CA1315968C

CA1315968C - Substrate and process for making a substrate

Info

Publication number: CA1315968C
Application number: CA000564411A
Authority: CA
Inventors: David Colin Sherrington; Philip William Small
Original assignee: Unilever PLC
Current assignee: Unilever PLC
Priority date: 1987-04-24
Filing date: 1988-04-18
Publication date: 1993-04-13
Anticipated expiration: 2010-04-13
Also published as: EP0288310A3; GR3006883T3; AU1505988A; DE3875515T2; NO170428C; NZ224284A; JPS6463858A; NO881761D0; EP0288310B1; DK223388A; AU618530B2; NO170428B; US5066784A; NO881761L; DK223388D0; ES2036679T3; DE3875515D1; US4965289A; EP0288310A2; JPH0743359B2

Abstract

T.3038 ABSTRACT OF THE DISCLOSURE
Substrate and process for making a substrate A substrate comprises a porous polymeric material having a porosity of at least 75% and comprising pores having a diameter within the range 1 to 100 µm 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 materials is contained and retained within the pores of the polymeric material and is adapted in use to interact with a reactive species and can be made by depositing and retaining the gel or a material adapted in use to form the gel within the pores of the porous polymeric materia. The high porosity of the porous polymeric material in combination with the retention of the gel within the pores permit high loading capacities, particularly in the area of peptide synthesis to be achieved. The substrate can be employed in chemical synthesis, chromatography techniques, ion exchange and separation techniques.

Description

1 3 ~ 8 - 1 - T. 3038 SUBSTRATE AND PROCESS FOR ~qAKING A SUBSTRATE

The present invention relates to novel substrates, processes for making them and uses for them, including S synthesising chemical compounds and chromatography.

A variety of chromatographic techniques and methods of chemica] 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 t~hrough-flow ~hrough; the column. Such ~problems can be particularly acute where the substrate involves some form of gel. ~`di~scussion of these problems in the area of solld-phase synthesis is conkained in : :

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- 2 - T.3038 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.
US3991017 (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 A units. The macroreticular polymers are conventionally in bead form usually in an overall particle size of about lO 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.

UK1574414 (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 ;

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- 3 - T.3038 such a substrate allowing improved loading factors to be achieved.

Broadly, the present invention contemplates 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 ~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 synthesised 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 ~; ,~.
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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 preferre~ ratio being about 80:~0.
The porous material can be in particulate form~
preferably of a particle size between 125 and 1500~m, more preferably between 25Q and 8SO~m, and can for example be the cross-linked vinyl material or a highly porous cross-linked poly-condensation polymeric material described in our co-pending application CA 564413. Both of these porous material~ have a high pore volume and can have pores within the range oi approximately 1 to lOO~m, preferably 1 to 50~m. Naterials made by the processes described in CA 564413 are particularly suitable for use in the present invention as they are highly porous and can consist of reqularly formed fully interconnecting cells. Such a combination of features provides a structure tha~ can show rapid uptake o~ iluids and relatively unobstructed flow through the matrix. These porous structures are suitably made by means o~ a high internal phase emulsion and thus havo 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 ^qwell to more than twice their dry bed volume in use. Throughou~ the present specification porosity values and pore size measuremen~
refer to the porous pol~meric 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 intercvnnecting pores of the particulate material and forms therein the gel. The resulting material is ~, : . . . , ~ . . .
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- 5 - T.3038 1 3 ~ g preferably placed5 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 ma~erial can be ~n monolithic block form and the gel can be formed in situ ollowing permeation lnto the intexconnec ing pores of the block.

More specifically, the present invention provides a substrate comprising a porous cross-linked vinyl polymeric material ha~ing a pore structure providing an open poro ity of at least 75% and comprising cells having a diameter in the range l`to 100 ~m interconnected by holes, and a ~el contained and retained in said pore structure by chemical binding to the ~urfaces of the polymeric material, or by interaction ~ith the sur~aces of the polymeric material. The gel i8 polymeric and has xeac~ive functionality and the cells and holes of the polymoric material have a shape and conformation resulting from polymerization of vinyl material in a high internal phase emulsion syst~m.
In another aspect~ the invention provides a process for preparing a substrate for use in chemic~l ~ynthesis, ; comprising the ~teps o~
a) ~orming a porous cros~-linked vinyl polymeric material having a pore structure providing an open porosity : of at least 75~ and comprising cells having a diameter in the range 1 to 100 ~m interconnected by holesj by polymerization of vinyl material in a high internal phase emulsion system, b) providing a polymeric gel having reactive functionality available for u~e in chemical ~ynthesis in said pore structure of æaid porous polymeric material, said gel I `

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interacting with said porou~ polymeric material so as to be retained in said pore structure.
In an alternative process aspect of the invention, reactive groups are provided at the surface of the porous polymeric material and the gel chemically binds ~o the reactive groups of the porous polymeric material 80 a to be chemically retained in the porou~ structure.
Preerably 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 mat~rial and simultaneously retaining the gel during its Pormation within the pores of the porous material. The gel can for example be made by the conventional polymerisation and co-polymerisation routes to form gels, for example free-radical vinyl polymerisation, poly-condensation reactions, and cross-linking of soluble linear polym~rs.

; The gel and the porous polymeric materials are suitably those men~ioned above. In particular we have found that u~e of porous polymeric material~ a3 described in our CA 564413 having interconnocted pores allows ready access of the gel materials intc within the pores and subsequent ready access of reactive species in 2~ use. L~

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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 poly~eric ; 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 reterltion 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 ~ ' ' .

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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 polymerise 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 polymerisation 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 echanism 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 ~' .
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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 retentio~ 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.

The pre5ent invention also contemplates a substrate comprising a highly porous polymeric material having a porosity of at least 75% and comprising pore~ having a diameter within the range 1 to lOO~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, eg. by binding chemically, with a reactive species. Suitable porous materials are disclosed in our CA 564413 and have pore siæes preferably in the range 1 to 50~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%.
Naterials made by the process described in our CA 564413 specifications are particularly suitable fox use in the present invention as they are highly porous and can consist of regularly formed fully interconnecting cells. Such a comblnation of features provides a struct~re 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 reproclucibly 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 porou~ material can be made with the reactive groups already in situ e.g. vinyl groups on a polyvinyl porous material or can be treate - g - T.3038 1 3 ~
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.

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A use of the present substrates involves pa~sing a reactive species through the substrates preferably under flow conditions, and interactions of the species with th~
~ 10 reastive ~ubstrate.
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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 chamical ~ 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 ~; 30 be attached with a high load leading to hi~h yields. In peptide synthesls yields of 0.1 to 5mmol 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 ~-i substantially rigid, incompressible and homogeneous.
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1315 ~ ~ 8 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 emploved.

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 unders~ood that the present invention extends to the products of the present processes and uses.

Embodiments of the present inven~ion 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 ~0 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 IX) is attached to the polymer and is reacted with the first amino acid of the sequence to be synthesised. 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 resul~ing peptide is th n detached from the ~polymer suppo=t and -an if desir~d be purified.

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- 11 - T.3038 ~ 3 ~
Diagrammatically the peptide synthesis scheme can be shown as fol low5:

PG-NH-CH(R )-COOH + X-POLYMER

~1 attach PG-NH~CH(R )COO-POLYMER

~` 10 1 deprotect NH2~CH(Rl)-COO-POLYMER

PG-NH-CH(R )-COX 1 couple PG-NH-CH(Rll)-CONH-CH(Rl)-COO-POLYMER

repeat deprotection and coupling steps ~ f Peptide-COO-POLYMER

cleave ~ . ~ .
Peptide + POLYMER

In one embodlment the synthesis takes place within a highly swollen deformable polyamide gel which is polymerised 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 pld~ce 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 ~onditions of flow~is preferred as, in general, flow systems offer yreater opportunities for analytical :
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- 12 - T.3038 131~
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.

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Preparation of Substrate .~ 10 A cross-linked polyvinyl porous polymeric material formed by the high internal phase emulsion method described in our own EP patent specification no. 0060138 was employed as the structural carrier. It had 90~ pore volume, and employed 10~ cross~linking agent divinyl benzene and had a density of 0.047g cm 3. The polymeric material was in the form of a milled and sieved powder I having a particulate size within the range 850 to 1200~m.
;~ Its pore size was within the range 1 to 50~m.
The polymeric gel matrix was poly (N-(2-l4-acetoxy-` phenyl)ethyl)acrylamide). A solution of 2.5g of the monomer N-(2-(4-acetoxyphenyl)ethyl)acrylamide, 0.075g of the cross-linking agent ethylene bis (acrylamide), O.lg of the initiator azobisisobutyronitrile was prepared in lOcm3 dichloroethane and deoxygenated by purging with nitrogen.

The milled and sieved particulate polymeric material (0.7g) was added to the solution and polymerisation of the acrylamide was initiated by heating the mixture at 60~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 polymerislng polyamide by the porous polymeric material.
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13 - T.3038 1 3 ~ 8 After 1 hour reaction time the resulting compositP
was washed exhaustively with dimethylformamide and diethyl ether and then vacuum dried. The yield of resulting composite was 2.7g, the gel being retained within the porous polymeric material due to chain entanglement.

0.25g of the composite was treated with 5~ solution of hydrazine hydrate in dimethylformamide for 5 minutes.
This treatment pxovided free phenolic functionalities within the secondary gel matrix which act as reactive groups (X).

Pe~tide Synthesis To commence a peptide synthesis 0.95g (5.Ommol) t-butyloxycarbonyl alanine and 1.24g (6mmol) dicyclohexylcarbodiimide were dissolved in lOcm3 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 (0.25g), followed by 0.24g (2.0mmol) dimethylaminopyridine, and the esteri~ication 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 0.52g.

To remove the protection group (PG =
t-butyloxycarbonyl) 0.50g of the loaded composite substrate was retained in the solid phase reactor and 9cm3 benzyl alcohol was added. The suspension was nitrogen stlrred~for 1 hour allowing sufficient time for the secondary gel matrix to swell in~the benzyl alcohol. lcm3 of the deprotection reagent boron trifluoride etherate was : ~

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- 14 - T.3038 131~
added and the reaction nitrogen stirred for 3 hours. The composite was washed exhaustively with dimethylacetamide and diethyl ether.

In order to Garry 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 synthesiser. The column was initially purged with a solution of 0.2g (2mmol) N methylmorpholine in 50cm 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 0.80g Fmoc-Pro-OH (2.4mmol) with 0.23g dicyclohexylcar-bodiimide in dichloromethane for 30 minutes. The resulting precipitate was removed by filtration. The solvent was evapoxated under reduced pressure and the ; resulting solid dissolved in 3cm 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 utilising dimethylformamide.
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- 15 - T.3038 1 3 ~

Removal of the Fmoc group was performed by flowing 20% diethylamine in dimethylformamide through the composite for 10 minutes, followed by a wash mode utilising dimethylformamide.

Two further coupling steps were carried out according to the following sequence of events:
(i) couple Fmoc-Alanine (0.74g 2.4mmol) (ii) deprotect with 20~ piperidine in dimethylformamide (iii) couple Boc-Alanine (0.44g 2.4mmol).

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 ; lOOmg sample was subjected to hydrazinolysis by reaction with O.lcm3 hydrazine hydrate in Scm3 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-N2H3 in a yield of 61mg.
An ~ Checks The product was subject to: thin layer chromatography (Silica gel 60254): Propanol: H2O (3:1) Rf = 0.78;
Chloroform: Methanol (4:1) Rf = 0.71 (both homogeneous, single component); and high performance liquid : `
' , - 16 - T.3038 )~31~8 A chromatography (Waters Novapak C-18 column): RT = 12.5 min t~90%) solvent B water containing 0.1~ trifluoroacetic acid; solvent C Acetonitrile containing 0.1% TFA, gradient 1 O O %B to 70%C over 30 minutes.

The Amino acid analysis gave a molar ratios of Ala (2.9) and Pro (1.0).

The present Example~ relate to the preparation of a substrate comprising a functionalised 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, CF3SO3H) 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 ~5 the surface of the pores. On introduction of pre~gel material in the form of monomers into the structure, followed by inltiation 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 chemlcally-linked to the porous polymeric material.
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Preparation of Substrate A cross-linked polyvinyl porous polymeric material formed by the high internal phase emulsion method described in our own EP patent specification no. 0060138 was employed as the starting material for the structural carrier. It had 90~ pore volume and a density of 0.047g cm and was made from a 10:90 mixture of commercial divinylbenzene and styrene. It had pore sizes within the range 10 to 20 ~m. The polymeric material was in the form of a milled and sieved powder having a particulate size within the range 425 to 850~m.
The powdered polymeric material (lOg, 10mmol), prewashed and ground to size (425 to 850 ~m), and N-hydroxymethylphthalimide (5.85g, 0.03 mol) were placed in a three neck round bottom flask (500cm3). The resulting resin was suspended in a mixed solvent system of trifluoroacetic acid: dichloromethane (1:2) (total volume 300cm3)~ Trifluoromethane sulphonic acid (0.9cm3, 0.01 mol) was added, 510wly, 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.
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The mixture was allowed to stand at room temperature overnight (ie. 16 houxs).
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The resin was transferred to a sintered funnel and washed with dichloromethane (2 x 200cm3) and ethanol (2 x 200cm3).
The damp phthalimido resin was placed into a three neck round bottom flask) (1 litre). Ethanol (422.5 ml~

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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 terminatad after sixteen hours by filtering the resin, whiist hot, and washing with hot ethanol (4 x 100 ml) and cold methanol (4 x 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 colour in a final ninhydrin test, indicating a high level of amino groups present.

ACRYL TION OF AMINO METHYL POROUS POLYMERIC MATERIAL

USING_ACRLOYL CEI~ORIDE ~- ~
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The amino methylated polymeric material (2.0 g, 0.20 mmol) was placed into a round-bottom flask t50 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 (THF3 t2.5 ml). The mixed solvent system, containing sodium hydroxide, was added to the polymeric material in the 35 flask. Acryloyl chloride (10 ml, 0.12 mol) was added dropwise to the mixture. During tlis addi ion, the pH was . ~
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monitoxed 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 x 50 ml) followed by methanol (3 x 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) Usin ~ e Hydrochloride ~ C~ CR- H Nt~ ,~ H

S t~9 ~ I ~ A ~ r ~ ~ C
O
2 s C~ C--N
C~ ~o--C~
sv~ ~(1 a~ ~cc~ls k StQ9~ llfQ~) T~ S C
~ )(~ 3- C O)~- O
~b ~ C - ~* 3 t c~a ~ e (P~ T ~) Sodium hydroxide (57.6 ml), 12 mol.dm3, 0.69 mol3 was poured into a three neck roundbottom flask (500 ml~, :

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- 20 ~ T.3038 1 3 1 ~
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 0C, 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 minute~ 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 3, 0.32 mol) was added and the reaction flask cooled to 0C, using a salt/ice bath. Rapid stirring was used to achieve e~fective mixing of the two phases. Acetic anhydride (32.7 ml, 0.35 mol) was added, to the rapidly stirring rPaction 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 hy the addition of sodium ~, ., : ~:
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' ' " ' '.

- 21 - T.3038 1 3 ~
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 5 allowed to settle into two phases. The lower aqueous phase was discaxded 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%)o The product was subjected to lH nmr analysis. The resulting spec~rum showed all the peaks and integral heights expected for acryloyl tyramine acetate.

IMPREGNATION OF DERIVATISED POROUS POLYMER MATERIAL
WITH ACRYLOYL TYRAMINE ACETATE
.

2 0 ~, Vl 1,~ 0~ , C C~ L (~'1 r ~) t Cy, ~ c ~ a~ ~ -c - c~ = c~ 2 ~ /60c/ o ~ 1~ o C~--C--1~1 = N--C--C1J C
c~ c~
Highly insoluble, cross-linked gel containing the following types of functional group.
O ~ o C--~ - C~ - Ck+1 ~ >~ _ C ~ 3 - .
' ' :- : , ; : , ' .
, :.''- .: ' . ~ .
. . :

- 22 - T. 3038 1 3 ~
Derivatised porous polymeric material (1.0g, 0.10 mmol), acryloyl tyramine acetate (ATA) (5g, 0.03 mol), each as prepared above, N,N'-ethylene bis-acrylamide (EBA, cross-linking monomer) (0.15g, 0.9 mmol) and azo bis-(iso butyronitrile) (AIBN, free radical initiator) (0.10g) were placed into a round bottom flask (50 ml) and suspended in the minimum volume of dimethyl formamide (DMF) (15cm3).
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 ev~porator, with a vacuum, allowed to rotate and maintained in a water bath at 60C 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.

5YNTHESIS OF ACRYLOYL SARCO_INE METHYL ESTER

S ~ ~ - X - C~L C-~ C~3--~ - C~, C 3 C~ C~ ~ ~ CSI`1EH) 3) 3 /
~` ~3 ~ , C~t C~3 ~ - -C~ C-~3 ~ C- ~ ~3 k) CA~ C~/
~3 .~`

~ ' -- 23 - T.3038 ~3~ ~9~
~ ethanol (400 ml) was poured into a threeneck, round bottom flask (1 litre) which was placed in a salt/ice bath. Thionyl chloride (32.6 ml, 0.44 mol) was added dropwise to the stirred methanol, over a period of ten minutes. Sarcosine (36 g, 0.4 mol) was added over a period o~ fifteen minutes and the mixture stirred for twenty minutes before it was allowed to come to room temperature. A condensor was fitted to the flask and the mixture was refluxed for two hours. After cooling, the remaining methanol solvent was removed, using a rotary evaporator, and the residual solid (sarcosine methyl ester hydrochloride, yield, 62 g, 98~) was dissolved in chloroform (500 ml) and dried over magnesium sulphate.
The wet magnesium sulphate was removed by filtration and the filtrate divided into two equal volumes (2 x 250 ml).

One of these portions was poured into a round-bottom flask ~1 litre) which was placed in a salt/ice bath.
Triethylamine (62 ml, 0.45 mol) was added dropwise to the stirred solution, over a period of ten minutes. Acryloyl chloride (18 ml, 0.22 mol) in chloroform (150 ml) was added to the stirred solution, over a period of fifteen minutes. The mixture was allowed to come to room temperature and the solution was stirred overnight. The mixture was filtered to remove reaction by-products such as triethylamine hydrochloride, and the filtrate diluted (to a total volume of 500 ml) with chloroform. This solution was washed with 10~ citric acid (10 ml), 5%
sodium bicarbonate (100 ml) and water (100 ml). If the monomer solution was to be retained for any length of time a quinhydrone (0.2 g) stabiliser was added to prevent polymerization. The chloroform solution was dried over magnesium sulphate, filtered to remove the wet magnesium sulphate and the chloroform solvent removed using a rotary evaporator. The product was vacuum distilled at 96.4C
and lmm Hg, taking care to discard the ini~ial few ml of ,, ~ .

,.

.
, ., . .; " ' . , .

- 24 - T.3038 :~ 3 ~ o distillate. The final product was a viscous orange/brown liquid which was analysed by 1H nmr. The 1H nmr spectrum showed all the peaks and their integral heights to be as anticipated for acryloyl sarcosine methyl ester.

IMPREGNATION OY DLRIVATISED POROUS POLYMERIC MATERIAL
WITH ACRYLOYL SARCOSINE METHYL ESTER
1~ ~ 3 "O
~ ~ t C*L_C~-C~ -a= C~ C~ )z + (C~,~ C- N
(~) O 1 /6vC
C)~3 ~3 ~3- ~ - ~ = N - C~ 3 Highly insoluble, cross-linked gel containing the following types of functional group.

~3 ~O
~ C ~ C~ L ~ C~ -Two types of resin were produced of different loading capacities.

, - .
.~ .
- -- 25 - T.3038 ~3~9~8 Resin I

Derivatized porous polymeric material (lg, 0.01 mmol), ASME (0.5g, 3 mmol), N-N-dimethylacrylamide (DMA) (5g, 50 mmol), EBA (cross-linking monomer) (0~5g, 3 mmol) and AIBN (O. lg) were placed into a round bottom Elask (100 ml) and suspended in the minimum volume of dimethyl formamide (DMF) (15cm ). 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 onto a rotary evaporator, with a vacuum, allowed to rotate and lowered into the water bath at 60C for two hours. The flask was rotated to hinder polymerization on the surface between adjacent porous polymeric particles and to promote polymerization of ~SME
within the pores of the porous polymeric material.

The final product was filtered, washed with DMF (3 x 50 ml), ethanol (3 x 50 ml) then ether (3 x 50 ml) and was finally dried in the vacuum oven at room temperature.
Since the resulting material consisted of a mixture of different slzed particles, the product was ground, using a mortar and pestle and sieved to produce particles in the range 250 to 500 microns in diameter.
~5 i Resin II

- The method for production is identical to that just described for Resin I above except that the following quantities were used:
' derivatized porous material ~lg, 0.01 mmol) ASME ~2g, 0.01 mmol) ` 35DMA (3.5g, Q.04 mmol) - 26 - T.3038 ~ 3 ~
EBA (0.5g, 3 mmol) AIBN (0.lg) Both the resins were chemically and physically suitable for use as solid phase supports in peptide synthesis as outlined above.

Resin I had a synthetic capacity of 0.25 mmolg 1 and Resin II had a synthetic capacity of 1.00 mmolg both measurements being with respect to mmol of peptide synthesised per g of composite substrate. It is of interest to compare these loading capacities wi~h commercially available Kieselguhr based resins having a synthetic capacity of about 0.1 mmolg 1.
Each of the present Resin I (0~25mmol g 1) and Resin II (1.00mmol g 1) composite substrates was employed in the synthesis of leucine enlcephalin (H-Tyr-Gly-Gly-Phe-l,eu-OH). In each case the composite containing reactive sarcosine methyl ester groups, was allowed to react overnight with ethylenediamine to provide primary amine groups throughout the gel.

A reference amino acid derivative Fmoc-Norleucine was coupled to the amine groups by condensation in the presence of dicyclohexylcarbodiimide. Following deprotection (removal of Fmoc) in the presence of 20 piperidine in dimethylformamide, the linkage agent 4-hydroxymethylphenoxyacetic acid was coupled to the ~ exposed amino groups.

`~ The C-terminal amino acid of the desired sequence Fmoc-Leucine was attached to the support by carbodiimide mediated esterification in the presenc~ of a catalytic amount of 4-dimethylaminopyridine. Further cycles of -~ deprotection and coupling were carried out utilising .~ .

~: :

~`

~31~8 Fmoc-Phe, Fmoc~Gly, Fmoc-Gly and Fmoc-Tyr (OtBu) to complete the assembly. All of the steps were performed under continuous flow conditions with the composite packed in a column employing a Pepsynthesiser Mk II ex Cambridge Research Biochemicals.

The following results were obtained:

Loading capacity composite (mmol g ~ 0.25 1.00 Detachment yield Iper 1.0g composite) 0.10g 0.48g Purity (by hplc) 95.3% 38.2%

hplc - high performance liquid chromatography.
By comparison the theoretical maximum yield from Kieselguhr based supports (eg ~Pepsyn KA available from Cambridge Research Biochemicals) is 0.05g per 1.0g composite based on a maximum loading capacity of 0.1 mmol g 1 and using the same consumption of solvents and reagents.
The followiny peptides have also been synthesised using the above described protocol and the composite substrate as described above (Resin II) having a capacity of 1.00 mm~l g 1.
i) H-Asp-Asn-Trp-Arg-Ser-Glu-Leu-Tyr-Lys-Tyr-OH
; yield: 1.15g per lg composite purity: 98.0~

ii~ H-Val-Pro-Val-Trp-Lys-Glu-Ala-Thr-Thr-Thr-OH yield:
0.98g per lg composite purity: 95.1%

iii) H-Cys-Val-Pro-Thr-Asp-Pro-Asn-Pro-Glu-Glu-Val-Val-OH
yield: 1.02g per lg composite purity: 97.2%
~ c~e~f~s ~f~Q m~k ~`

.

- 28 - T.3038 ~ . ~ 3 ~
iv) ACP ~65 - 74) ie. a segment from acyl carrier protein yield: O.91g per lg composite purity: 97,0~

By way of comparison ACP (65 - 74) was additionally synthesised on control composites of Pepsyn-KA (a kieselguhr based composite having a maximum theoretical loading capacity of O.lmmol g 1) and batch polystyrene having a maximum theoretical loading capacity of 0.7mmol g 1, rhe results were as follows:

Substrate Yield as in (iv) O.91g 97.0~
Pepsyn-KA 0.07g 96.0%
lS Batch polystyrene 0.35g 35.0 -; A cross-linked polyvi~yl porous polymeric ma~erial formed by the high internal phase emulsion method described in oux own EP patent specification no. 0060138 was employed as the substrate. It had 90~ pore volume, a density o~ 0.047gcm 3 and employed in its preparation 10 cross-linking agent commercial divinyl benzene. The polymerLc material was in the form of a milled and sieved powder having a particulate size within the range 850 to 1200~m. It had a pore size within the range l to 50~m.

The surface of the porous polymeric material was modified as described above by the attachment of the reactive group ~ CH2-NH2, which then served as reactive group X in the peptide synth~sis scheme outlined above.
Following substantially this peptide synthesis scheme ~(Fmoc-Leu)2O was employed as the firs~ source of amino acid to be coupled to the substrate. The resulting loading of the amino acid Leu was 0.12 mmolg 1 substrate.
, ~ . : ; :

i.; .~.. , ~. , ~

~ .
.
, . . ~ .

- 29 - T.3038 ~- 131~8 Removal of the Fmoc group to cause deprotection occurred by use of 20% piperidine in DMF. The acid labile linker HO CH2 ~ CH2CO2H was next coupled to the deprotected amino acid as an active hydroxybenzotriazole ester.
Esterification then occured between the assembly on the substrate and (Fmoc-Gly)2-O in the presence of 0.1 eq. of DMAP. Amino acid analysis of the Gly loading of the substrate was 0.09 mmolg 1 substrate. Deprotection next occurred to remove the end Fmoc group and permit lC c.ubse~ ent coupling to Ala hy use of (Fmoc-Ala)2O.

The resulting peptide assembly had a Gly:Ala ratio of 1:1 indicating a quantitative formation of the desired peptide.

.

., .

~' ` .

;` :: : : :

, :~ :

:

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A substrate comprising a porous cross-linked vinyl polymeric material having a pore structure providing an open porosity of at least 75% and comprising cells having a diameter in the range 1 to 100 µm interconnected by holes and a gel contained and retained in said pore structure by chemical binding to or interaction with the surfaces of the said polymeric material, said gel being polymeric and having reactive functionality, said cells and holes of said polymeric material having a shape and conformation resulting from polymerization of vinyl material in a high internal phase emulsion system.

2. The substrate according to claim 1, wherein said cells have a diameter in the range 1 to 50 µm.

3. The substrate according to claim 1, wherein said porous polymeric material has an open porosity in the range 85 to 98%.

4. The substrate according to claim 1, wherein the porous polymeric material is cross-linked to an extent that in use it swells to up to twice its dry bed volume.

5. The substrate according to claim 1, wherein the gel is capable of a loading of reactive residue of from 0.1 to 5 mmol chemical compound synthesized per g of substrate.

6. The substrate according to claim 1, wherein said gel is chemically bound to said porous polymeric material by an amide linkage.

7. The substrate according to claim 1, wherein the ratio by weight of swollen gel to porous polymeric material lies in the range 60:40 to 95:5.

8. A substrate comprising a porous cross-linked vinyl polymeric material having a pore structure providing an open porosity of at least 75% and comprising cells having a diameter in the range 1 to 100 µm interconnected by holes and a gal contained and retained in said pore structure by interaction with the surfaces of the said polymeric material, said gel being polymeric and having reactive functionality, said cells and holes of said polymeric material having a shape and conformation resulting from polymerization of vinyl material in a high internal phase emulsion system.

9. The substrate according to claim 8, wherein said cells have a diameter in the range 1 to 50 µm.

10. The substrate according to claim 8, wherein said porous polymeric material has an open porosity in the range 85 to 98%.

11. The substrate according to claim 8, wherein the porous polymeric material is cross-linked to an extent that in use it swells to up to twice its dry bed volume.

12. The substrate according to claim 8, wherein the gel is capable of a loading of reactive residue of from 0.1 to 5 mmol chemical compound synthesized per g of substrate.

13. The substrate according to claim 8, wherein the gel is retained within the pores of the porous polymeric material by chain entanglement between the gel and the porous polymeric material.

14. The substrate according to claim 8, wherein the ratio by weight of swollen gel to porous polymeric material lies in the range 60:40 to 95:5.

15. A process for preparing a substrate for use in chemical synthesis, comprising the steps of a) forming a porous cross-linked vinyl polymeric material having a pore structure providing an open porosity of at least 75% and comprising cells having a diameter in the range 1 to 100 µm interconnected by holes, by polymerization of vinyl material in a high internal phase emulsion system, b) providing a polymeric gel having reactive functionality available for use in chemical synthesis in said pore structure of said porous polymeric material, said gel interacting with said porous polymeric material so as to be retained in said pore structure.

16. A process according to claim 15 wherein said gel is retained in said pore structure by effecting chain entanglement of said gel and said porous polymeric material.

17. A process according to claim is including depositing and retaining the gel within the pore structure of the porous polymeric material by subjecting porous polymeric material to a solution comprising gel precursor materials and a swelling solvent for the porous polymeric material, allowing the gel precursor materials to permeate the swellen porous polymeric material and forming the gel from the gel precursor materials within the pore structure.

18. A process for preparing a substrate for use in chemical synthesis, comprising the steps of a) forming a porous cross-linked vinyl polymeric material having a pore structure providing an open porosity of at least 75% and comprising cells having a diameter in the range 1 to 100 µm interconnected by holes, by polymerization of vinyl material in a high internal phase emulsion system, b) providing reactive groups at the surface of said porous polymeric material c) providing a polymeric gel having reactive functionality available for use in chemical synthesis in said pore structure of said porous polymeric material, said gel chemically binding to said reactive groups of said porous polymeric material so as to be chemically retained in said pore structure.

19. A process according to claim 18 including depositing and retaining said gel within said pore structure of the porous polymeric material having said 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 pre structure and simultaneously allowing the gel and/or gel precursor to react with the reactive groups on the pores of the porous polymeric material to effect -the chemical binding.

20. A process according to claim 19 wherein the gel precursor materials include at least one of a monomer and a prepolymer and the gel is formed by a polymerisation reaction.

21. A process according to claim 18 wherein said chemical binding of said gel to said porous polymeric material is achieved by forming an amide linkage between them.

22. A process of peptide synthesis using as a substrate for the synthesis a substrate comprising a porous cross-linked vinyl polymeric material having a pore structure providing an open porosity of at least 75% and comprising cells having a diameter in the range 1 to 100 µm interconnected by holes and a gel contained and retained in said pore structure by chemical binding to the surfaces of the said polymeric material, said gel being polymeric and having reactive functional groups used in the peptide synthesis, said cells and holes of said polymeric material having a shape and conformation resulting from polymerization of vinyl material in a high internal phase emulsion system, said peptide synthesis comprising sequentially passing reagents through the pore structure of said substrate so as to perform the steps of a) attaching a first amino acid residue to said gel via said reactive functional groups of said gel b) attaching a second amino acid residue to said first amino acid residue to form 2 peptide chain c) attaching further amino acids sequentially to increase said peptide chain, thereby to obtain a pre-determined peptide d) detaching the pre-determined peptide from said gel.

23. A process according to claim 22 wherein said cells of said porous polymeric material have a diameter in the range 1 to 50 µm.

24. A process according to claim 22 wherein said porous polymeric material has an open porosity in the range 85 to 98%.

25. A process according to claim 22 wherein the amount of peptide synthesized is in the range from 0.1 to 5 mmol per g of said substrate.

26. A process according to claim 22 wherein said gel is chemically bound to said porous polymeric material by an amide linkage.

27. A process of peptide synthesis using as a substrate for the synthesis a substrate comprising a porous cross-linked vinyl polymeric material having a pore struature providing an open porosity of at least 75% and comprising cells having a diameter in the range 1 to 100 µm interconnected by holes and a gel contained and retained in said pore structure by interaction with the surfaces of the said polymeric material, said gel being polymeric and having reactive functional groups used in the peptide synthesis, said cells and holes of said polymeric material having a shape and conformation resulting from polymerization of vinyl material in a high internal phase emulsion system, said peptide synthesis comprising sequentially passing reagents through the pore structure of said substrate so as to perform the steps of a) attaching a first amino acid residue to said gel via said reactive functional groups of said gel b) attaching a second amino acid residue to said first amino acid residue to form a peptide chain c) attaching further amino acids sequentially to increase said peptide chain, thereby to obtain a pre-determined peptide d) detaching the pre-determined peptide from said gel.

28. A process according to claim 27 wherein said cells of said porous polymeric material have a diameter in the range 1 to 50 µm.

29. A process according to claim 27 wherein said porous polymeric material has an open porosity in the range 85 to 98%.

30. A process according to claim 27 wherein the amount of peptide synthesized is in the range from 0.1 to 5 mmol per g of said substrate.

31. A process according to claim 27 wherein said gel is retained within the pores of the porous polymeric material by chain entanglement between the gel and the porous polymeric material.