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Description  |
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The present invention relates to novel substrates, processes for making
them and uses for them, including synthesizing chemical compounds and
chromatography.
A variety of chromatographic techniques and methods of chemical synthesis
employ some form of substrate. In a simple batchwise operation the
substrate is contained in a vessel and interacted sequentially with added
reagents which are then removed by filtration and thorough washing. In a
continuous or semi-continuous process the substrate is in the form of a
bed such as a column and various reagents are sequentially passed through
the bed.
Continuous and semi-continuous techniques thus usually offer advantages
over batchwise operation in terms of ease of operation, but can
nonetheless suffer problems related to volume change in the bed resulting
in pressure changes in the through-flow through the column. Such problems
can be particularly acute where the substrate involves some form of gel. A
discussion of these problems in the area of solid-phase synthesis is
contained in Dryland and Sheppard J. Chem. Soc. Perkin Trans. I 1986 p.125
to 137. An additional relevant publication in this area is Epton, Marr,
McGinn, Small, Wellings and Williams Int. J. Biol. Macromol 1985 7 p.289
to 298. It is known as explained in the first of these publications to
provide in the case of gel substrate a rigid framework to enclose the gel
polymer, so constructed so as to maintain channels for liquid flow and yet
permit diffusion of reactants into and out of the gel matrix.
U.S. Pat. No. 3991017 (Rohm and Haas Company) describes a substrate for use
in ion exchange resins in which a gel type, crosslinked copolymer at least
partially fills the macropores of a macroreticular ion exchange resin.
Typically the macroreticular polymers have a surface area of at least 1
sq. meter per gram, more generally at least 5 sq. meters per gram, and
have pores larger than about 15 to 20 .ANG. units. The macroreticular
polymers are conventionally in bead form usually in an overall particle
size of about 10 to 900 microns. At least 5 parts by weight of gel forming
components up to a maximum of 300 parts by weight of gel copolymer per 100
parts by weight of macroreticular base polymer are suitably used.
UK No. 1574414 (United Kingdom Atomic Energy Authority) describes a
composite material comprising a plurality of discrete particles of a
porous rigid support material having a deformable gel within the pore
structure of the particles. The particles are discrete porous particles of
inorganic material such as natural diatomaceous earth.
It is an object of the present invention to provide an improved substrate
for use in for example solid phase synthesis, chromatography and ion
exchange applications. It is a further object of the present invention to
provide such a substrate allowing improved loading factors to be achieved.
According to a first aspect of the present invention there is provided a
substrate comprising a porous polymeric material having a porosity of at
least 75% and comprising pores having a diameter within the range 1 to 100
.mu.m and being interconnected by a plurality of holes, and a gel or
material adapted in use to form a gel which gel or pre-gel material is
contained and retained within the pores of the polymeric material and is
adapted in use , to interact with a reactive species.
The interaction between the gel and a reactive species will be selected
having regard to the desired use of the composite substrate. In the case
of chemical synthesis the interaction is suitably that of chemical binding
and the gel is suitably adapted.
By use of the present invention a substrate is provided in which as in the
case of synthesis the gel is capable of a loading of reactive residues up
to 5mmol of chemical compound synthesized per g of composite substrate.
The gel is suitably a highly solvent swollen cross-linked gel and can for
example be a soft deformable polyamide gel. Examples of other gels that
can be employed include polystyrenes, saccharose, dextrans,
polyacryloylmorpholine, polyacrylates, polymethylacrylates,
polyacrylamides, polyacrylolpyrrolidone, polyvinylacetates,
polyethyleneglycol, agaroses, sepharose, other conventional chromatography
type materials and derivatives and mixtures thereof. Preferably the highly
porous material has a pore volume of 75 to 98%, more preferably 85 to 98%,
even more preferably 90 to 95%. Suitably the material is a cross-linked
polymeric material. On a weight for weight basis the ratio of swollen gel
to porous material can range from 60:40 to 95:5 swollen gel:porous
material more preferably from 75:25 to 95:5, with a preferred ratio being
about 80:20.
The porous material can be in particulate form, preferably of a particle
size between 125 and 1500 .mu.m, more preferably between 250 and 850
.mu.m, and can for example be the cross-linked vinyl material described in
U.S. Pat. No. 4,522,953 or a highly porous cross-linked poly-condensation
polymeric material described in our co-pending application GB No. 8709688.
Both of these porous materials have a high pore volume and can have pores
within the range of approximately 1 to 100 .mu.m, preferably 1 to 50
.mu.m. Materials made by the processes described in U.S. Pat. No.
4,522,953 or GB No. 8709688 are particularly suitable for use in the
present invention as they are highly porous and can consist of regularly
formed fully interconnecting cells. Such a combination of features
provides a structure that can show rapid uptake of fluids and relatively
unobstructed flow through the matrix. These porous structures are suitably
made by means of a high internal phase emulsion and thus have the
advantage that they can be reproducibly engineered to provide a range of
cell sizes and interconnecting holes. Preferably the porous polymeric
materials are cross-linked to an extent such that they do not swell to
more than twice their dry bed volume in use. Throughout the present
specification porosity values and pore size measurements refer to the
porous polymeric material in the unswollen state.
Preferably the gel is formed in situ in the pores of the porous material.
The particulate porous material can be admixed with a solution which
permeates the open interconnecting pores of the particulate material and
forms therein the gel. The resulting material is preferably placed, or can
be made in, in a column in order to provide an appropriate through flow
system for example in performing a chemical synthesis. Alternatively the
porous material can be in monolithic block form and the gel can be formed
in situ following permeation into the interconnecting pores of the block.
According to a second aspect of the present invention, there is provided a
process for preparing a substrate comprising depositing and retaining a
gel or a material adapted in use to form a gel within the pores of a
porous polymeric material having a porosity of at least 75% and comprising
pores having a diameter within the range 1 to 100 .mu.m and being
interconnected by a plurality of holes, the gel being adapted in use to
interact with a reactive species.
Preferably the process includes forming the gel within the pores of the
porous material. More preferably the process includes forming the gel
within the pores of the porous material and simultaneously retaining the
gel during its formation within the pores of the porous material. The gel
can for example be made by the conventional polymerization and
co-polymerization routes to form gels, for example free-radical vinyl
polymerization, poly-condensation reactions, and cross-linking of soluble
linear polymers.
The gel and the porous polymeric materials are suitably those mentioned
above. In particular we have found that use of porous polymeric materials
as described in U.S. Pat. No. 4,522,953 or GB No. 8709688 having
interconnected pores allows ready access of the gel materials into within
the pores and subsequent ready access of reactive species in use.
Preferably the process includes forming the gel within the pores of the
porous material. Preferably retention of the gel within the pores is by a
chain entanglement and/or interpenetration between the gel and the surface
of the porous polymeric material and/or by a process that is believed to
involve chemically binding the gel to the surface of the pores of the
porous material. Thus preferably the process includes depositing and
retaining the gel within the pores of the porous polymeric material by
subjecting porous cross-linked polymeric material to a solution comprising
a swelling solvent for the porous polymeric material and gel precursor
materials, allowing the gel precursor materials to permeate the swollen
polymeric material and forming the gel from the gel precursor materials
within the pores. Preferably the process additionally or alternatively
includes depositing and retaining the gel within pores of the porous
polymeric material having reactive groups thereupon by allowing gel
precursor materials to permeate the pores of the polymeric material and
forming the gel from the gel precursor materials within the pores and
simultaneously allowing the gel and/or gel precursor to react with the
reactive groups on the pores of the porous polymeric material.
In the mode of retention comprising chain entanglement/interpenetration a
porous cross-linked polymeric material is suitably employed which is mixed
with the precursors for forming the gel in the presence of a swelling
agent for the polymeric material. As the gel precursors permeate the
porous polymeric material, the porous material swells and entraps the
contactable portion of the forming cross-linked swollen gel material by
polymer chain interpenetration between the swollen polymeric polymer
material and the forming cross-linked swollen gel. Cross-linking of the
porous polymeric material to the extent that it can swell up to twice its
dry bed volume has been found appropriate. Suitable swelling solvents will
depend on the nature of the porous polymeric material. For polystyrene for
example suitable solvents would be halocarbons such as dichloroethane,
dichloromethane, chloroform, and toluene and tetrahydrofuran. The gel
material is suitably the monomer precursors which permeate the pores and
polymerize in situ leading to the chain entanglement and interpenetration.
Where retention is believed to occur by chemical bonding between the highly
porous material and the material described in use to be in the form of a
gel the chemical bonding can be achieved by reaction between the gel ready
formed and reactive groups on the porous material and/or reaction with
reactive groups on the porous material during gel formation. An example of
this latter technique is vinyl polymerization to form the gel and
simultaneous attachment via a reactive group on the porous material.
References throughout the specification to chemical binding between the
gel and the porous polymeric material are to be interpreted as the
believed mechanism having regard to the evidence given below.
The porous material can be made with the reactive groups ready in situ or
can be treated subsequent to preparation to contain the reactive groups.
Appropriate reactive groups include vinyl, aminomethyl and carboxyl.
Evidence indicating a slight difference in performance between these two
modes of effecting retention is given as follows, showing a preference for
the binding route and indicating that the binding is probably chemical
co-valent binding. For each embodiment a sample of substrate was prepared
and the yield of composite substrate relative to the starting materials
was calculated. For the chain entanglement mode with swelling of the
porous polymeric material 70% retention of gel in the composite substrate
was achieved. For the presumed binding route 100% inclusion of the gel was
achieved, indicating complete retention of the gel by the porous material.
For comparison mere permeation of the gel into the porous polymeric
material with no active steps to effect its retention resulted in 0%
inclusion of gel following conventional washing steps.
According to another aspect of the present invention there is provided a
substrate comprising a highly porous polymeric material having a porosity
of at least 75% and comprising pores having a diameter within the range 1
to 100 .mu.m and being interconnected by a plurality of holes, wherein
reactive groups are chemically bound to the pore surfaces and are adapted
in use to interact, e.g. by binding chemically, with a reactive species.
Suitable porous materials are disclosed in U.S. Pat. No. 4,522,953 and in
our co-pending application mentioned above and have pore sizes preferably
in the range 1 to 50 .mu.m. Preferably the materials are cross-linked and
have a porosity of 75 to 98%, more preferably 85 to 98%, even more
preferably 90 to 95%. Materials made by the process described in U.S. Pat.
No. 4,522,953 or GB No. 8709688 specifications are particularly suitable
for use in the present invention as they are highly porous and can consist
of regularly formed fully interconnecting cells. Such a combination of
features provides a structure that can show rapid uptake of fluids and
relatively unobstructed flow through the matrix. These porous structures
are suitably made by means of a high internal phase emulsion and thus have
the advantage that they can be reproducibly engineered to provide a range
of cell sizes and interconnecting holes. The porous polymeric material can
be employed in particle, sheet or monolithic block form. The porous
material can be made with the reactive groups already in situ e.g. vinyl
groups on a polyvinyl porous material or can be treated subsequent to
preparation to provide the reactive groups e.g. aminomethyl groups. If
desired the reactive groups can be further reacted to provide spacer
groups which subsequently interact with the reactive species.
According to another aspect of the present invention there is provided a
use of the present substrates wherein a reactive species is passed through
the substrate, preferably under flow conditions, and interacts with the
reactive substrate.
Examples of use of the present method include: chemical synthesis including
peptide synthesis, oligonucleotide synthesis, oligosaccharide synthesis,
and monoclonal synthesis; chromatography; ion exchange; and separation
techniques including gel electrophoresis. In chemical synthesis a first
species can be passed through the substrate and then further reactive
species can be passed sequentially through the substrate so as to react
with the reactive residue then present and chemically attached to the
substrate. Eventually the final chemical assembly can be detached and
removed from the substrate. The present process can thus be particularly
suitable for the synthesis of peptides.
The substrate can be any of those described above. By means of the present
use sequential synthesis can occur at high yield. The chemical nature of
the highly swollen gel in a flow through system allows reactive residues
to be attached with a high load leading to high yields. In peptide
synthesis yields of 0.1 to 5 mmol per g of composite substrate can be
achieved.
In the preferred embodiment in which gel is contained and retained within
the pores of a highly porous polymeric material the overall substrate can
nonetheless be substantially rigid, incompressible and homogeneous. With
such a substrate in the form of a packed column flow rates suitable for
flow operation can be achieved. Batchwise operation can alternatively be
employed.
Moreover in the more preferred embodiment in which the gel is believed
chemically bound to the porous polymeric material suitable flow rates can
be achieved without the gel being washed out of the porous material or
lost into solution.
It is to be understood that the present invention extends to the products
of the present processes and uses.
Embodiments of the present invention will now be described by way of
example only with reference to the following Examples.
The present invention can be applied to a variety of systems. One system
however which is of particular interest is peptide synthesis. The present
system is especially applicable to peptide synthesis as it lends itself to
repeated sequential reactions at a relatively high throughput rate.
Thus in a peptide synthesis scheme the reactive group (X) is attached to
the polymer and is reacted with the first amino acid of the sequence to be
synthesized. This first amino acid contains its own protecting group (PG).
After deprotection, a further protected amino acid is attached and then
the process of deprotection and coupling is repeated until the desired
amino acid sequence is produced. The resulting peptide is then detached
from the polymer support and can if desired be purified.
Diagrammatically the peptide synthesis scheme can be shown as follows:
##STR1##
In one embodiment the synthesis takes place within a highly swollen
deformable polyamide gel which is polymerized within the pore structure of
the polymeric structural material, which is rigid. Diffusion of reactants
into and out of the polyamide gel where the reaction takes place can be
rapid and negligible pressure develops when the system is for example in
the form of a column under normal flow conditions. Synthesis under
conditions of flow is preferred as, in general, flow systems offer greater
opportunities for analytical control. For example the continuous
monitoring of effluent streams by UV-VIS spectrophotometry and other
continuous monitoring techniques can be readily achieved and offers the
potential for automated feedback control of each synthesis cycle.
EXAMPLE 1
Preparation of Substrate
A cross-linked polyvinyl porous polymeric material formed by the high
internal phase emulsion method described in our U.S. Pat. No. 4,522,953
was employed as the structural carrier. It had 90% pore volume, and
employed 10% cross-linking agent divinyl benzene and had a density of
0.047 g cm.sup.-3. The polymeric material was in the form of a milled and
sieved powder having a particulate size within the range 850 to 1200
.mu.m. Its pore size was within the range 1 to 50 .mu.m.
The polymeric gel matrix was poly (N-(2-(4-acetoxyphenyl)ethyl)acrylamide).
A solution of 2.5 g of the monomer N-(2-(4-acetoxyphenyl)ethyl)acrylamide,
0.075g of the cross-linking agent ethylene bis (acrylamide), 0.1 g of the
initiator azobisisobutyronitrile was prepared in 10 cm.sup.3
dichloroethane and deoxygenated by purging with nitrogen.
The milled and sieved particulate polymeric material (0.7 g) was added to
the solution and polymerization of the acrylamide was initiated by heating
the mixture at 60.degree. C. while rotating the sample on a rotary
evaporator modified for reflux. The dichloroethane served to swell the
porous polymeric material and allow ready penetration of the polyamide
monomer and subsequent entrapment and interpenetration of the polymerizing
polyamide by the porous polymeric material.
After 1 hour reaction time the resulting composite was washed exhaustively
with dimethylformamide and diethyl ether and then vacuum dried. The yield
of resulting composite was 2.7 g, the gel being retained within the porous
polymeric material due to chain entanglement.
0.25 g of the composite was treated with 5% solution of hydrazine hydrate
in dimethylformamide for 5 minutes. This treatment provided free phenolic
functionalities within the secondary gel matrix which act as reactive
groups (X).
Peptide Synthesis
To commence a peptide synthesis 0.95 g (5.0 mmol) t-butyloxycarbonyl
alanine and 1.24 g (6 mmol) dicyclohexylcarbodiimide were dissolved in
10cm.sup.3 dimethylformamide and allowed to react for 30 minutes with
stirring. This activated form of the thus produced protected amino acid
(O-acyl urea) was added to the dried composite substrate (0.25 g),
followed by 0.24 g (2.0 mmol) dimethylaminopyridine, and the
esterification reaction was allowed to proceed for 24 hours during which
time the mixture was agitated by passing through nitrogen in a solid phase
reactor. At the end of this time the composite was washed exhaustively
with dimethylformamide and diethyl ether. The weight of the loaded
composite following the reaction was 0.52 g.
To remove the protection group (PG=t-butyloxycarbonyl) 0.50 g of the loaded
composite substrate was retained in the solid phase reactor and 9cm.sup.3
benzyl alcohol was added. The suspension was nitrogen stirred for 1 hour
allowing sufficient time for the secondary gel matrix to swell in the
benzyl alcohol. 1cm.sup.3 of the deprotection reagent boron trifluoride
etherate was added and the reaction nitrogen stirred for 3 hours. The
composite was washed exhaustively with dimethylacetamide and diethyl
ether.
In order to carry out continuous flow synthesis the resulting composite was
transferred to the column of a Pepsynthesiser Mk2 (ex Cambridge Research
Biochemicals), which is a semi-automatic continuous flow peptide
synthesizer. The column was initially purged with a solution of 0.2 g (2
mmol) N-methylmorpholine in 50cm.sup.3 dimethylformamide to release the
free amino terminal groups, followed by a wash through with
dimethylformamide.
Further Chain Elongation
The symmetrical anhydride of Fmoc- Proline (PG=Fmoc
=fluorenylmethoxycarbonyl) was prepared by reacting 0.80 g Fmoc-Pro-OH
(2.4 mmol) with 0.23 g dicyclohexylcarbodiimide in dichloromethane for 30
minutes. The resulting precipitate was removed by filtration. The solvent
was evaporated under reduced pressure and the resulting solid dissolved in
3cm.sup.3 dimethylformamide.
The solution was drawn into the column of the Pepsynthesiser, which was set
to operate in a recirculation mode. After 25 minutes a small sample of the
composite substrate was removed from the column, washed with
dimethylformamide and ether and subjected to the kaiser test (ninhydrin)
for detection of primary amine. The test was negative and therefore the
Pepsynthesiser was switched to wash mode utilizing dimethylformamide.
Deprotection
Removal of the Fmoc group was performed by flowing 20% diethylamine in
dimethylformamide through the composite for 10 minutes, followed by a wash
mode utilizing dimethylformamide.
Two further coupling steps were carried out according to the following
sequence of events: (i) couple Fmoc-Alanine (0.74 g 2.4 mmol) (ii)
deprotect with 20% piperidine in dimethylformamide (iii) couple
Boc-Alanine (0.44 g 2.4 mmol).
The amino acids were reacted, following pre-activation as the symmetrical
anhydride, using the procedure given previously and the quantities given
above.
Detachment
The composite was removed from the instrument and a 100mg sample was
subjected to hydrazinolysis by reaction with 0.1cm.sup.3 hydrazine hydrate
in 5cm.sup.3 dimethylformamide for 2 minutes. The reaction solution was
drawn into chilled diethyl ether and the precipitate collected by
filtration. The precipitate was washed exhaustively with diethyl ether and
vacuum dried. The washed and dried precipitate comprised
Boc-Ala-Ala-Pro-Ala-N.sub.2 H.sub.3 in a yield of 61 mg.
Analytical Checks
The product was subject to: thin layer chromatography (Silica gel
60.sub.254) Propanol: H.sub.2 O (3:1) Rf=0.78; Chloroform: Methanol (4:1)
Rf=0.71 (both homogeneous, single component); and high performance liquid
chromatography (Waters Novapak C-18 column): R.sub.T =12.5 min (>90%)
solvent B water containing 0.1% trifluoroacetic acid; solvent C
Acetonitrile containing 0.1% TFA, gradient 100%B to 70%C over 30 minutes.
The Amino acid analysis gave a molar ratios of Ala (2.9) and Pro (1.0).
EXAMPLES 2 AND 3
The present Examples relate to the preparation of a substrate comprising a
functionalized porous polymeric material chemically reacted with a gel
during the preparation of the gel.
In outline, the preformed porous polymeric material was reacted with
N-hydroxymethylphthalimide in the presence of a catalyst (trifluoromethane
sulphonic acid, CF.sub.3 SO.sub.3 H) to yield a phthalimide derivative
which on nucleophilic scission with hydrazine provides the aminomethyl
porous polymeric material.
This derivative on reaction with acryloyl chloride provides a porous
polymeric material with double bonds at the surface of the pores. On
introduction of pre-gel material in the form of monomers into the
structure, followed by initiation of polymerization (heat) the surface
double bonds of the porous material are assumed also take part in the
reaction, producing what is believed to be a gel chemically-linked to the
porous polymeric material.
EXAMPLE 2
Preparation of Substrate
A cross-linked polyvinyl porous polymeric material formed by the high
internal phase emulsion method described in U.S. Pat. No. 4,522,953 was
employed as the starting material for the structural carrier. It had 90%
pore volume and a density of 0.047 g cm-.sup.3 and was made from a 10:90
mixture of commercial divinylbenzene and styrene. It had pore sizes within
the range 10 to 20 .mu.m. The polymeric material was in the form of a
milled and sieved powder having a particulate size within the range 425 to
850 .mu.m.
The powdered polymeric material (10 g, 10 mmol), prewashed and ground to
size (425 to 850 .mu.m), and N-hydroxymethylphthalimide (5.85 g, 0.03 mol)
were placed in a three neck round bottom flask (500cm.sup.3). The
resulting resin was suspended in a mixed solvent system of trifluoroacetic
acid: dichloromethane (1:2) (total volume 300cm.sup.3). Trifluoromethane
sulphonic acid (0.9cm.sup.3, 0.01 mol) was added, slowly, to the rapidly
stirred reaction mixture. Once uniform mixing had been achieved, and the
reaction mixture appeared consistent, the stirring was ceased to prevent
further fragmentation of the polymeric particles.
The mixture was allowed to stand at room temperature overnight (i.e. 16
hours).
The resin was transferred to a sintered funnel and washed with
dichloromethane (2.times.200cm.sup.3) and ethanol (2.times.200cm.sup.3).
The damp phthalimido resin was placed into a three neck round bottom flask)
(1 liter). Ethanol (422.5 ml) containing 5% hydrazine (22.5 ml) (total
volume 450 ml) was added to the resin and the mixture allowed to reflux,
with stirring for sixteen hours. A ninhydrin test after five hours gave a
positive result, however, the reaction was allowed to continue. The
reaction was terminated after sixteen hours by filtering the resin, whilst
hot, and washing with hot ethanol (4.times.100 ml) and cold methanol
(4.times.100 ml). The resin was placed into a vacuum oven at room
temperature and amino methyl polymeric material (10.21 g) of a particulate
nature was obtained. The material gave an intense blue color in a final
ninhydrin test, indicating a high level of amino groups present.
ACRYLATION OF AMINO METHYL POROUS POLYMERIC MATERIAL USING ACRLOYL CHLORIDE
##STR2##
The amino methylated polymeric material (2.0 g, 0.20 mmol) was placed into
a round-bottom flask (50 ml), which was situated in a salt/ice bath.
Sodium hydroxide (9.28 mg, 0.029 mmol) was dissolved in distilled water
(2.5 ml) and this solution was mixed with tetrahydrofuran (THF) (2.5 ml).
The mixed solvent system, containing sodium hydroxide, was added to the
polymeric material in the flask. Acryloyl chloride (10 ml, 0.12 mol) was
added dropwise to the mixture. During this addition, the pH was monitored
by spotting the reaction mixture on to full range indicator paper, and
maintained at pH>11 by the addition of sodium hydroxide solution, when
necessary. After 4 hours a ninhydrin test on the resin was negative,
indicating an absence of primary amine.
The reaction was terminated by filtering the reaction mixture and washing
with methanol:water (1:1) (3.times.50 ml) followed by methanol (3.times.50
ml). The resulting solid was placed in the vacuum oven at room temperature
until constant weight had been obtained. A white solid (2.05 g) was
obtained.
SYNTHESIS OF N-(2-(4-ACETOXYPHENYL)-ETHYL) ACRYLAMIDE (OR ACRYLOYL TYRAMINE
ACETATE)
Using Tyramine Hydrochloride
##STR3##
Sodium hydroxide (57.6 ml), 12 mol.dm.sup.3, 0.69 mol) was poured into a
three neck roundbottom flask (500 ml), equipped with a dropping funnel,
overhead electric stirrer and guard tube. Tyramine hydrochloride (25 g,
0.144 mol) was added to the rapidly stirring caustic and an aliquot of the
slurry was removed. This sample as subjected to a ninhydrin test, the
result of which was positive, as expected, indicating the presence of
primary amine groups.
The reaction flask was cooled to 0.degree. C., using a salt/ice bath, prior
to the dropwise addition of acryloyl chloride (14 ml, 0.17 mol) over a
period of fifteen minutes. During this addition, the pH of the reaction
was monitored by spotting the reaction mixture on to full range indicator
paper and maintained at pH 10 by the addition of sodium hydroxide solution
as required. The pH was controlled at this level to prevent formation of
the diacrylate waste product as much as possible. The mixture was stirred
for thirty minutes and another aliquot of the slurry was removed and
subjected to a ninhydrin test. Again, the result was positive indicating
that the first stage of the reaction had not gone to completion. A second
portion of acryloyl chloride (14 ml, 0.17 mol) was added, under controlled
pH conditions, as above. The mixture was stirred for a further thirty
minutes and subjected to a ninhydrin test for primary amine, which proved
to be negative. An equal volume of ethyl acetate was added to the mixture.
Sodium hydroxide solution (26.4 ml, 12 mol.dm-.sup.3, 0.32 mol) was added
and the reaction flask cooled to 0.degree. C., using a salt/ice bath.
Rapid stirring was used to achieve effective mixing of the two phases.
Acetic anhydride (32.7 ml, 0.35 mol) was added, to the rapidly stirring
reaction mixture, over a period of five minutes. During the acetylation,
the pH of the reaction was monitored by spotting the reaction mixture onto
full range indicator paper and maintained at pH>11 by the addition of
sodium hydroxide solution, as required. The pH was controlled at this
level in order to prevent back hydrolysis of the acryloyl tyramine acetate
(ATA) product. After all the acetic anhydride had been added, the reaction
mixture was allowed to settle into two phases. The lower aqueous phase was
discarded whereas the upper ethyl acetate was allowed to stand over
anhydrous magnesium sulphate for a period, filtered and the solvent
removed, using a rotary evaporator. A white solid was produced and washed
several times with ether. The final product was a white powder in a yield
of 21 g (65%).
The product was subjected to .sup.1 H nmr analysis. The resulting spectrum
showed all the peaks and integral heights expected for acryloyl tyramine
acetate.
IMPREGNATION OF DERIVATIZED POROUS POLYMER MATERIAL WITH ACRYLOYL TYRAMINE
ACETATE
##STR4##
Highly insoluble, cross-linked gel containing the following types of
functional group.
##STR5##
Derivatized porous polymeric material (1.0 g, 0.10 mmol), acryloyl tyramine
acetate (ATA) (5 g, 0.03 mol), each as prepared above, N,N'-ethylene
bis-acrylamide (EBA, cross-linking monomer) (0.15 g, 0.9 mmol) and azo
bis-(iso butyronitrile) (AIBN, free radical initiator) (0.10 g) were
placed into a round bottom flask (50 ml) and suspended in the minimum
volume of dimethyl formamide (DMF) (15cm.sup.3). The reaction mixture was
purged with nitrogen for thirty minutes to remove any traces of oxygen
which would inhibit the subsequent polymerization. The flask was placed on
to a rotary evaporator, with a vacuum, allowed to rotate and maintained in
a water bath at 60.degree. C. for one hour. The flask was rotated to
hinder polymerization on the surface between adjacent polymeric particles
and to promote polymerization of ATA within the pores of the polymeric
material.
The final product was filtered, washed with DMF (3 times), then ether and
was finally dried in the vacuum oven at room temperature.
EXAMPLE 3
The procedure of Example 2 was followed with the exception that acryloyl
sarcosine methyl ester was employed in place of acryloyl tyramine acetate
SYNTHESIS OF ACRYLOYL SARCOSINE METHYL ESTER
##STR6##
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