easily and effectively immobilized.
30,785~-F -lO-
Preferably, the polyalkylenepolyamine or gelatin,
polyisocyanate and protein are contacted in the suitable
solution at the suitable pH and subjected to the
appropriate agitation. Typically, the amount of protein
which is employed ranges from 0.1 to 10 milligrams per
milliliter of polyalkylenepolyamine or gelatin which is
employed.
When employed as a biosuppo~t for enzyme
immobilization, the reaction conditions under which the
gels are formed can be modified in accordance with the
requirements necessary for the individual enzyme.
Covalent attachment of the enzyme to the gel or foam
is believed to occur during the mixing and gellation or
foamation stages. It is believed that immobilization
occurs via the reaction of free isocyanate moieties of
the gel or foam with amino moieties of the protein. If
desired, the enzyme active site can be protected from
reaction by the presence of suitable enzyme substrates
and/or products. The amount of immobilized protein can
vary. The enzyme activity of the immobilized enzyme
can also vary and is typically from 2a to 80 percent of
its original value.
The gels which are prepared are very hydro-
philic, though essentially water-insoluble. When dry,
the gels are amorphous, hard, solid materials which can
be easily handled. When fully hydrated, the gels are
transparent to translucent, soft, compressible solids
which are insoluble in water.
The foams which are prepared are very hydro-
philic, though essentially water-insoluble. When dry,
the foams can be hard and/or friable solid materials
30,785A-F -11-
-12-
~264~
which maintain foam structure and can be easily handled.
When fully hydrated, the foams are transparent to
transucent, soft, compressible solids which are insoluble
in water. The foams are typically closed cell foams.
The gels or foams can be employed as water
adsorbent materials, insoluble chelants for metals,
insol~ble modifiers of pH for aqueous systems, biosupports,
and other such uses.
The following examples are presented to
further illustrate the scope of this invention.
Example 1
A 50 percent aqueous solution of pol~mer was
prepared by dispersing 5 parts by weight of a high
molecular weight polyethyleneimine in water. The pH of
the solution was adjusted to 6.5 using concentrated
hydrochloric acid. The solution was diluted with water
to yield a solution containing 30 percent polyethylene-
imine. To this solution was added 0.2 parts by weight
of hexamethylene-diisocyanate. The mixture was
homogenized using a blender (agitation rate was about
10,000 rpm) for 5 seconds. The mixture was allowed to
stand for 20 minutes. The resulting firm gel was broken
into pieces, stirred with 100 ml water and centrifuged
for S minutes. The supernatant liquid was decanted.
- 25 The resulting gel has a volume of about 50 ml at a pH
- of about 5.
Example 2
A 25 percent aqueous solution of polymer was
prepared by dispersing 5 g of said polymer in wa-ter in
an amount sufficient to provide a 20 ml aqueous solution.
30,7~5A-F -12-
- -13-
~l26a~
The polymer was a 50 percent hydrolyzed polyethyl-
oxazoline having a molecular weight of 50,000. The pH
of the solution was adjusted to 8 using hydrochloric
acid. The solution was magnetically stirred in a
beaker and 15 ml of methylene chloride and 0.4 ml of
1,6-hexamethylene diisocyanate was added thereto. The
mixture was rapidly stirred for 15 minutes using a
magn~tic stirrer set at the highest obtainable spe~d
(estimated at 200 rpm). The mixture was allowed to
stand for 1 hour. The product was triturated 3 times
with 40 ml each of acetone. The resulting white,
acetone-swollen gel was dried to give 4 g of white
granular solid.
Example 3
Thè i~nobilization of glucose oxidase (GOD)
using polyethyleneimine~was performed as follows.
To 1 ml of an aqueous polyethylimine solution
(obtained from Aceto Chemicals, under the specification
PEI-1,000) and adjusted to pH 6.1 and concentration of
0.25 g/ml was added 1 ml of aqueous solution containing
50 units (U) GOD and 20 ~1 of 1,6,-hexamethylene
dissocyanate and the mixture was homogenized for 5
seconds using a laboratory model Ross homogenizer at
its maximum speed. The gel was allowed to sit for lS
minutes, diluted to 20 ml with water and dispersed by
stixring at low speed in a 3-speed Waring Blender. The
total gel, (20 ml) which assayed for 35 U was centrifuged
and the supernatant ~6 ml) was decanted. The supernatant
assayed for 4 U and the washed gel containing the
immobilized ~OD assayed for 33 U. After sitting at
room temperature for 1 week, the gel assayed for 38 U.
30,785A-F -13-
-14-
~648~3
Example 4
The immobilization of esterase using hydrolyzed
polyethyloxazoline was performed as follows.
To 5 ml of a 15 percent solution of 50 percent
hydrolyzed polyethyloxazoline (M.W. = 50 M, pH 7.6) was
added 100 ~1 of esterase (about 100 U~ and 200 ~1
1,6-hexamethylene dissocyanate and the mixture was
homogenized for 10 seconds on a Vertis Model 45
homogenizer set at medium speed and left for 1 hour.
The firm gel was chopped up in a Waring Blender with 10
ml of water, made up to 20 ml, stirred for 30 minutes
and then centrifuged. The supernatant had no activity
and the ge~l assayed for 50 U. The yield of i~nobilized
esterase is 50 percent.
ExamPle 5
15 ml of a 7.5 percent solution of 300 bloom
swine skin gelatin was adjusted to a pH of 8. To this
mixture was added 0.1 ml hexamethylene-2,4-diisocyanate
and the mixture was homogenized for 30 seconds at high
speed using a Ross Laboratory Homogenizer (12 mm head).
The resulting foam which has a volume of about 25 ml
was set in about one minute. The foam was air dried to
yield about 15 ml of a firm white foam.
Example 6
- 15 ml of a 7.5 percent solution of 200 bloom
technical grade gelatin containing 0.15 ml of alkaline
phosphatase (Sigma ~o. P4502) was adjusted to a pH of
8. To this mixture was added 0.1 ml hexamethylene-
2,4-diisocyanate and the mixture was homogenized as in
Example 5. The foam gels slowly enough to be cast as a
film. After air drying for three days and reswelling
30,785A F -14-
-15-
in water, the washed film assays for most of the original
enzyme activity.
Exam~le 7
S g of a 10 perce~t solution of 200 bloom
gelatin was diluted with lO ml water and the pH of the
mixture was adjusted to about 8.5. To this was added
one drop of a liquid emulsifier. To this was added
0.1 ml of hexamethylene-2,4-diisocyan~te and the mixture
was immediately homogenized as described in Example 5.
The mixture sets to a white, soft foam with a volume of
about 50 ml. After thorough drying at room temperature,
the volume of the white collapsed foam was about 20 ml
and the f~am weighs 0.7 g.
30,785A-F -15-
