WO2004113389A2 - High capacity poly(alkylene)glycol based amino polymers - Google Patents

Abstract

The present invention relates to a cross-linked and beaded, stable and high loading capacity polymer matrix for affinity chromatography applications and for solid phase synthesis. The polymer matrix can be obtained by a method comprising the steps of providing a plurality of macromonomers each comprising a poly(oxalkylene) chain terminated with an acylamide functional group, polymerising said macromonomers using a free radical initiator or an ionic initiator, optionally with the addition of copolymerizing agents, and converting in the beaded polymer matrix at least 50% of the amide groups to amine functional groups by reduction of the amide groups with a suitable reducing agent.

Description

High capacity poly(alkylene)glycol based amino polymers

This application is a non-provisional of U.S. provisional application Serial No. 60/482,452 filed on 26 June 2004, which is hereby incorporated by reference in its entirety. All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention relates to the development of a beaded, stable and high loading capacity resin for affinity chromatography applications and for solid phase synthesis. The exhaustive reduction of amide groups in highly crosslinked amide based polymers comprising poly(oxyalkylene) chains terminated with acrylamide functional groups copolymerized with other modifying agents form a stable polymer resin with a high amine functional loading.

The polymer comprises a crosslinked poly(alkylene)glycol network which has a unique, three dimensional structure and can be applicable e.g. as a chroma- tographic resin or as a solid support for the synthesis of peptides, oligonucleotides, oiigosaccharides, or as a substrate for the immobilization of proteins.

Background of invention

Wide ranges of conventional homogeneous solution phase organic reactions are now successfully performed on solid supports. However this has required suitable polymeric materials. The development and exploitation of solid-phase combinatorial chemistry technology has rapidly evolved based on the pioneering work of Merrifield [Merrifield, J. Am. Chem. Soc, 85, 2149 (1963)].

Merrifield introduced the divinyl benzene crosslinked polystyrene for solid phase synthesis and it has been widely used until now with some refinements. The initial revelations of its use focused on the solid-phase synthesis of oligomers of amino acids or nucleotides, or on unnatural oligomers of other chemical building blocks like peptoids [Geysen et al., J. Bioorg. Med. Chem. Lett., 3, 397 (1993); Egholm et al., J. Am. Chem. Soc, 114, 1895 (1992); Simon et al., Proc. Natl. Acad. Sci. USA, 89, 9367 (1992)]. Recently, the library synthesis of nonoligomeric small molecules has become an area of intense research activity [Wang et al., J. Med. Chem., 38, 2995 (1995)].

In any approach to produce chemical library, whether it is solid-phase or solution- phase, is the need of rapid purification, isolation, and manipulation of chemical library members during their intermediate and final synthetic steps of preparation. The solid-phase technology offers advantages like ease of separating the products from the reaction medium and the manipulation of the beads using volumetric tech- niques. Due to the high rigidity and hydrophobicity the PS-DVB resin it is not suitable for all conventional organic synthesis and is often not suitable for the subsequent screening of the libraries on solid support.

In order to overcome the above-mentioned difficulties, a series of resins such as poly(ethyleneglycol) polystyrene (PS-PEG) [Zalipsky et al., React. Polym., 22, 243

(1994)], tentagel graft resin [Hellerman et al., Makromol Chem., 184, 2603 (1983)], alkanediol diacrylate crosslinked polystyrene [Renil et al., Tetrahedron, 50, 6681 (1994); Varkey et al., J. Peptide Res., 51, 49 (1998); Roice et al., Macromolecules, 32, 8807 (1999)], polyamides [Arshady et al., J. Chem. Soc, Perkin Trans. 1 , 529 (1981)], cotton and other carbohydrates [Englebresten et al., Int. J. Peptide Protein

Res., 48, 546 (1994)], PEGA [Meldal, Tetrahedron Lett., 33, 3077 (1992)], CLEAR [Kempe et al., J. Am. Chem. Soc, 118, 7083 (1996)], POEPS [Renil et al., Tetrahedron Lett., 36, 4647 (1995)], POEPOP [Renil et al., Tetrahedron Lett., 34, 6185 (1996)] and SPOCC [Rademann et al., J. Am. Chem. Soc, 121 , 5459 (1999)] resins were developed and tested successfully for solid phase synthesis. One of the limitations of these resins is the imbalance of extent of swelling and functional group capacity.

Summary of the Invention

The present invention provides an efficient, high capacity resin for the solid phase synthesis, immobilization and chromatographic separation as specific applications.

The present invention in one aspect is directed to novel po!y(alkylene)glycol based high loading, high swelling polymer beads with a high mechanical and chemical sta bility. The polymer matrices according to the invention can be prepared from poly(alkylene)glycol acrylamide resins by the exhaustive reduction of amide groups.

The polymer matrices according to the present invention is highly flow stable and polar which assists the peptide solvatization, allowing the diffusion of polar components into the interior of the beads. Also, the polymer beads are transparent with no absorbance in the aromatic region to allow the spectroscopic monitoring of reaction within the resin. The polymer is highly swellable in various solvents and there is no considerable change in the swelling property even after a series of reactions per- formed on the resin. Also the density of the resin permit multiple column peptide synthesis.

The present invention in one preferred aspect provides a beaded or granulated polymer matrix formed by reduction of a substantial amount of amide groups, such as more than 50% of amide groups, in a cross-linked polymer obtainable by polymerization of a poly(oxyalkylene) chain terminated with an acylamide functional group using radical or ionic initiators.

The cross-linked polymer can be obtained by polymerization of a poly(oxyalkylene) chain terminated with an acylamide functional group using radical and/or ionic initiators, and the beading can be achieved by inverse suspension polymerization

In one embodiment, the cross-linked polymer is obtained by a method comprising the steps of

i) providing a plurality of macromonomers each comprising a poly(oxyalkylene) chain terminated with an acylamide functional group,

ii) polymerising said macromonomer using a free radical initiator or an ionic initiator, optionally with the addition of copolymerizing agents, and

iii) converting in the beaded polymer matrix more than 50% of the amide functional groups to amine functional groups by reduction of the amide functional groups with a suitable reducing agent. The polymerization can be achieved by e.g. inverse suspension polymerization leading to the formation of a beaded resin, or a granulated matrix can be obtained.

Definitions

Beaded polymer matrix: A beaded polymer matrix is a multitude of beads of crosslinked polymer formed by beading according to principles of suspension or inverse suspension polymerization, by spray polymerization, or by droplet polymerization.

Substantial amount of amide groups in a resin: At least more than 50%, such as more than 60%, for example more than 70%, such as more than 80%, for example more than 90%, such as more than 95%, for example more than 99% of the amide groups present in the resin.

Solid phase synthesis: Synthesis where one of several of the reactants forming the target molecule is attached to a solid support e. g. a beaded polymer

Swelling: When beads or granules or particles are capable of swelling, any physical measurement of the afore-mentioned, including size determinations and volume determinations, refer to measurements conducted for the swelled bead or granule or particle. Swelling of the beads are for practical reasons measured as the volume of a packed bed of beads swollen in a specific solvent and divided by the dry weight of the beads. The unit is given as ml/g. Typical solvents are water, methanol and di- chloromethane, but any suitable solvent may be chosen.

Degree of polymerization: The number of monomeric units in a macromolecule or oligomeric molecule, a block or a chain.

Brief Description of the Drawings

Fig. 1: Synthesis of high capacity poly(alkylene)glycol-based resin (Resin B) from poly(alkylene)glycol acrylamide resin (Resin A)

Fig. 2: Optical micrograph of the high capacity poly(ethylene)glycol-based resin

(Resin B) Fig. 3: Swelling character of high capacity poly(ethylene)glycol-based resin (Resin B) and PEGA (Resin A) in various solvents

Fig. 4: IR spectroscopy of (a) PEGA (Resin A) and (b) high capacity poly(ethylene)glycol based resin (Resin B)

Fig. 5: Stability comparison of high capacity poly(ethylene)glycoI based resin (Resin B) by IR spectroscopy after treatment with various reagents (a) original (b) 20% Pi- peridine/DMF (c) Saturated aq. NaOH (d) DBU (100%) (e) triflic anhydride (100%)

(f) BF₃ Et₂0 (100%) (g) BuLi (2.7 M solution in heptane, 100%) and (h) TFA (100%)

Fig. 6: Swelling character of the high capacity poly(ethylene)glycol-based resin (Resin B) after 7 days treatment with various reagents

Fig. 7: Mechanical testing studies (a) high capacity poly(ethylene)glycol based resin (Resin B) (b) PEGA resin (Resin A)

Fig. 8: Synthesis of Ac-His-(D)Phe-Arg-Trp-NH₂ [Peptide (1)]

Fig. 9: Synthesis of Fmoc-Dap(N₃)

Fig. 10: Synthesis of cyclic peptidomimetic (3)

Fig. 11 : High performance liquid chromatogram of pure peptide (1 )

Fig. 12: High performance liquid chromatogram of pure linear peptidomimetic (2)

Fig. 13: High performance liquid chromatogram of pure cyclic peptidomimetic (3)

Detailed Description of the Invention

The polymer matrix according to one embodiment of the invention is illustrated as formula 1 herein below. The polymer matrix can be prepared by exhaustive reduc- tion of amide groups in the polymer matrix of formula 2.

Z = H or CH₃ or C₂H₅

R = H or CH₃ or CH₂OH or C₂H₅OH or i-C₃H₇ or n-C₃H₇ or i-C₄H₉ or n-C₄rf₉

R' = H or CH₃ or i-C₃H₇ or n-C₃H₇ or i-C₄H₉ or n-C₄H₉

R" = H or CH₃

R^m = H or CH₃

(1 )

r C₂H₅OH or i-C₃H₇ or n-C₃H₇ or n-C₃H₇ or i-C₄H₉ or n-C₄H₉

(2)

wherein n is a real number and designates the average degree of polymerization (dp) of poly(alkylene)glycol in the range of from 3 to 2000.

n can be a real number in the range of from 3 to 2000, such as from 3 to 800, for example from 3 to 600, such as from 3 to 400, for example from 3 to 300, for exam pie from 3 to 200, such as from 3 to 100, for example from 3 to 90, such as from 3 to 80, for example from 3 to 70, such as from 3 to 60, for example from 3 to 50, such as from 3 to 45, for example from 3 to 40, such as from 3 to 30, for example from 3 to 25, such as from 10 to 25, for example from 10 to 20, such as from 10 to 15, for example from 15 to 20, such as from 11 to 19, for example from 12 to 18, such as from 13 to 17, for example from 10 to 12, such as from 12 to 14, for example from 14 to 16, such as from 16 to 18, for example from 18 to 20.

A preferred value for n is between about 4 and about 180, such as about 10, for example about 20, such as about 30, for example about 40, such as about 50, for example about 60, such as about 70, for example about 80, such as about 90, for example about 100, such as about 110, for example about 120, such as about 130, for example about 140, such as about 150, for example about 160, such as about 170, i.e. the compound is preferably a derivative of PEG₁₉ to PEG ₈ooo or of PPG ₆o to PPG ₄₈₀₀.

The polymer matrices according to the invention can be obtained by the reduction of a polymer obtained by the polymerization of a 50-100% partially or fully acryloylated 0,0 -bisaminoprop-1-yl)PEGιgoo and 0-50% acrylamide by weight, with the pre- ferred amount of acrylamide being less than 15%.

The polymer matrix employed in the invention comprises or consists of insoluble, crosslinked poly(alkylene)glycols. The polymer is highly crosslinked and suitable for use in a wide range of applications. The polymer of the present invention can be used as a solid support in a range of solid phase synthetic applications, as a stationary phase in chromatography, and matrices for immobilization of macromole- cules. The polymers of the present invention can be synthesized in a wide range of molecular weights and crosslinking. The polymer can be homopolymers or copolymers and can be substituted or unsubstituted.

The polymer can be a homopolymer or a copolymer of one or more amine containing monomers in combination with one or more non-amine containing monomers. The polymer is prepared from monomers either by bulk, suspension or inverse suspension polymerization techniques. Examples of non-amine containing monomers include vinyl alcohol such as vinyl benzyl alcohol; vinyl carboxylic acids such as acrylic acid, methacrylic acid, itaconic acid, and vinyl benzoic acid; vinyl esters such as vinyl acetate, vinyl propionate; allyl esters such as allyl acetate; allyl amines such as allyl ethyl amine; allyl alcohols such as allyl alcohol, 1-buten-3-ol, 1-penten-3-ol, 1-hexen-3-ol, 1-hydroxy-1 -vinyl cyclohexane, 2-bromoallyl alcohol, 2-chloroallyl al- cohol; hydroxy containing vinyl ethers such as hydroxyethyl vinyl ether; vinyl acid halides such as acryloyl chloride and methacryloyl chloride; styrenes and substituted styrenes such as 4-ethyl styrene, 4-amino styrene, dichlorostyrene, chlorostyrene, 4-hydroxystyrene, hydroxymethyl styrene, and 4-hydroxy-3-nitro styrene; vinyl toluene; hetroaromatic vinyl such as 1-vinylimidazole, 4-vinyl pyridine, and 2-vinyl pyri- dine; acrylamide; dimethyl acrylamide; and hydroxy containing (meth)acrylamides such as N-(hydroxymethyl) (meth)acrylamide, N-(1 -hydroxyethyl) (meth)acrylamide, N-methyl-N-(2-hydroxyethyl) (meth)acrylamide, N-(1-hexyl-2-hydroxy-1-methylethyl) (meth)acrylamide, N-propyl-N-(2-hydroxyethyl) (meth)acrylamide. Examples of amine containing monomers preferably bisamino polyethyleneglycol with various chain length. Most preferably the polymer is a copolymer of bisamino polyethyleneglycol with acrylamide, such as a high-density amine functionality is accessible after the exhaustive reduction.

Preferably the polymer is insoluble by crosslinking. The cross-linking agent can be characterized by functional groups, which react with amino group of the bisamino poly(alkylene)glycol. Alternatively, the crosslinking group can be characterized by vinyl groups, which can be polymerized by free radical polymerization with amine monomer.

The level of crosslinking makes the polymer differ in their swelling behaviour, which directly affect the reactivity of the polymer. The free radical initiators useful in the present invention include azo compounds, tertiary amines, organic and inorganic peroxides and peroxodisulphates. The preferred free radical initiator is ammonium peroxodisulfate. The commercial products include VAZO 67, VAZO 64 and VAZO 52 can also be used as the initiator.

The number average molecular weight (M_n) of the polymer matrix is in the range of from 200 to 60000, such as from 200 to 45000, for example from 200 to 30000, such as from 200 to 25000, for example from 200 to 20000, such as from 200 to 15000, for example from 200 to 10000, such as from 200 to 8000, for example from 200 to 6000, such as from 200 to 5000, for example from 200 to 4500, such as from 200 to 4000, for example from 200 to 3500, such as from 200 to.3000, for example from 200 to 2500, such as from 200 to 2400, for example from 200 to 2300, such as from 200 to 2200, for example from 200 to 2100, such as from 200 to 2000, for example from 200 to 1900, such as from 200 to 1800, for example from 200 to 1700, such as from 200 to 1600, for example from 200 to 1500, such as from 200 to 1400, for example from 200 to 1300, such as from 200 to 1200, for example from 200 to 1100, such as from 200 to 1000, for example from 200 to 900, such as from 200 to 800, for example from 200 to 700, such as from 200 to 600, for example from 400 to 3000, such as from 400 to 2500, for example from 400 to 2000, such as from 400 to 1800, for example from 400 to 1600, such as from 400 to 1400, for example from 400 to 1200, such as from 400 to 1000, for example from 400 to 800, such as from 400 to 600, for example from 600 to 3000, such as from 600 to 2500, for example from 600 to 2000, such as from 600 to 1800, for example from 600 to 1600, such as from 600 to 1400, for example from 600 to 1200, such as from 600 to 1000, for example from

600 to 800, such as from 800 to 3000, for example from 800 to 2500, such as from 800 to 2000, for example from 800 to 1800, such as from 800 to 1600, for example from 800 to 1400, such as from 800 to 1200, for example from 800 to 1000, such as from 1000 to 3000, for example from 1000 to 2500, such as from 1000 to 2000, for example from 1000 to 1800, such as from 1000 to 1600, for example from 1000 to

1400, such as from 1000 to 1200.

The amine group loading capacity of the polymer matrix is preferably in the range of from 0.01 to 14 mmol/gram, such as from 0.01 to 13 mmol/gram, for example from 0.01 to 12 mmol/gram, for example from 0.01 to 11 mmol/gram, such as from 0.01 to 10 mmol/gram, for example from 0.01 to 9 mmol/gram, such as from 0.01 to 8 mmol/gram, for example from 0.01 to 7 mmol/gram, such as from 0.01 to 6 mmol/gram, for example from 0.01 to 5 mmol/gram, such as from 0.01 to 4 mmol/gram, for example from 0.01 to 3 mmol/gram, such as from 0.01 to 2 mmol/gram, for example from 0.01 to 1 mmol/gram, such as from 0.01 to 0.5 mmol/gram, for example from 0.01 to 0.4 mmol/gram, such as from 0.02 to 2 mmol/gram, for example from 0.04 to 2 mmol/gram, such as from 0.06 to 2 mmol/gram, for example from 0.08 to 2 mmol/gram, such as from 0.1 to 13 mmol/gram, for example from 0.1 to 12 mmol/gram, such as from 0.1 to 11 mmol/gram, for example from 0.1 to 10 mmol/gram, such as from 0.1 to 5 mmol/gram, for example from 0.1 to 4 mmol/gram, such as from 0.1 to 3 mmol/gram, for example from 0.1 to 2 mmol/gram, such as from 0.1 to 1.5 mmol/gram, for example from 0.1 to 1 mmol/gram, such as from 0.1 to 0.8 mmol/gram, for example from 0.1 to 0.6 mmol/gram, such as from 0.1 to 0.5 mmol/gram, for example from 0.1 to 0.4 mmol/gram, such as from 0.1 to 0.3 mmol/gram, for example from 0.1 to 0.2 mmol/gram, such as from 0.2 to 2 mmol/gram, for example from 0.4 to 2 mmol/gram, such as from 0.6 to 2 mmol/gram, for example from 0.8 to 2 mmol/gram, such as from 0.9 to 2 mmol/gram, for example from 1.5 to 2 mmol/gram, such as from 0.4 to 1.3 mmol/gram, for example from 0.6 to 1.3 mmol/gram, such as from 0.8 to 1.3 mmol/gram, for example from 1 to 2 mmol/gram, such as from 1.2 to 2 mmol/gram, for example from 1.4 to 2 mmol/gram, such as from 1.6 to 2 mmol/gram, for example from 1.8 to 2 mmol/gram, such as from 0.01 to 0.05 mmol/gram, for example from 0.05 to 0.1 mmol/gram, such as from 0.1 to 0.2 mmol/gram, for example from 0.2 to 0.4 mmol/gram, such as from 0.4 to 0.6 mmol/gram, for example from 0.6 to 0.8 mmol/gram, such as from 0.8 to 1 mmol/gram, such as from 1 to 1.2 mmol/gram, for example from 1.2 to 1.4 mmol/gram, such as from 1.4 to 1.6 mmol/gram, for example from 1.6 to 1.8 mmol/gram.

The swelling volume of the polymer matrix in an aqueous liquid, including water, is in the range of from 1 ml/gram to preferably less than 32 ml/gram, such as from 1 ml/gram to 24 ml/gram, for example from 1 ml/gram to 20 ml/gram, such as from 1 ml/gram to 18 ml/gram, for example from 1 ml/gram to 16 ml/gram, such as from 1 ml/gram to 14 ml/gram, for example from 1 ml/gram to 12 ml/gram, such as from 1 ml/gram to 10 ml/gram, for example from 1 ml/gram to 9 ml/gram, such as from 1 ml/gram to 8 ml/gram, for example from 1 ml/gram to 7 ml/gram, such as from 1 ml/gram to 6 ml/gram, for example from 1 ml/gram to 5 ml/gram, such as from 1 ml/gram to 4 ml/gram, for example from 1 ml/gram to 3 ml/gram, such as from 1 ml/gram to 2 ml/gram, for example from 4 ml/gram to 20 ml/gram, such as from 4 ml/gram to 18 ml/gram, for example from 4 ml/gram to 16 ml/gram, such as from 4 ml/gram to 14 ml/gram, for example from 4 ml/gram to 12 ml/gram, such as from 4 ml/gram to 10 ml/gram, for example from 4 ml/gram to 8 ml/gram, such as from 4 ml/gram to 6 ml/gram, for example from 6 ml/gram to 20 ml/gram, such as from 6 ml/gram to 18 ml/gram, for example from 6 ml/gram to 16 ml/gram, such as from 6 ml/gram to 14 ml/gram, for example from 6 ml/gram to 12 ml/gram, such as from 6 ml/gram to 10 ml/gram, for example from 6 ml/gram to 8 ml/gram, such as from 8 ml/gram to 20 ml/gram, for example from 8 ml/gram to 16 ml/gram, such as from 8 ml/gram to 12 ml/gram, for example from 2 ml/gram to 4 ml/gram, such as from 8 ml/gram to 10 ml/gram, for example from 10 ml/gram to 12 ml/gram, such as from 12 ml/gram to 14 ml/gram, for example from 14 ml/gram to 16 ml/gram, such as from 16 ml/gram to 18 ml/gram, for example from 18 ml/gram to 20 ml/gram.

The ratio R between i) the amine group loading capacity and ii) the swelling volume of the matrix in an aqueous liquid, such as e.g. water, is in the range of from 10^"4 to 0.5, such as from 10^"4 to 0.4, for example from 10^"4 to 0.3, such as from 10^"4 to 0.2, for example from 10^"4 to 0.1, such as from 10^"4 to 0.09, for example from 10^"4 to 0.08, such as from 10^"4 to 0.07, for example from 10^"4 to 0.06, such as from 10^"4 to 0.05, for example from 10^"4 to 0.04, such as from 10^"4 to 0.03, for example from 10^"4 to 0.02, such as from 10^"4 to 0.01, for example from 10^"4 to 0.009, such as from 10^"4 to 0.005, for example from 10^"3 to 0.5, such as from 10^"3 to 0.4, for example from

10^"3 to 0.3, such as from 10^"3 to 0.2, for example from 10^"3 to 0.1 , such as from 10^"3 to 0.09, for example from 10^"3 to 0.08, such as from 10^"3 to 0.06, for example from 10^"3 to 0.04, such as from 0.01 to 0.5, for example from 0.1 to 0.5_; such as from 0.01 to 0.4, for example from 0.02 to 0.04, such as from 0.04 to 0.08, for example from 0.05 to 0.5, such as from 0.08 to 0.5.

The polymer matrix can be beaded or granulated. When the polymer matrix is beaded, it has an essentially spherical form, and preferably a diameter in the range of from 0.1 μm to preferably less than 3000 μm. A more preferred range of diameter is between 10 μm and 1000 μm.

The beaded, cross-linked polymer matrix can be formed by polymerization of droplets in an inert phase, such as unreactive oil, for example paraffin oil. The polymer resin can also be formed by bulk polymerization followed by granulation.

There is also provided a composition comprising a plurality of cross-linked polymer beads according to the invention. The composition preferably comprises more than 10³ beads, for example more than 10⁵ beads, such as more than 10⁷ beads, for example more thanlO⁹ beads. The average diameter of the beads of the composition is preferably in the range of from 0.1 μm to less than 3000 μm.

In a further aspect there is provided a functional surface comprising the polymer matrix according to the invention, obtained e.g. by bulk or moulded polymerization, and attached thereto at least one functional moiety. The functional moiety can be a bioactive species preferably selected from a scaffold moiety comprising at least one site for functionalization, a RNA moiety, a DNA moiety, a peptide moiety, or an amino acid residue. The functional surface can be planar, tubular, spherical or a porous material.

The functional surface can further comprise a linker residue linking the functional moiety to the functional surface.

Methods for generating the polymer matrix according to the invention

There is provided a method for generating a beaded polymer matrix according to the invention, said method comprising the steps of

synthesizing a monomer and/or macromonomer and a crosslinker for polymeriza- tion, and,

polymerizing the macromonomer by either i) suspension polymerization and/or; ii) inverse suspension polymerization and/or iii) bulk polymerization followed by granulation and/or iv) droplet polymerization and/or v) emulsion polymerization and/or vi) seeded polymerization,

and obtaining a polymer matrix according to the invention.

The polymerization reaction can be a radical initiated chain polymerization reaction as disclosed by Meldal in US 5,352,756.

In preferred embodiments, there is provided a method for preparing a beaded polymer matrix according to the invention, said method comprising the steps of providing a macromonomer comprising a bisamino poly(alkylene)glycol functional- ized with at least one fragment comprising a conjugated vinyl group,

mixing the conjugated vinyl macromonomer with acrylic amide derivatives,

copolymerizing the vinyl groups of said macromonomers using radical initiators or ionic initiators,

forming a beaded, cross-linked polymer matrix comprising a plurality of amide func- tionalities,

reducing the amide functionalities, and

obtaining a beaded polymer matrix wherein the majority of the amide functionalities are reduced to primary and secondary amine functionalities.

The exhaustive reduction of amide groups in the above described polymers can be achieved by the treatment with reducing agents like borane-THF reagent in presence of boric acid and trimethyl borate, arsenic trioxide in aqueous alcoholic HCI, antimony pentoxide in aqueous alcoholic HCI, LiAIH₄, H₂0₂, BF₃.Et₂0 in presence of sodium borate, lithium tri-(tert)-butoxyaluminium hydride (LiAIH(OtBu)₃), DIBAL-H, NaBH₄, NaBH₃CN and NaH . The preferred reducing agent for the amide groups are borane-THF reagent in presence of boric acid and trimethyl borate [Yu et al, J. Org. Chem., 67,3138 (2002)]. The reaction has been optimized by using different con- centration of reagent cocktail such as (i) borane-THF (2 equiv)/boric acid (1 equiv)/tr ϊimethyl borate (1 equiv); (ii) borane-THF (5 equiv)/boric acid (1.5 equiv)/tr iimethyl borate (1.5 equiv); (iii) borane-THF (7 equiv)/boric acid (2.5 equiv)/tr iimethyl borate (2.5 equiv); (iv) borane-THF (10 equiv)/boric acid (3 equiv)/tr iimethyl borate (3 equiv); (v) borane-THF (20 equiv)/boric acid (6 equiv)/tr iimethyl borate (6 equiv); (vi) borane-THF (40 equiv)/boric acid (12 equiv)/tr iimethyl borate (12 equiv). A quantitative amide bond reduction to amine is observed in all reactions except for the reactions using less than 10 equiv of reducing agent. Applications of the polymer matrix

The polymer of the present invention can be used a solid support for a range of applications including solid phase synthesis, chromatography and immobilization of macromolecules.

The polymers according to the invention may again be derivatized with any of the commercial available linkers for solid phase synthesis, such as e.g. linkers comprising functional groups comprising or consisting of one or more of amino, alkyla- mino, hydroxy, carboxyl, mercapto, sulfeno, sulfino, sulfo, and derivatives of these. The resin can also be used for the combinatorial library synthesis. The resin is also suitable for syntheses involving enzymatic reactions.

The invention also relates to a solid support for enzymatic synthesis of oligosaccha- rides with glycosyltransferases said support including a polymer according to the invention as described above.

The invention also relates to a solid support for the immobilization of proteins said support involving a polymer according to the invention as described above.

Again, the invention relates to a resin for chromatographic separations such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, ion pair chromatography, normal phase chromatography and reversed phase chromatography said resin involving a polymer according to the invention as described above.

The invention also relates to a method of continuous flow or batchwise synthesis of peptides, oligonucleotides or oligosaccharides during the synthesis is attached to a solid support involving a polymer according to the invention as described above. Due to the particular features of the polymer according to the invention this method also can extend to the synthesis involving enzymatic reactions.

The invention relates to a method of immobilizing a protein wherein a protein is attached to a solid support involving a polymer according to the invention as described above. The invention also relates to a method of performing chromatographic separations which comprises the use of a chromatographic resin involving a polymer according to the invention as described above.

Further, the invention relates to a solid support for scavenging the excess reagents in solution phase synthesis involving a polymer according to the invention as described above. In preferred embodiments, there is provided the use of the polymer matrix according to the invention for scavenging excess acyl compounds or excess carbonyl compounds from a composition comprising a mixture of molecular entities.

Also provided is a partially acryloylated bisamino poly(alkylene)glycol for use in the preparation of a beaded, cross-linked polymer matrix according to the invention, said preparation preferably comprising the step of inverse suspension polymerization.

There is also provided the following methods:

A method for preparing a functional surface said method comprising the steps of

i) providing a macromonomer comprising a bisamino poly(alkylene)glycol func- tionalized with at least one fragment comprising a vinyl group,

ii) mixing the conjugated vinyl macromonomer with acrylic amide derivatives

iii) polymerizing the vinyl groups of said macromonomers using radical initiators or ionic initiators,

iv) forming a beaded, cross-linked polymer matrix comprising a plurality of amide functionalities,

v) reducing the amide functionalities,

vi) obtaining a beaded polymer matrix wherein the majority of the amide functionalities are reduced to primary and secondary amine functionalities, and vii) contacting the beaded polymer matrix obtained in step e) with at least one functional moiety and obtaining the functional surface.

A method for targeting a functional moiety attached to a functional surface, said method comprising the steps of

i) providing a functional surface according to the invention, and

ii) targeting said functional moiety with at least one targeting species having a non-covalent affinity, for said functional moiety, or

iii) targeting said functional moiety with at least one targeting species forming a covalent bond with the said functional moiety.

A method for identifying and/or purifying a targeting species having an affinity for a functional moiety, said method comprising the steps of

i) providing a functional surface according to the invention, and

ii) targeting said functional moiety with at least one targeting species having an affinity for said functional moiety, and

iii) identifying and/or purifying the at least one targeting species having an affinity for said functional moiety.

Targeting species identified and/or purified by the above method are also within the scope of the invention as are methods for therapy of a human or animal body when said methods comprise the step of administering to said human or animal body a targeting species identified as described herein above in a pharmaceutical effective amount.

There is also provided the use of the polymer matrix according to the invention as a support for combinatorial chemistry reactions and the synthesis of combinatorial libraries. Accordingly, one preferred application for the polymer resins according to the invention is in the synthesis of libraries using combinatiorial chemistry. A combinatorial library is a collection of multiple species of chemical compounds comprised of smaller subunits or monomers. Combinatorial libraries come in a variety of sizes, ranging from a few hundred to many hundreds of thousand different species of chemical compounds. There are also a variety of library types, including oligomeric and polymeric libraries comprised of compounds such as peptides, carbohydrates, oligonucleotides, and small organic molecules, etc. Such libraries have a variety of uses, such as immobilization and chromatographic separation of chemical com- pounds, as well as uses for identifying and characterizing ligands capable of binding an acceptor molecule or mediating a biological activity of interest.

The library compounds may comprise any type of molecule of any type of subunits or monomers, including small molecules and polymers wherein the monomers are chemically connected by any sort of chemical bond such as covalent, ionic, coordination, chelation bonding, etc, which those skilled in the art will recognize can be synthesized on a solid-phase support

The term polymer as used herein includes those compounds conventionally called heteropolymers, i.e., arbitrarily large molecules composed of varying monomers, wherein the monomers are linked by means of a repeating chemical bond or structure. The polymers of the invention of this types are composed of at least two sub- units or monomers that can include any bi-functional organic or herteronuclear molecule including, but not limited to amino acids, amino hydroxyls, amino isocya- nates, diamines, hydroxycarboxylic acids, oxycarbonylcarboxylic acids, aminoalde- hydes, nitroamines, thioalkyls, and haloalkyls.

In the disclosure of the present invention, the terms "monomer," "subunits" and "building blocks" will be used interchangeably to mean any type of chemical building block of molecule that may be formed upon a solid-phase support. The libraries are not limited to libraries of polymers, but is also directed to libraries of scaffolded small molecules.

Various techniques for synthesizing libraries of compounds on solid-phase supports are known in the art. Solid-phase supports are typically polymeric objects with sur faces that are fu notional ized to bind with subunits or monomers to form the compounds of the library. Synthesis of one library typically involves a large number of solid-phase supports.

To make a combinatorial library, solid-phase supports are reacted with a one or more subunits of the compounds and with one or more numbers of reagents in a carefully controlled, predetermined sequence of chemical reactions. In other words, the library subunits are "grown" on the solid-phase supports. The larger the library, the greater the number of reactions required, complicating the task of keeping track of the chemical composition of the multiple species of compounds that make up the library. Thus, it is important to have methods and apparatuses which facilitate the efficient production of large numbers of chemical compounds, yet allow convenient tracking of the compounds over a number of reaction steps necessary to make the compounds.

Combinatorial libraries represent an important tool for the identification of e.g. small organic molecules that affect specific biological functions. Due to the interaction of the small molecules with particular biological targets and their ability to affect specific biological functions, they may also serve as candidates for the development of therapeutics. Accordingly, small molecules can be useful as drug leads eventually resulting in the development of therapeutic agents.

Because it is difficult to predict which small molecules will interact with a biological target. Intense efforts have been directed towards the generation of large numbers, or "libraries", of small organic compounds. These libraries can then be linked to sensitive screens to identify the active molecules.

Libraries have been designed to mimic one or more features of natural peptides. Such peptidomimetic libraries include phthalimido libraries (WO 97/22594), thio- phene libraries (WO 97/40034), benzodiazopene libraries (U.S. Pat. No. 5,288,514), libraries formed by sequential reaction of dienes (WO 96/03424), thiazolidinone libraries, libraries of metathiazanones and their derivatives (U.S. Pat. No. 5,549,974), and azatide libraries (WO 97/35199) (for peptidomimetic technologies, see Gante, J., Angew. Chem. Int. Ed. Engl. 1994, 33, 1699-1720 and references cited therein). Examples

General Methods

Reagents were obtained from Aldrich and used without any purification. All solvents used were of HPLC grade kept over molecular sieves. The PEGA beads were prepared in a 250 ml baffled glass reactor equipped with a dispersion stirrer.

Synthesis of partially acryloylated (NH₂)₂PEGι₉oo

Acryloyl chloride (1.267 ml, 14 mmol) in DCM (12 ml) was added dropwise to a so- lution of (NH₂)₂PEG₁₉oo (20 g, 10 mmol) in DCM (18 ml) at 0 °C with stirring. The reaction mixture was kept for 1 h at 20 °C. The DCM was evaporated and drying in vacuo at 20 °C yielded the 70% acyloyiated (NH₂)₂PEGιg₀oas colourless thick oil.

Preparation of PEGA_ig0o beads (Resin A) The PEGAi9oo polymer beads were prepared by inverse suspension polymerization method. In order to prepare the beads having a size 500 μm, a 1.4 wt % of sorbitan monolaurate with the macromonomer was used as the suspension stabilizer. The n- heptane was used as the suspension medium and was degassed with argon for 1 h before the addition of monomers. In a typical synthesis procedure, a solution of (Acr)ι.4 (NH₂)₂PEG₁₉oo (7.3 g, 3.54 mmol) in water (21 ml) was degassed with argon for 30 min. Acrylamide (0.36 g, 5 mmol) in water (0.5 ml) was added to the degassed solution and the purging of argon was continued for 5 min. A solution of sorbitan monolaurate (0.1 ml) in DMF (1 ml) and the free radical initiator ammonium persulfate (300 mg) in water (2 ml) were added to the monomer mixture. The reac- tion mixture was then rapidly added to the suspension medium and stirred at 600 rpm at 70 °C. After one min, TEMED (1.5 ml) was added to the reactor. The reaction was allowed to continue for 3h, the beads formed were filtered through the sieves and the 500 μm fraction was collected. The beads were washed thoroughly with ethanol (10χ), water (10χ), ethanol (10χ) and dried under high vacuum to provide Resin A.

Synthesis of high loading poly(ethylene)glycol based resin (Resin B) by exhaustive reduction

The exhaustive reduction of amide groups in PEGA resin (Resin A) was carried out in a 100 ml glass tubes under argon. a) The resin (500 mg, 0.85 mmol carbonyl) and boric acid (1 equiv, 52.55 mg, 0.85 mmol) were taken in the glass tube. Trimethyl borate (1 equiv, 100 μl, 0.85 mmol) was added followed by the addition of 1M borane-THF complex (2 equiv, 1.7 ml, 1.7 mmol). After cessation of hydrogen evolution, the tubes were capped tightly and kept in an oil bath at 65°C for 72 h. The resin was then filtered, washed with DMF (10 ml x 4) and MeOH (10 ml x 4). The resin was then suspended in piperidine (100%, 10 ml) and heated at 65°C for 20 h to disproportionate the borane complexes. Following the decantation of the piperidine- borane solution, the resin was washed with DMF (10 ml x 4), DCM (10 ml x 4) and MeOH (10 ml x 4) and dried under vacuum to provide Resin B with 30 % of amide reduction. b) The resin (500 mg, 0.85 mmol carbonyl) and boric acid (1.5 equiv, 78.83 mg, 1.27 mmol) were taken in the glass tube. Trimethyl borate (1.5 equiv, 150 μl,

1.27 mmol) was added followed by the addition of 1 M borane-THF complex (5 equiv, 4.25 ml, 4.25 mmol). After cessation of hydrogen evolution, the tubes were capped tightly and kept in an oil bath at 65°C for 72 h. The resin was then filtered, washed with DMF (10 ml x 4) and MeOH (10 ml x 4). The resin was then suspended in piperidine (100%, 10 ml) and heated at 65°C for 20 h to disproportionate the borane complexes. Following the decantation of the piperidine- borane solution, the resin was washed with DMF (10 ml x 4), DCM (10 ml x 4) and MeOH (10 ml x 4) and dried under vacuum to provide Resin B with 50 % of amide reduction. c) The resin (500 mg, 0.85 mmol carbonyl) and boric acid (2.5 equiv, 131.4 mg, 2.13 mmol) were taken in the glass tube. Trimethyl borate (2.5 equiv, 250 μl, 2.13 mmol) was added followed by the addition of 1 M borane-THF complex (7 equiv, 5.95 ml, 5.95 mmol). After cessation of hydrogen evolution, the tubes were capped tightly and kept in an oil bath at 65°C for 72 h. The resin was then filtered, washed with DMF (10 ml x 4) and MeOH (10 ml x 4). The resin was then suspended in piperidine (100%, 10 ml) and heated at 65°C for 20 h to dispropor- tionate the borane complexes. Following the decantation of the piperidine- borane solution, the resin was washed with DMF (10 ml x 4), DCM (10 ml x 4) and MeOH (10 ml x 4) and dried under vacuum to provide Resin B with 75 % of amide reduction. d) The resin (500 mg, 0.85 mmol carbonyl) and boric acid (3 equiv, 157.66 mg, 2.55 mmol) were taken in the glass tube. Trimethyl borate (3 equiv, 300 μl, 2.55 mmol) was added followed by the addition of 1M borane-THF complex (10 equiv, 8.5 ml, 8.5 mmol). After cessation of hydrogen evolution, the tubes were capped tightly and kept in an oil bath at 65°C for 72 h. The resin was then filtered, washed with DMF (10 ml x 4) and MeOH (10 ml x 4). The resin was then suspended in piperidine (100%, 10 ml) and heated at 65°C for 20 h to disproportionate the borane complexes. Following the decantation of the piperidine- borane solution, the resin was washed with DMF (10 ml x 4), DCM (10 ml x 4) and MeOH (10 ml x 4) and dried under vacuum to provide Resin B with a quantitative conversion of amide to amine. e) The resin (500 mg, 0.85 mmol carbonyl) and boric acid (6 equiv, 315.32 mg, 5.1 mmol) were taken in the glass tube. Trimethyl borate (6 equiv, 600 μl, 5.1 mmol) was added followed by the addition of 1 M borane-THF complex (20 equiv, 17 ml, 17 mmol). After cessation of hydrogen evolution, the tubes were capped tightly and kept in an oil bath at 65°C for 72 h. The resin was then filtered, washed with DMF (10 ml x 4) and MeOH (10 mix 4). The resin was then suspended in piperidine (100%, 10 ml) and heated at 65°C for 20 h to disproportionate the borane complexes. Following the decantation of the piperidine-borane solution, the resin was washed with DMF (10 ml x 4), DCM (10 ml x 4) and MeOH (10 ml x 4) and dried under vacuum to provide Resin B with a quantitative conversion of amide to amine. f) The resin (500 mg, 0.85 mmol carbonyl) and boric acid (12 equiv, 630.64 mg, 10.2 mmol) were taken in the glass tube. Trimethyl borate (12 equiv, 1.2 ml, 10.2 mmol) was added followed by the addition of 1 M borane-THF complex (40 equiv, 34 ml, 34 mmol). After cessation of hydrogen evolution, the tubes were capped tightly and kept in an oil bath at 65°C for 72 h. The resin was then filtered, washed with DMF (10 ml x 4) and MeOH (10 ml x 4). The resin was then suspended in piperidine (100%, 10 ml) and heated at 65°C for 20 h to dispropor- tionate the borane complexes. Following the decantation of the piperidine- borane solution, the resin was washed with DMF (10 ml x 4), DCM (10 ml x 4) and MeOH (10 ml x 4) and dried under vacuum to provide Resin B with a quantitative conversion of amide to amine. Characterization Loading:

The amino functional loading was determined from the Fmoc-Gly derivatized Resin B. The resin (3-5 mg) was treated with piperidine-DMF solution (20% v/v, 8 ml) for 30 min. The amino capacity of the resin was calculated from the OD value of piperi- dine-dibenzofulvene solution at 290 nm. The amino functional loading of Resin B is measured as 1.6 mmol/g where starting with a resin obtained from the reduction of a polymer obtained by the polymerization of a 95% of partially acryloylated 0,0^'- bisaminoprop-1-yl)PEG₁₉₀o and 5% acrylamide (Resin A).

Swelling:

The swelling capabilities of the resin in different solvents were determined by the syringe method. In a typical procedure, the resin (100 mg) was taken in a 2 ml syringe fitted with a Teflon filter at the bottom. The solvent was sucked in to the sy- ringe and after 3 h, excess solvent was removed by applying force on the piston.

The extent of swelling of the resin in each solvent was determined from the volume of the resin before and after the solvent incubation.

Mechanical stability: The mechanical stability of the high capacity poly(ethylene)glycol based resin (Resin

B) was compared with PEGA resin (Resin A). The compressive properties describe the behaviour of the bead when it is subjected to a compressive stress. The bead (300-500 μm) is placed between compressive plates parallel to the surface and then compressed at a constant rate. The compressive modulus can be calculated from the ratio of compressive stress (the force per area of cross section of the bead at low strain) over compressive strain (ratio of the diameter over original diameter). Compressive modulus of high capacity poly(ethylene)glycol based resin (Resin B) is 0.5 MPa and that of PEGA resin (Resin A) is 0.4 MPa, which indicates that even after the exhaustive reduction, the resin does not change its mechanical properties considerably.

Chemical stability:

-The stability studies of the resin were carried out in different reagents like trifluoro acetic acid (100%), 20% piperidine in DMF, 1,8-diazobicyclo[5.4.0]-undec-7-ene (DBU) (100%), butyl lithium (2.7 M solution in heptane, 100%), saturated NaOH, and BF₃ Et₂0 (100%). The resin samples (100 mg) were separately stirred with the reagents. After 48 h, the resin was filtered, washed, dried and IR spectra were recorded and compared with original. The swelling properties of the resin after treatment with the reagents for two weeks were also compared. The resin did not dissolve in any of these conditions and showed no changes in colour or swelling indicating no bond cleavage.

Use of the high capacity PEG-based resin for solid phase synthesis

Synthesis of Ac-His-(D)Phe-Arg-Trp-NH₂ [Peptide (1)]

The peptides were synthesized in a plastic syringe fitted with a Teflon filter at the bottom.

The high capacity poly(ethylene)glycol based resin (35 mg, 0.056 mmol) was swollen in dry DMF (5 ml) and treated with Fmoc-Rink amide linker (90.65 mg, 0.168 mmol, 3 equiv) in presence of TBTU (51.77 mg, 0.224 mmol, 2.88 equiv) and NEM

(28.3 μl, 0.224 mmol, 4 equiv). After 3 h at room temperature, the resin was washed with DMF (10χ), MeOH (10χ), DCM (10χ) and dried in vacuo. The resin was negative to Kaiser amine test and a quantitative reaction was observed by measuring the Fmoc group on the resin (5 mg) with 20% Piperidine/DMF solution (8 ml) for 30 min at room temperature.

The resin was swollen in dry DMF (5 ml) and the Fmoc group was removed by 20% Piperidine/DMF (1 ml) for 20 min at room temperature. The resin was washed with DMF (10χ) and the amino acids Fmoc-Trp(Boc), Fmoc-Arg(Pmc), Fmoc-(D)Phe and Fmoc-His(Trt) (3 equiv) were attached successively in presence of TBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of all amino acids, the Fmoc protection was removed by 20% piperidine in DMF (1 ml, 20 min) and the resin was washed with DMF (10χ). The peptide on the resin was then acetylated with aceti- canhydride/pyridine/DMF (2:4:4) (1 ml) and washed with DMF (10χ), MeOH (10χ), DCM (10χ) and dried in vacuo. The peptide was cleaved from the resin by treating with a solution of TFA (90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%) and thioanisole (1%) for 3 h at room temperature. The resin was filtered off and washed with TFA (2 x) and DCM (2 x). The combined filtrate was concentrated under vacuum and the peptide was precipitated by ether. The peptide was washed with ether (10 x) and dried in vacuo to afford 36.93 mg (96%) of pure peptide. HPLC: -_R = 9.71 min

ESI-MS: calcd (M+H)⁺ = 686.78 Da; found (M+H)⁺ = 686.4 MALDI TOF MS: calcd (M+H)⁺ = 686.78 Da; found (M+H)⁺ = 686.98 ¹H NMR (600 MHz, MeOH-d₄): δ = 1.38-1.64 (m, 2H, Arg H^β), 1.10-1.15 (m, 2H, Arg H ), 2.00 (s, 3H, Acetyl CH₃), 2.96 (m, 2H Arg H^δ), 3.00-3.09 (m, 2H Phe H^p), 3.24-

3.41 (m, 2H Trp H^p), 3.04-3.23 (m, 2H His H^β), 4.01 (m, 1H Arg H^α), 4.73 (m, 1H His H^α), 4.51 (m, 1H Phe H^α), 4.71 (m, 1H Trp H^α), 7.04-7.67 (br 5H Trp ring protons), 7.21 , 8.76 (2H, His ring protons), 7.25-7.33 (br, 5H Phe ring protons).

Synthesis of Fmoc-Dap(N₃)OH

Fmoc-Dap-OH (980 mg, 3 mmol) was dissolved in 80% aqueous acetic acid (9 ml) and CuS0₄.5H₂0 (15 mg, 0.06 mmol, 0.02 equiv) in water (1 ml) was added. The pH of the solution was adjusted to 9-10 with K₂C0₃. Water (15 ml), MeOH (32 ml) and trifluoromethanesulfonyl azide (6 mmol) in DCM (25 ml) was added and the pH was readjusted to 9-10 with K₂C0₃. The two-phase system was stirred vigorously for

20 h. The layers were separated by addition of DCM and the organic phase was washed with water (2 x 40 ml) and then the combined aqueous phases were acidified with 3 M HCI (aqueous) to a pH 2. The aqueous phase was extracted with DCM (4 x 50 ml) and the combined organic phases were dried over sodium sulfate, Til— tered and concentrated under vacuo (0.934 g, 88.2%).

HPLC: t_R = 10.08 min

ESI-MS: calcd (M+H)⁺ = 353.34 Da; found (M+H)⁺ = 353.1 ¹H NMR (250 MHz, CDCI₃): δ = 3.75 (d, 2H), 4.14-4.9 (t, 1H), 4.36-4.39 (d, 2H), 4.50-4.54 (m, 1H), 5.50-5.54 (2H, NH and OH), 7.22-7.28 (4H, aromatic ring), 7.51- 7.54 (d, 2H, aromatic ring), 7.68-7.71 (d, 2H, aromatic ring).

Synthesis of Fmoc-Lys(Boc)-Dap(N₃)-His(Trt)-(D)Phe-Arg(Pmc)-Trp(Boc)-Pra- Met-HMBA-Gly-Resin B

The high capacity poly(ethylene)glycol based resin (150 mg, 0.24 mmol) was swol- len in dry DMF (5 ml) and treated with Fmoc-Gly (215 mg, 0.72 mmol, 3 equiv) in presence of TBTU (222 mg, 0.69 mmol, 2.88 equiv) and NEM (121.8 μl, 0.96 mmol, 4 equiv). After 3 h at room temperature, the resin was washed with DMF (10χ), MeOH (10χ), DCM (10χ) and dried in vacuo. The resin was negative to Kaiser amine test and a quantitative reaction was observed by measuring the Fmoc group on the resin (5 mg) with 20% Piperidine/DMF solution (8 ml) for 30 min at room temperature.

The resin was swollen in dry DMF (5 ml), Fmoc group was removed by 20% Piperi- dine/DMF and treated with HMBA linker (109.5 mg, 0.72 mmol, 3 equiv) in presence of TBTU (222 mg, 0.69 mmol, 2.88 equiv) and NEM (121.8 μl, 0.96 mmol, 4 equiv). After 3 h at room temperature, the resin was washed with DMF (10χ), MeOH (10χ), DCM (10χ) and dried in vacuo. The resin was negative to Kaiser amine test

The resin was swollen in dry DCM (2 ml), Fmoc-Met (267.5 mg, 0.72 mmol, 3 equiv), MSNT (213.4 mg, 0.72 mmol, 3 equiv) and Melm (43 μl, 0.54 mmol, 2.25 equiv) were added. After 1 h, the resin was filtered and washed with DCM (10χ), MeOH (10χ) and DMF (10χ). The Fmoc group was removed by 20% Piperidine/DMF (1 ml) for 20 min at room temperature. The resin was washed with DMF (10χ) and the amino acids Fmoc-Pra, Fmoc-Trp(Boc), Fmoc-Arg(Pmc), Fmoc-

(D)Phe, Fmoc-His(Trt), Fmoc-Dap(N₃) and Fmoc-Lys(Boc) (3 equiv) were attached successively in presence of TBTU (2.88 equiv) and NEM (4 equiv). After the incorporation of all amino acids, the resin was washed with DMF (10χ), MeOH (10χ), DCM (10χ) and dried in vacuo.

Cleavage of linear peptidomimetic (2) (Lys-Dap(N₃)-His-(D)Phe-Arg-Trp-Pra- Met) from the Resin B

The N-terminal Fmoc protection of the peptidyl resin (8.7 mg) was removed by 20% piperidine/DMF solution (2 ml, 30 min) and the resin was washed with DMF (10χ), MeOH (10χ), DCM (10χ) and dried. The resin was treated with a solution of TFA

(90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%) and thioanisole (1%) for 3 h at room temperature for removing all the side chain protection groups. The resin was washed with DCM (10χ), MeOH (10χ) and DCM (10χ). The peptide was cleaved from the resin by treating with 0.1 M NaOH (100 μl) for 2 h at room temperature. The resin was filtered and the filtrate was neutralized with 0.1

M HCI (100 μl) yielding 4.1 mg (96%) of pure peptide. HPLC: t_R = 10.63 min ES MS/MS: calcd (M+H)⁺ = 1112.29 Da; found (M-N₃) = 1068.63 Da Cyclization of Fmoc-Lys(Boc)-Dap(N₃)-His(Trt)-(D)Phe-Arg(Pmc)-Trp(Boc)-Pra- Met-HMBA-Gly-Resin B

(a) The peptidyl resin (20 mg) was treated with a solution of TFA (90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%) and thioanisole (1%) for 3 h at room temperature for removing all the side chain protection groups. The resin was washed with DCM (10χ), MeOH (10χ) and DMF (10χ). The Fmoc group was removed by 20% Piperidine/DMF (2 ml) and the resin was washed with DMF (10χ), MeOH (10χ), DCM (10χ) and THF (10χ). DIPEA (61 μl, 0.35 mmol, 50 equiv) and Cul (2.66 mg, 0.014 mmol, 2 equiv) in THF (300 μl) were added to the resin. The reaction was left for 16 h and then washed with THF, water, DMF, MeOH, DCM and dried in vacuo.

(b) The peptidyl resin (20 mg) was treated with DIPEA (61 μl, 0.35 mmol, 50 equiv) and Cul (2.66 mg, 0.014 mmol, 2 equiv) in THF (300 μl) were added to the resin. The reaction was left for 16 h and then washed with THF, water, DMF, MeOH, DCM and dried in vacuo. A solution of TFA (90%), water (5%), ethanedithiol (2%), triisopropyl silane (2%) and thioanisole (1 %) were added to the resin for removing all the side chain protection groups (3 h at room temperature). The resin was washed with DCM (10χ), MeOH (10χ) and DMF (10χ). The Fmoc group was removed by 20% Piperidine/DMF (2 ml) and the resin was washed with DMF (10χ), MeOH (10χ),

DCM (10χ) and dried in vacuo.

Cleavage of cyclic peptidomimetic (3) from the Resin B

The resin was treated with 0.1 M NaOH (100 μl) for 2 h at room temperature. The resin was filtered and the filtrate was neutralized with 0.1 M HCI (100 μl).

(a) Yield= 8.1 mg (82.5%)

(b) Yield= 7.8 mg (79%) HPLC: t_R = 10.89 min

ES MS/MS: calcd (M+H)⁺ = 1112.29 Da; found (M+H)⁺ = 1112.56 Da ¹H NMR (600 MHz, DMSO-d₆): 1.259-1.273 (m, 2H Arg H^γ), 1.311-1.332 (m, 2H Lys

H^γ), 1.508-1.514 (m, 2H Lys H^δ), 1.416-1.616 (m, 2H Arg H^β), 1.650-1.669 (m, 2H Lys H^β), 1.852-1.979 (m, 2H Met H^β), 2.022 (s, 3H Met -CH₃), 2.461 (t, 2H Met H^γ), 2.484-2.577 (m, 2H Pra H^β), 2.671-2.848 (m, 2H His H^β), 2.721-2.944 (m, 2H Phe H^β), 2.728-2.734 (t, 2H Lys H^ε), 2.961-3.157 (m, 2H Trp H^β), 2.998-3.004 (m, 2H Arg H^δ), 3.366-3.568 (m, 2H Dap H^β), 3.794 (m, 1H Lys H^α), 4.282 (m, 1H Arg H^α), 4.309 (m, 1 H Met H^α), 4.417 (m, 1H Pra H^α), 4.521 (m, 1H Dap H^α), 4.568 (m, 1 H Trp H^α), 4.584 (m, 1H His H^α), 4.662 (m, 1 H Phe H^α), 7.164-7.239 (br, 5H Phe ring protons), 7.201 , 8.211 (2H, His ring protons), 7.447 (s, 1 H Arg -NH), 6.955-7.312 (br, 5H Trp ring protons), 8.240 (s, 1 H Triazole ring proton), 8.094 (1 H Trp amide H), 8.185 (1 H

Met amide H), 8.213 (1 H Phe amide H), 8.250 (1H Pra amide H), 8.329 (1H Arg amide H), 8.408 (1H His amide H), 8.805 (1H Dap amide H), 10.697 (1H Trp ring NH).

Claims

Claims

1. A beaded or granulated polymer matrix formed by a reduction of more than 50% of the amide groups in a cross-linked polymer obtainable by polymerization of a poly(oxyalkylene) chain terminated with an acylamide functional group.

2. The polymer matrix according to claim 1 , wherein the cross-linked polymer is obtained by polymerization of a poly(oxyalkylene) chain terminated with an acylamide functional group using radical or ionic initiators.

3. The polymer matrix according to any of claims 1 and 2, wherein the cross-linked and beaded polymer is obtained by a method comprising the steps of

a) providing a plurality of macromonomers each comprising a poly(oxyalkylene) chain terminated with an acylamide functional group,

b) polymerising said macromonomer using a free radical initiator or an ionic initiator, optionally with the addition of copoiymerizing agents, and

c) converting in the beaded polymer matrix at least 50% of the amide groups to amine functional groups by reduction of the amide groups with a suitable reducing agent.

4. The polymer matrix according to claim 3, wherein the polymerization is an in- verse suspension polymerization.

5. The polymer matrix according to any of claims 3 and 4 wherein a free radical initiator is used.

6. The polymer matrix according to any of claims 3 an 4 wherein a ionic initiator is used.

7, The polymer matrix according to any of claims 1 to 6, wherein the reduction of . amide groups is achieved by using a reducing agent selected from the group consisting of borane, arsenic trioxide in aqueous alcoholic HCI, antimony pen toxide in aqueous alcoholic HCI, LiAIH₄, H₂0₂, BF₃.Et₂0 in the presence of sodium borate, lithium tri-(tert)-butoxyaluminium hydride (LiAIH(OtBu)₃), DIBAL-H, NaBH₄, NaBH₃CN and NaH.

8. The polymer matrix according to any of claims 1 to 7 comprising the structure

Z = H or CH₃ or C₂H₅

R = H or CH₃ or CH₂OH or C₂H₅OH or i-C₃H₇ or n-C₃H₇ or i-C₄H₉ or n-C₄rf₉

R' = H or CH₃ or i-C₃H₇ or n-C₃H₇ or i-C₄H₉ or n-C₄H₉

R" = H or CH₃

R"' = H or CH₃ n is a real number in the range of from 3 to 2000

9. The polymer matrix according to claim 8, wherein

Z is selected from H, CH₃ and C₂H₅, and independently thereof,

R is selected from H, CH₃, CH₂OH, C₂H₅OH, i-C₃H₇, n-C₃H₇, i-C₄H₉ and n-C₄H₉, including any combination thereof, and independently thereof,

R^Λ is selected from H, CH₃, i-C₃H₇, n-C₃H₇, i-C₄H₉ and n-C₄H₉, including any combination thereof, and independently thereof,

R^" is selected from H and CH_3ι or the combination of both, and independently thereof,

R^'" is selected from H and CH_3| or the combination of both, and wherein n is a real number in the range of from 4 to 180.

10. The polymer matrix according to any of claims 8 and 9, wherein R is H.

11. The polymer matrix according to any of claims 8 and 9, wherein R is CH₃.

12. The polymer matrix according to any of claims 8 and 9, wherein R is CH₂OH.

13. The polymer matrix according to any of claims 8 and 9, wherein R is C₂H₅OH

14. The polymer matrix according to any of claims 8 and 9, wherein R is n-C₃H₇

15. The polymer matrix according to any of claims 8 and 9, wherein R is n-C₃H₇

16. The polymer matrix according to any of claims 8 and 9, wherein R is n-C₄H₉

17. The polymer matrix according to any of claims 8 and 9, wherein R is i-C₄H₉

18. The polymer matrix according to any of claims 8 to 17, wherein R^' is selected from H, CH₃, i-C₃H₇, n-C₃H₇, i-C H₉ and n-C₄H₉.

19. The polymer matrix according to any of claims 8 to 17, wherein R^" is H

20. The polymer matrix according to any of claims 8 to 17, wherein R^' is CH₃

21. The polymer matrix according to any of claims 8 to 17, wherein R^Λ is n-C₃H₇

22. The polymer matrix according to any of claims 8 to 17, wherein R" is i-C₃H₇

23. The polymer matrix according to any of claims 8 to 17, wherein R" is n-C₄H₉

24. The polymer matrix according to any of claims 8 to 17, wherein R^' is i-C₄H₉

25. The polymer matrix according to any of claims 8 to 24, wherein R" is H or CH₃

26. The polymer matrix according to any of claims 8 to 24, wherein R^" is H

27. The polymer matrix according to any of claims 8 to 24, wherein R" is CH₃

28. The polymer matrix according to any of claims 8 to 27, wherein R^'" is H or CH₃

29. The polymer matrix according to any of claims 8 to 27, wherein R'^" is H.

30. The polymer matrix according to any of claims 8 to 27, wherein R^"' is CH₃

31. The polymer matrix according to any of claims 8 to 30, wherein Z is selected from H and CH₃ and C₂H₅, including any combination thereof.

32. The polymer matrix according to any of claims 8 to 30, wherein Z is H

33. The polymer matrix according to any of claims 8 to 30, wherein Z is CH₃

34. The polymer matrix according to any of claims 8 to 30, wherein Z is C₂H₅

35. The polymer matrix according to claim 8, wherein ή is a real number in the range of from 3 to 800, for example from 3 to 600, such as from 3 to 400, for example from 3 to 300, for example from 3 to 200, such as from 3 to 100, for example from 3 to 90, such as from 3 to 80, for example from 3 to 70, such as from 3 to 60, for example from 3 to 50, such as from 3 to 45, for example from 3 to 40, such as from 3 to 30, for example from 3 to 25.

36. The polymer matrix according to any of the previous claims, wherein the number average molecular weight (M_n) is in the range of from 200 to 60000, such as from 200 to 45000, for example from 200 to 30000, such as from 200 to 25000, for example from 200 to 20000, such as from 200 to 15000, for example from 200 to 10000, such as from 200 to 8000, for example from 200 to 6000, such as from 200 to 5000, for example from 200 to 4500, such as from 200 to 4000, for example from 200 to 3500, such as from 200 to 3000, for example from 200 to 2500, such as from 200 to 2400, for example from 200 to 2300, such as from 200 to 2200, for example from 200 to 2100, such as from 200 to 2000, for example from 200 to 1900, such as from 200 to 1800, for example from 200 to 1700, such as from 200 to 1600, for example from 200 to 1500, such as from

200 to 1400, for example from 200 to 1300, such as from 200 to 1200, for example from 200 to 1100, such as from 200 to 1000, for example from 200 to 900, such as from 200 to 800, for example from 200 to 700, such as from 200 to 600, for example from 400 to 3000, such as from 400 to 2500, for example from 400 to 2000, such as from 400 to 1800, for example from 400 to 1600, such as from 400 to 1400, for example from 400 to 1200, such as from 400 to 1000, for example from 400 to 800, such as from 400 to 600, for example from 600 to 3000, such as from 600 to 2500, for example from 600 to 2000, such as from 600 to 1800, for example from 600 to 1600, such as from 600 to 1400, for example from 600 to 1200, such as from 600 to 1000, for example from 600 to 800, such as from 800 to 3000, for example from 800 to 2500, such as from 800 to 2000, for example from 800 to 1800, such as from 800 to 1600, for example from 800 to 1400, such as from 800 to 1200, for example from 800 to 1000, such as from 1000 to 3000, for example from 1000 to 2500, such as from 1000 to 2000, for example from 1000 to 1800, such as from 1000 to 1600, for example from 1000 to 1400, such as from 1000 to 1200.

37. The polymer matrix according to any of the previous claims, wherein the amine group loading capacity is in the range of from 0.01 to 14 mmol/gram, such as from 0.01 to 13 mmol/gram, for example from 0.01 to 12 mmol/gram, for example from 0.01 to 11 mmol/gram, such as from 0.01 to 10 mmol/gram, for example from 0.01 to 9 mmol/gram, such as from 0.01 to 8 mmol/gram, for example from 0.01 to 7 mmol/gram, such as from 0.01 to 6 mmol/gram, for example from 0.01 to 5 mmol/gram, such as from 0.01 to 4 mmol/gram, for example from 0.01 to 3 mmol/gram, such as from 0.01 to 2 mmol/gram, for example from 0.01 to 1 mmol/gram, such as from 0.01 to 0.5 mmol/gram, for example from 0.01 to 0.4 mmol/gram, such as from 0.02 to 2 mmol/gram, for example from 0.04 to 2 mmol/gram, such as from 0.06 to 2 mmol/gram, for example from 0.08 to 2 mmol/gram, such as from 0.1 to 13 mmol/gram, for example from 0.1 to 12 mmol/gram, such as from 0.1 to 11 mmol/gram, for example from 0.1 to 10 mmol/gram, such as from 0.1 to 5 mmol/gram, for example from 0.1 to 4 mmol/gram, such as from 0.1 to 3 mmol/gram, for example from 0.1 to 2 mmol/gram, such as from 0.1 to 1.5 mmol/gram, for example from 0.1 to 1 mmol/gram, such as from 0.1 to 0.8 mmol/gram, for example from 0.1 to 0.6 mmol/gram, such as from 0.1 to 0.5 mmol/gram, for example from 0.1 to 0.4 mmol/gram, such as from 0.1 to 0.3 mmol/gram, for example from 0.1 to 0.2 mmol/gram, such as from 0.2 to 2 mmol/gram, for example from 0.4 to 2 mmol/gram, such as from 0.6 to 2 mmol/gram, for example from 0.8 to 2 mmol/gram, such as from 0.9 to 2 mmol/gram, for example from 1.5 to 2 mmol/gram, such as from 0.4 to 1.3 mmol/gram, for example from 0.6 to 1.3 mmol/gram, such as from 0.8 to 1.3 mmol/gram, for example from 1 to 2 mmol/gram, such as from 1.2 to 2 mmol/gram, for example from 1.4 to 2 mmol/gram, such as from 1.6 to 2 mmol/gram, for example from 1.8 to 2 mmol/gram, such as from 0.01 to 0.05 mmol/gram, for example from 0.05 to 0.1 mmol/gram, such as from 0.1 to 0.2 mmol/gram, for example from 0.2 to 0.4 mmol/gram, such as from 0.4 to 0.6 mmol/gram, for example from 0.6 to 0.8 mmol/gram, such as from 0.8 to 1 mmol/gram, such as from 1 to 1.2 mmol/gram, for example from 1.2 to 1.4 mmol/gram, such as from 1.4 to 1.6 mmol/gram, for example from 1.6 to 1.8 mmol/gram.

38. The polymer matrix according to any of the previous claims, wherein the swelling volume of the matrix in an aqueous liquid, including water, is in the range of from 1 ml/gram to preferably less than 32 ml/gram, such as from 1 ml/gram to 24 ml/gram, for example from 1 ml/gram to 20 ml/gram, such as from 1 ml/gram to 18 ml/gram, for example from 1 ml/gram to 16 ml/gram, such as from 1 ml/gram to 14 ml/gram, for example from 1 ml/gram to 12 ml/gram, such as from 1 ml/gram to 10 ml/gram, for example from 1 ml/gram to 9 ml/gram, such as from 1 ml/gram to 8 ml/gram, for example from 1 ml/gram to 7 ml/gram, such as from 1 ml/gram to 6 ml/gram, for example from 1 ml/gram to 5 ml/gram, such as from 1 ml/gram to 4 ml/gram, for example from 1 ml/gram to 3 ml/gram, such as from

1 ml/gram to 2 ml/gram, for example from 4 ml/gram to 20 ml/gram, such as from 4 ml/gram to 18 ml/gram, for example from 4 ml/gram to 16 ml/gram, such as from 4 ml/gram to 14 ml/gram, for example from 4 ml/gram to 12 ml/gram, such as from 4 ml/gram to 10 ml/gram, for example from 4 ml/gram to 8 ml/gram, such as from 4 ml/gram to 6 ml/gram, for example from 6 ml/gram to

20 ml/gram, such as from 6 ml/gram to 18 ml/gram, for example from 6 ml/gram to 16 ml/gram, such as from 6 ml/gram to 14 ml/gram, for example from 6 ml/gram to 12 ml/gram, such as from 6 ml/gram to 10 ml/gram, for example from 6 ml/gram to 8 ml/gram, such as from 8 ml/gram to 20 ml/gram, for example from 8 ml/gram to 16 ml/gram, such as from 8 ml/gram to 12 ml/gram, for example from 2 ml/gram to 4 ml/gram, such as from 8 ml/gram to 10 ml/gram, for example from 10 ml/gram to 12 ml/gram, such as from 12 ml/gram to 14 ml/gram, for example from 14 ml/gram to 16 ml/gram, such as from 16 ml/gram to 18 ml/gram, for example from 18 ml/gram to 20 ml/gram.

39. The polymer matrix according to any of the previous claims, wherein the ratio R between i) the amine group loading capacity and ii) the swelling volume of the matrix in an aqueous liquid, including water, is in the range of from 10^"4 to 0.5, such as from 10^"4 to 0.4, for example from 10^~4 to 0.3, such as from 10^"4 to 0.2, for example from 10^"4 to 0.1 , such as from 10^"4 to 0.09, for example from 10^~4 to

0.08, such as from 10^"4 to 0.07, for example from 10^"4 to 0.06, such as from 10^"4 to 0.05, for example from 10^"4 to 0.04, such as from 10^"4 to 0.03, for example from 10^"4 to 0.02, such as from 10^"4 to 0.01 , for example from 10^"4 to 0.009, such as from 10^"4 to 0.005, for example from 10^"3 to 0.5, such as from 10^"3 to 0.4, for example from 10^"3 to 0.3, such as from 10^~3 to 0.2, for example from 10^~3 to

0.1, such as from 10^~3 to 0.09, for example from 10^~3 to 0.08, such as from 10^"3 to 0.06, for example from 10^"3 to 0.04, such as from 0.01 to 0.5, for example from 0.1 to 0.5, such as from 0.01 to 0.4, for example from 0.02 to 0.04, such as from 0.04 to 0.08, for example from 0.05 to 0.5, such as from 0.08 to 0.5.

40. The polymer matrix according to any of the previous claims, wherein said matrix is beaded and has an essentially spherical form.

41. The polymer matrix according to claim 40 having a diameter in the range of from 0.1 μm to preferably less than 3000 μm, preferably a diameter in the range of between 10 μm and 1000 μm.

42. The polymer matrix according to any of claims 40 and 41 , formed by polymerization of droplets in an inert phase, such as unreactive oil, for example paraffin oil.

43. The polymer matrix according to any of claims 1 to 39 formed by bulk polymerization followed by granulation.

44. A method for preparing the beaded polymer matrix according to any of claims 1 to 42, said method comprising the step of

a) providing a macromonomer comprising a bisamino poly(a!kylene)glycol func- tionalized with at least one fragment comprising a conjugated vinyl group, b) mixing the conjugated vinyl macromonomer with acrylic amide derivatives,

c) copolymerizing the vinyl groups of said macromonomers using radical initiators or ionic initiators,

d) forming a beaded, cross-linked polymer matrix comprising a plurality of amide functionalities,

e) reducing at least 50% of the amide functionalities, and

f) obtaining a beaded polymer matrix wherein the majority of the amide functionalities are reduced to primary and secondary amine functionalities.

45. The method of claim 44, wherein the reduction of the amide functionalities in step e) is performed using a reducing agent selected from the group consisting of borane, arsenic trioxide in aqueous alcoholic HCI, antimony pentoxide in aqueous alcoholic HCI, LiAIH , H₂0₂, BF₃Εt₂0 in the presence of sodium borate, lithium tri-(tert)-butoxyaluminium hydride (UAIH(0tBu)₃), DIBAL-H, NaBH₄, NaBH₃CN and NaH.

46. Use of the polymer matrix according to any of claims 1 to 43 for a support for the synthesis of an organic molecule.

47. Use of the polymer matrix according to any of claims 1 to 43, for scavenging excess acyl compounds from a composition comprising a mixture of molecular entities.

48. Use of the polymer matrix according to any of claims 1 to 43, for scavenging excess carbonyl compounds from a composition comprising a mixture of mo- lecular entities.

49. Use of the polymer matrix according to any of claims 1 to 43, for solid phase enzyme reactions.

50. Use of the polymer matrix according to any of claims 1 to 43, as a support for the chemical and/or enzymatic synthesis of a peptide, a protein, a DNA, a RNA or an oligosaccharide.

51. Use of the polymer matrix according to any of claims 1 to 43, for protein immobilization.

52. Use of the polymer matrix according to any of claims 1 to 43, for chromatographic separation or purification.

53. The use of claim 52, wherein the chromatographic separation or purification comprises at least one step employing affinity purification.

54. Use of the polymer matrix according to any of claims 1 to 43, as a support for combinatorial chemistry.

55. Use of a partially acryloylated bisamino poly(alkylene)glycol in the preparation of the beaded, cross-linked polymer matrix according to any of claims 1 to 43.

56. The use of claim 55, wherein the preparation comprises the step of inverse suspension polymerization.

57. A composition comprising a plurality of cross-linked polymer beads according to any of claims 1 to 42.

58. The composition according to claim 57, wherein the average diameter of the beads is in the range of from 0.1 μm to preferably less than 3000 μm.

59. A functional surface comprising the polymer matrix according to any of claims 1 to 43, and attached thereto at least one functional moiety.

60. The functional surface according to claim 59, wherein the functional moiety is a bioactive species preferably selected from a scaffold moiety comprising at least one site for functionalization, a RNA moiety, a DNA moiety , a peptide moiety, or an amino acid residue.

61. The functional surface according to claim 59 or 60, wherein said surface is attached to a solid support.

62. The functional surface according to claim 59 to 61 , wherein the said surface is planar, tubular, spherical or a porous material.

63. The functional surface according to claim 60 to 62, wherein said surface further comprises a linker residue, preferably a linker residue comprising functional groups consisting of amino, alkylamino, hydroxy, carboxyl, mercapto, sulfeno, sulfino, sulfo, and derivatives of these.

64. A method for preparing a functional surface according to any of claims 59 to 63, said method comprising the steps of

a) providing a macromonomer comprising a bisamino poly(alkylene)glycol functionalized with at least one fragment comprising a vinyl group,

b) mixing the conjugated vinyl macromonomer with acrylic amide deriva- tives

c) polymerizing the vinyl groups of said macromonomers using radical initiators or ionic initiators,

d) forming a beaded, cross-linked polymer matrix comprising a plurality of amide functionalities,

e) reducing the amide functionalities and obtaining a beaded polymer matrix wherein the majority of the amide functionalities are reduced to primary and secondary amine functionalities, and

f) contacting the beaded polymer matrix obtained in step e) with at least one functional moiety, and

g) obtaining the functional surface.

65. A method for targeting a functional moiety attached to a functional surface, said method comprising the steps of

a) providing a functional surface according to any of claims 59 to 63, and

b) targeting said functional moiety with at least one targeting species having a noncovalent affinity, for said functional moiety, or

c) targeting said functional moiety with at least one targeting species forming a covalent bond with the said functional moiety.

66. A method for identifying and/or purifying a targeting species having an affinity for a functional moiety, said method comprising the steps of

a) providing a functional surface according to any of claims 59 to 63, and

b) targeting said functional moiety with at least one targeting species having an affinity for said functional moiety, and

c) identifying and/or purifying the at least one targeting species having an affinity for said functional moiety.

67. A targeting species identified and/or purified by the method of claim 66.

68. A method for therapy of a human or animal body, said method comprising the step of administering to said human or animal body a targeting species according to claim 67 in a pharmaceutical effective amount.