WO1998055533A1

WO1998055533A1 - Zwitterionic compounds and their use to cross-link collagenous materials

Info

Publication number: WO1998055533A1
Application number: PCT/GB1998/001646
Authority: WO
Inventors: Richard Neil Templar Freeman; Simon William Leppard; Jeremy Colin Russell
Original assignee: Biocompatibles Limited
Priority date: 1997-06-04
Filing date: 1998-06-04
Publication date: 1998-12-10
Also published as: AU7781498A; ZA984836B

Abstract

Cross-linking of collagenous material may be achieved by treatment with compounds of the formula (I) Xm(R1)(CHO)¿n?, wherein R?1¿ is an organic group having n+m functionality, X is a zwitterionic pendant group having an overall neutral charge, m is at least 1 and n is at least 2 (or a gem-diol, hemiacetal or acetal thereof). This results in the pendant amine groups of lysine residues present on the surface of the collagenous material to undergo a condensation reaction with the aldehyde groups present in formula (I) to form an imine linkage, thus fixing the biological moieties. The incorporation of the zwitterionic groups, suitably phosphoryl choline derivatives may have significant benefits in extending the life and reducing calcification of collagenous materials. One specific compound is 3-(oxyethyl-2'-(trimethylammoniumethyl)phosphate)-pentan-1,5-dial.

Description

ZWITTERIONIC COMPOUNDS AND THEIR USE TO CROSS-LINK COLLAGENOUS MATERIALS

The present invention relates to the synthesis of phosphate compounds and to their use to form biocompatible materials, especially to react with proteinaceous materials to cross link and biocompatibilise them.

It is known that compounds having phosphoryl choline groups and other zwitterionic groups, have useful biocompatibilising properties. Coatings of polymers with pendant phosphoryl choline groups have been shown to be useful as coatings to render blood contacting devices non-thrombogenic. Contact lenses formed from hydrogel polymers with pendant PC groups are less subject to protein deposition, lipid deposition and bacterial adhesion than contact lenses having similar water contents.

We have also described a variety of reagents which are useful for derivatising preformed surfaces, for instance of polymeric substrates, to introduce PC groups. Such reagents are generally mono functional, that is each reagent molecule includes a single PC group and a single reactive group. Examples of some such reagents are described in EP-A-0157469, EP-A-0515895 andEP-A-0556216. In EP-A-0515895 reagents which are capable of reacting with amino groups at surfaces to give amine linkages are described. InEP-A-0556216 compounds which react with surface amino groups include activated amine groups.

In WO-A-9301221 we described copolymers of ethylenically unsaturated PC group containing monomers and copolymerisable comonomers selected so as to give suitable surface binding characteristics. One class of comonomers includes a reactive group by which covalent bonding to an underlying surface may be carried out. Examples of covalent reactive groups include an aldehyde group.

It has been reported that PC may have significant benefits in extending the life and reducing calcification of tissue valves, although the means by which PC had been incorporated into such heart valve was not disclosed.

Monomers for use in forming condensation polymers, such as polyesters and polyurethanes have been described. For instance in EP-A-0199790 and EP-A-0275293 , monomers comprising two hydroxyl groups are used to react with di-isocyanates and dicarboxylic acids respectively to form polyurethanes and polyesters. Processes for synthesising dialdehyde compounds have been described. For instance ribose derivatives when reacted with sodium periodate are oxidised at the 3 and 4 hydroxyl groups with the C₃-C₄ bond being cleared to generate two aldehyde groups.

In US-A-4203893 (& EP-A-000T 197) cytidine diphosphocholine, which comprises a ribose moiety, is periodated and the dialdehyde reaction product reacted with compounds having free primary amine groups, such as polypeptides or proteins comprising lysine moieties or amino polysaccharides. The diphosphocholine moiety is zwitterionic but has an overall anionic charge (it is not neutral). In EP-A-0140640 inositol phosphate dialdehyde derivative is reacted with an amine containing hemoglobin derivative.

In J.C.S. Perkin II (1976) 1162-1165 Astin, K. et al describe phosphate derivatives of various terpene derivatives, including some cyclic alkenes and acyclic alkadienes, for solvolysis. Biological tissues can be "fixed" by allowing them to soak for some minutes in a dilute solution of glutaraldehyde. The glutaraldehyde is used to cross link proteins by their amine groups. Lysine residues in the proteins are the most common source of these amine groups. The aldehyde and amine undergo a condensation reaction to form an imine with the subsequent loss of water. This process is described further in Cheung, D. T. and Nimni, M. E., Connective Tissue Research, 10, 187-216 (1982).

The major constituent of bioprosthetic implants is the protein collagen. To increase the stability of such implants against biodegradation and to reduce the antigenicity, collagen is cross-linked with glutaraldehyde. The problem is that, after implantation, calcification onto the implant gradually occurs, rendering it necessary to replace the implant. The present inventors have established that a PC derivative having two aldehyde groups, or aldehyde group precursors, is usefiil to replace glutaraldehyde in tissue fixing.

The present inventors have synthesised novel functional phosphate diester compounds, which are suitably phosphoryl choline derivatives, which can be used to replace glutaraldehyde to fix tissue and have devised a synthetic method for producing such compounds. According to the invention there is provided a new process in which a zwitterionic crosslinker of the formula I

X-. (R¹) (CHO)_n I in which X is a zwitterionic pendant group having an overall neutral charge, R¹ is an organic group having n+m functionality, m is at least 1 and n is at least 2, or a gem-diol, hemiacetal or acetal derivative thereof, is contacted in aqueous solution with a substrate having pendant primary amine groups, under conditions allowing reaction of the CHO groups of the compound of the formula I with the primary amine groups of the substrate.

It is generally preferred for the reaction conditions to be such that all of the aldehyde groups (or acetal derivative thereof) react with primary amine groups. Thus the conditions under which the compound of the formula I is contacted with the substrate having pendant amine groups will include lower than stoichiometric amounts of the compound of the formula I. Alternatively, where, following contact of the compound of the formula I with substrate having pendant amine groups results in a product having residual aldehyde groups, the treated substrate may be subjected to a post-treatment step in which residual aldehyde groups are reacted with amine group containing compounds in solution.

Preferably the compound of the formula I is used to replace glutaraldehyde in a tissue fixing process. Thus the proteinaceous substrate preferably comprises primarily coUagenous material, of which the lysine residues are reacted with the aldehyde groups.

The substrate is preferably proteinaceous, and is most preferably a coUagenous substrate. The process of the invention provides crosslinking between pendant amine groups of lysine moieties of the substrate.

The coUagenous material may be skin, connective tissue, bone or the organic matter of teeth. Tissues which may usefully be treated in the invention include heart valves tissue and vessels, veins and arteries, ligaments, tendons and Farcia lata, dura matter, pericardium, nerves and cornea implants.

According to the present invention there is also provided crosslinked proteinaceous materials produced by the process of the present invention. The use of crosslinking compounds incorporating a zwitterionic pendant group has been found to produce crosslinked coUagenous products which retain good mechanical properties and are expected to have reduced tendency to calcification.

The group X may be a betaine group, for instance a sulpho-, carboxy- or phospho-betaine. A betaine group must have no overall charge and is preferably therefore a carboxy- or sulpho-betaine. If it is a phosphobetaine the phosphate terminal group must be a diester, ie be esterified with an alcohol. Such groups may be represented by the general formula II

-X³-R²⁴-N⁺(R²⁵)₂-R²⁶-V II

in which X³ is a valence bond, -O-, -S- or -NH-, preferably -O-;

V is a carboxylate, sulphonate or phosphate (diester-monovalently charged) anion; R²⁴ is a valence bond (together with X³) or alkylene -C(O)alkylene- or -

C(O)NHalkylene preferably alkylene and preferably containing from 1 to 6 carbon atoms in the alkylene chain; the groups R²⁵ are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms or the groups R²⁵ together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 atoms; and

R²⁶ is alkylene of 1 to 20, preferably 1 to 10, more preferably 1 to 6 carbon atoms provided that when V is a sulphonate anion, R²⁶ is alkylene of 6 or more carbon atoms. One preferred sulphobetaine monomer has the formula III

R¹⁶

I

-*N-(CH₂)_d-SO₃ ^e III

_R I I6

where the groups R¹⁶ are the same or different and each is hydrogen or C_M alkyl and d

Preferably the groups R¹⁶ are the same. It is also preferable that at least one of the groups R¹⁶ is methyl, and more preferable that the groups R¹⁶ are both methyl. Preferably d is 2 or 3, more preferably 3.

Alternatively the group X may be an amino acid moiety in which the alpha carbon atom (to which an amine group and the carboxylic acid group are attached) is joined through a linker group to the group R¹. Such groups may be represented by the general formula IV

NR²⁸ ₃

-X⁴. rv

^•R^{27 H} "C0₂

in which X⁴ is a valence bond, -O-, -S- or -NH-, preferably -O-, R²⁷ is a valence bond (optionally together with X⁴) or alkylene, -C(O)alkylene- or -C(O) Halkylene, preferably alkylene and preferably containing from 1 to 6 carbon atoms; and the groups R²⁸ are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or two of the groups R²⁸, together with the nitrogen to which they are attached, form a heterocyclic ring of from 5 to 7 atoms, or the three group R²⁸ together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring. X is preferably of formula V

oΘ

in which the moieties X¹ and X², which are the same or different, are -O-, -S-, - NH- or a valence bond, preferably

-O-, and W⁺ is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C^- alkylene group.

Preferably W contains as cationic group an ammonium group, more preferably a quaternary ammonium group. The group W⁺ may for example be a group of formula -W^!-N⁺R²³ ₃, -W'-P⁺R²³^ -W'-S⁺R²³^ or -W'-Het^* in which:

W¹ is alkylene of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W¹ optionally contains one or more fluorine substituents and/or one or more functional groups; and either the groups R²³ are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R²³ together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R²³ together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R²³ is substituted by a hydrophilic functional group, and the groups R^23a are the same or different and each is R²³ or a group OR²³, where

R²³ is as defined above; or

Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.

Preferably W¹ is a straight-chain alkylene group, most preferably 1,2-ethylene. Preferred groups X of the formula V are groups of formula VI:

where the groups R¹² are the same or different and each is hydrogen or C alkyl, and e is from 1 to 4.

Preferably the groups R¹² are the same. It is also preferable that at least one of the groups R¹² is methyl, and more preferable that the groups R¹² are all methyl.

Preferably e is 2 or 3, more preferably 2. Alternatively the ammonium phosphate estergroup VI may be replaced by a glycerol derivative of the formula VB, VC or VD defined in our earlier publication no WO-A-93/01221.

The compound of the formula I may be a small, non-polymeric compound. Such compounds may have more than one pendant group X, that is m may be more than 1, although more usually m is 1. In such compounds, although n may be more than 2, it is generally found that adequate crosslinking takes place, as for glutaraldehyde, where there are two aldehyde groups per molecule, that is n is 2. In such non-polymeric compounds, the group R¹ is generally a C₂.₁₂-n+m-functional optionally substituted alkane group. The group R¹ may be an alkyl or an aralkyl group and may be interrupted by heteroatoms such as oxygen atoms, amido and/or sulphonamido, groups, or by carbonyl groups and may be substituted by alkyl, alkoxy, aryl, aralkyl, aralkoxy, hydroxyl or alkylamido groups, or halogen atoms. Generically any substituents should have no ionic charge, under aqueous conditions at around pH 7, eg in the range 6-8 preferably 4-10. Most preferably R¹ is a C₃.₂₄-alkyl group, especially a C₃.₁₂-alkyl group.

Preferably the compound of the formula I is a compound of the formula VII

in which X is as defined above; R² is hydrogen or a C -alkyl group, each of the groups R³ and R⁴ is independently selected from hydrogen, halogen, optionally substituted Cι.₂₄-alkyl, and hydroxyl groups, or two groups R³ or two groups R⁴ or one group R³ and one group R⁴ together represent an optionally substituted C^- alkylene, an optionally substituted C₂._g-alkenylene or an optionally substituted C₂.₈-alkynylene group, or two groups R³ or two groups R⁴ attached to adjacent carbon atoms may, together with the carbon atoms to which they are attached form a 1 ,2-arylene group, p is 1-4; q is 1-4;

R⁵ and R⁶ are each independently selected from hydrogen and C₁.₄-alkyl, each R⁷ is the same and is hydroxyl or C^-alkoxy, each R⁸ is the same and is selected from hydroxyl and C^ alkoxy, or the groups R⁷ and R⁸ attached to the same carbon atom together are =O or C_j. -oxaalkyleneoxy, and

R⁹ is a bond or a C^ alkylene group.

Compounds of the formula VII are thus dialdehyde compounds or the corresponding gem-diol, hemiacetal or acetal derivatives. Gem-diols, hemiacetal and acetal compounds are precursors to aldehydes in that they can react easily to form the corresponding aldehydes. These compounds are believed to be novel and form a further aspect of this invention. p and q are preferably each 1. Thus the group R¹ is preferably a linear C3 alkane derivative, in which the CHO groups are joined at the 1 and 3-positions whilst the zwitterionic group is joined through the 2 position.

Preferably each group R³ and R⁴ is independently selected from hydrogen, optionally substituted C^-alkyl, hydroxyl, protected hydroxyl and amine.

Preferred substituents in alkyl groups R³ and R⁴ are hydroxyl, amino and halogen. Preferably each of R³ and R⁴ is unsubstituted C,.₄-alkyl or hydrogen, most preferably hydrogen.

Preferably R⁵ and R⁶ each represent hydrogen.

The novel compounds may be made by a process in which a compound of the formula VIII

in which a) either R¹⁰ is CR¹² ₂ and

R¹¹ is CR⁵=CR¹² ₂ or b) R¹⁰ and R^π together are CR⁵; and in which each R¹² is selected from hydrogen and C alkyl; R⁵ and R⁶ are each hydrogen; and

R³, R⁴, R⁹, X, q and p have the same meanings as in the compound of the formula VII, is oxidised to form the dialdehyde compound upon oxidation at the double bond(s). The dialdehyde compound may be reacted with a water, an alcohol or a glycol to form the corresponding gem-diol, hemiacetal or acetal, that is in which each of R⁷ and R⁸ represents hydroxyl or alkoxy. Such gem-diols, hemiacetals and acetals are found to be relatively stable to long term storage.

Compounds of the formula VIII believed to be novel are further claimed in our copending application filed even date herewith claiming priority from GB 97303847.4.

The oxidation reaction of the compound of the formula VIII to form the compounds of the formula VII has been found to produce stable products, where there are 3 carbon atoms between the groups R⁶C(R⁷)R⁸ and R⁵C(R⁷)(R⁸) (ie q=p=l ), or where there are more than 3 carbon atoms and the groups R⁶C(R⁷)R⁸ and R⁵C(R⁷)R⁸ are kept apart by steric means to prevent an intramolecular alcohol condensation taking place.

Where q+p>2, it is preferred for one group R³ and one group R⁴ together to represent a C_j.₂, preferably -alkylene, usually methylene. The dialdehyde product of the oxidation of such compounds is resistant to an internal aldol condensation and is relatively storage stable in the form of the gem-diol, hemiacetal or acetal derivative. The oxidation reaction may be carried out using an appropriate oxidising agent, such as ozone in conjunction with hydrogen and a palladium-carbon catalyst, with zinc and acetic acid, with iodide and acetic acid, with dimethyl sulphide, with thiourea, with triphenylphosphine, with trimethylphosphite, or with pyridine or periodate, for instance in the presence of a catalyst such as osmium tetroxide. Other oxidising agents known to generate aldehyde groups or precursors thereof from ethylenically unsaturated starting materials are potassium permanganate, sodium periodate with potassium permanganate catalyst, with ruthenium (III) chloride or ruthenium (VI) dioxide catalyst, (bi py)H₂CrOCl₅ and potassium permanganate and silica gel. These systems are further described in Comprehensive Organic Transformations, by Larock, R. C, VCH, 1989, pp595-596.

The oxidation reaction is preferably carried out with the compound of the formula VTII in solution, for instance in water or an organic solvent, or a mixture thereof. Suitable organic solvents are alcohols, dimethyl formamide or glacial acetic acid.

The oxidation reaction may be carried out with cooling. The reaction is generally carried out to completion, using an excess of oxidising agent.

The novel starting material of the formula VIII may be made from known starting materials by various routes. In the synthetic process the zwitterionic group X is introduced into the molecule prior to oxidation of the ethylenic bond(s), using suitable chemistry. The synthesis of sulphobetaine monomer may proceed by the reaction of a precursor having a reactive group and a preformed sulphobetaine group with an ethylenically unsaturated alcohol, amine or carboxylic acid. Alternatively a sulphobetaine may be formed by reaction of a tertiary amine with a 1,3 propane sulfone.

Amino acid type compounds may be made by processes analogous to those described in our earlier application no WO-A-9416749. Where, as in the preferred aspect of the invention, the zwitterionic group is a group of formula V in which W⁺ is W¹ N⁺ R²³ ₃ which W¹ is C₂ or C₃-alkylene (optionally substituted) and at least two of the groups R²³ are methyl, the compound of the formula VIII may be formed by the reaction of a compound of the formula IX

in which the groups R³, R⁴, R⁶, R⁹, R¹⁰, R¹¹ have the same meanings as in the compound of the formula Vm, X¹ and X² are as defined in the group of formula V is reacted of the compound with a phospholane reagent of the formula II 11

in which Hal is a halogen atom, preferably chlorine,

R¹³ is a bond or a group C(R¹⁴)R¹⁵, each group R¹⁴ is selected from hydrogen and C_M-alkyl groups; each group R¹⁵ is selected from hydrogen and C,.₄-alkyl, or two groups R¹⁵ may form a Cι.₅-alkylene group to produce a phospholane intermediate of the formula X

in which all the groups have the same meanings as in the respective compounds of the general formulae IX and X. Subsequently the intermediate of the formula X is subjected to a ring opening amination with an amine NR²³ ₃ reaction to produce the compound of the formula VIII.

In the phospholane reagent of the formula X, the groups R¹⁴ and R¹⁵ are preferably all the same, each preferably being hydrogen.

Where R⁵ is N⁺R⁶ ₃, the ring opening reaction is carried out under anhydrous conditions. Preferably each of the groups R²³ is methyl. This basic phospholane ring opening reaction has been described by Thuong and Chabrier in Bull. Soc. Chim. de France (1974) (3-4) 667-670 and in FR-A-2,270,887. The compound of the formula VII is preferably used as a crosslinking reagent for reacting with substrates having pendant primary amine groups, especially proteinaceous substrates. In this application of the compound of the formula VII, it is preferred for the reaction to be conducted in an aqueous environment. The invention is illustrated in the following examples:

Example 1 - Synthesis of 3-Methoxy-fethyl-2'-(trimethylammonium ethyl) phosphate) - hexan-1.6-dial

1.1: Synthesis of 3-Cvclohexen- 1 -methoxyethyl-2'-(trimethylammonium ethyl) phosphate 1.1 To a solution of +/- 3 -cylcohexen-1 -methanol (2.60g, 0.023mole) in dry acetonitrile (60ml) and triethylamine (4. OOg, 0.039mole) at - 10 ° C, a solution of 2-chloro-

2-oxo-l,3,2-dioxaphospholane (CCP) (4.37g, 0.030mole) in acetonitrile (15ml) was slowly added. The reaction mixture was allowed to warm to RT over a period of 1 h.

The solids were removed by filtration, and the solution added to a flask containing trimethylamine (2.4g, 0.041mole) in acetonitrile (50ml). The resulting mixture was heated at 50 °C overnight. The solvents were removed in vacuo to afford a viscous oil

(7.10g, 0.025mole), contaminated by slight amounts of trimethylamine. hydrochloride, and acetonitrile:

Η NMR (199.5MHZ, CDC1₃), 1.2 (1H, m, CH), 1.6-2.2 (6Η, m, 3xCH₂), 3.43 (9Η, s, NMe₃), 3.74 (2H, m, CH₂OP), 3.8-4.6 (4Η, m, OCH₂-CH₂-N), 5.65 (2Η, s, CH=CH).

¹³C NMR (50.1MHz, D₂O), 23(CHCH₂CH₂CH=), 25(CHCH₂CH₂CH=), 27(=CHCH₂CH), 33(CH), 53(NMe₃), 59(POCΗ₂), 65(CH₂N), 70(CH₂O), 125(=CHCH₂CH), 127(CHCH₂CH₂CH=).

An analogous process can be carried out using in place of the 3-cyclohexene-l- methanol, 3 -methyl-2-cyclohexen- 1 methanol or 2-cyclohexen- 1 -ol or 3 , -methyl 5 , 5 tri-2- cyclohexen- 1 -methanol . 1.2: Synthesis of 3-Methoxy-(ethyl-2'-ftrimethylammonium ethyl) phosphate) -hexan-

1.6-dial

1.2.1 - Osmium tetroxide.

To a solution of 3-cyclohexen-l-methoxyethyl-2'-(trimethylammonium ethyl) phosphate (2.771g, 0.0099M) in 1,4-dioxane / water (50ml, 1:1, v/v), a grain of osmium tetroxide ( lmg) was added, and the solution stirred for 30 min. Sodium periodate (6.727g, 0.030mole) was slowly added over 30 min and the reaction stirred for 6 h. The solid material was filtered off, and the solvent removed in vaccuo to give a white solid. The solid was dissolved in ethanol (50ml) and again filtered. The solution was concentrated to give a viscous oil (3.03g, 0.0097mole) still containing some solvent and sodium iodide salts:

ΗNMR (199.5MHz, D₂O), 1.3-1.9 (7H, m, CH₂CH₂CHCH₂), 3.23 (9Η, s, NMe₃), 3.7-

4.3 (6H, CH₂OPOCH₂CH₂N), 9.7(2Η, CH=O).

¹³C NMR (50.1MHz, D₂O) 30.8, 45.2, 53.4(ref NMe₃), 59.0, 60.3, 65.6, 66.7, 206.1.

IR(KBr) 3401, 2946, 1716, 1670, 1479, 1398, 1220, 1040, 971, 875, 765, 666, 502.

1.2.2 -Ozone. Ozone was bubbled through a solution 3-cyclohexen-l-methoxyethyl-2'-

(trimethylammonium ethyl) phosphate (7.52g, 0.027mole) in ethanol (200ml) at -78 °C, for 5 h. Triphenylphosphine (14. Og, 0.053mole) was added to quench the reaction mixture, whereupon the reaction darkened from colourless to yellow, and then a fine white precipitate was observed. The reaction mixture was stirred for 48 h. The reaction mixture was filtered, and the solvents removed in vacuo. Dissolution of the resulting oil proved problematic, and 'Η NMR analysis of the reaction mixture showed a complex mixture of signals indicating that there may be decomposition of the starting material. Example 2

Synthesis of 3-(Oxyethyl-2'-(trimethylammonium ethyl) phosphate)-pentan-l. 5-dial 2.1-2.2: Synthesis of 3-Cvclopenten-l-ol

2.1 To a stirred solution of cyclopentadiene (57.8g, 0.87mole), and sodium carbonate (400g) in dichloromethane ( 1000ml) at 0 ° C, peracetic acid ( 168ml, 40% in acetic acid), pre-treated with sodium acetate (0.2g) was added slowly, and the reaction allowed to stir for 4 h. The reaction mixture was filtered, and the solvent distilled off. [Η NMR (199.5MHz, CDC1₃) 2.0-3.0(m, CH-O-CH), 3.8(d, CH₂), 6.1(m, CH=CH)]

2.2 The residue was slowly added to a slurry of lithium aluminium hydride (13.0g, 0.36mole) in diethyl ether (400ml) at 0°C, and stirred overnight. The reaction was quenched by slow addition of water (50ml), and then after 10 min dried magnesium sulphate was added (50-70g). The solids were filtered off, and then washed with diethyl ether (2x200ml). The organic layers were combined, and the solvent was removed carefully in vacuo (no heating). 3-Cyclopenten-l-ol (20.38g, 0.243mole) was isolated from the mixture by distillation (27°C, ca 754 mmΗg).

ΗNMR (199.5MHz, CDC1₃) 1.9(1H, br.s, OH), 2.2-2.8(4Η, qd, CH₂CHCH₂), 4.5(1Η, br.m, CHOH), 5.7(2H, s, CH=CH).

¹³C NMR (50.1MHz, CDC1₃) 42.0 (CH₂), 10.8 (CHOH), 122.4(C=Q.

2.3: Synthesis of 3-Cvclopenten-l-oxyethyl-2'-(^"trimethylammonium ethyl) phosphate

To a solution of 3-cyclopenten-l-ol (8.04g, 0.091mole), and N,N,N,N- tetramethylethylenediamine (6.33g, 0.055mole) in acetonitrile (160ml) at -10°C, a solution of 2-chloro-2-oxo-l,3,2-dioxaphospholane (15.6g, 0.073mole) in acetonitrile (50ml) was added, and the reaction allowed to warm to RT over 1.5 h. The reaction mixture was filtered, and then trimethylamine (12.68g, 0.21 mole) was added. The reaction was heated at 50° C in a closed system for 18 h. The reaction was cooled, and solvents removed in vacuo. The reaction mixture was taken up into water, and washed with chloroform (2x 100ml) . The aqueous layer was concentrated to yield 3 -cyclopenten- l-oxyethyl-2'-(trimethylammonium ethyl) phosphate (15.3g, O.Oόlmole) as an oil.

¹HNMR(199.5MHz,D₂O) 2.1-2.4(4H, m, 2x-CH₂-), 3.0(9Η, s, NMβ_j), 3.3-4.1(5H, m, CHOP, OCH₂CH₂N), 5.5(2Η, s, CH=CH).

¹³C NMR (50.1MHz, D₂O) 39.5(CH₂), 53(ref NMe₃), 58.5&60.0, 65.0&66.5(OCH₂CH₂N), 75.5(CHOP), 127.2(C=C).

m/s (FAB⁺) 336(M^+«NMe₃ ^«H₂O), 250(M⁺), 185

Ir (KBr smear) 3401, 2959, 2510, 1653, 1483, 1220, 991, 769, 668, 510.

2.4: Synthesis of 3-(Oxyethyl-2 Wtrimethylammonium ethyl) phosphateVpentan - 1.5-dial (ethoxyacetalderivative)

To a solution of 3-cyclopenten-l-oxyethyl-2'-(trimethylammonium ethyl) phosphate (2.43g, 0.0097mole) in water (100ml), a grain of osmium tetroxide ( lmg) was added, and the reaction stirred at RT for 30 min. Sodium periodate (5.43g, 0.0254mole) was added slowly, and the reaction stirred at RT for 3 h, during which a colour change to dark orange, and then back to colourless was observed. Ethanol

(200ml) was added to quench the reaction, and the precipitate was filtered off. The solvent was removed in vacuo to afford quantitative yield of 1, 1,5,5 -tetraethoxy-3-

(oxyethyl-2'-(trimethylammonium ethyl) phosphate)-pentane (based on ¹³C NMR), i.e. the ethoxy acetal derivative of the dialdehyde.

¹H NMR (199.5MHz,D₂O) 1.1-2.0(4H, m, 2xCH₂), 3.0(9Η, s, NMe₃), 3.3-4.2(7H, m,

CHOP, OCH₂CH₂N, CH(O(D))₂).

¹ CNMR(50.1MHz, D₂O) 17(CΗ₃CΗ₂O), 35&37(-CH₂-), 53(refNMe₃), 57(CH₃CH₂O), 58&60(CH₂OP), 65&66(CH₂N), 70(CHOP), 90(CH(OEt)₂).

IR (KBr smear) 3401, 2505, 2360, 1653, 1481, 1218, 1085,971,759,513. Example 3 - Tissue interaction

3.1 Tissue Preparation:

The tissue was stored in phosphate buffer solution (PBS) for 3 hours and then washed in fresh PBS prior to cutting. The tissue was cut into strips of 1 cm by 6 cm.

Eighteen strips was used for the fixation solutions and the rest stored in PBS, in the fridge, as control samples. A small sample of tissue from each of the three solutions was investigated by Differential Scanning Calorimetry (DSC).

3.2 Preparation of Fixation solutions: The fixation solutions of 0.35 %w/v glutaraldehyde and 1.03 %w/v 3 -[(oxy)( 1,1,1 trimethyl ammonium) phosphoranate]- pentan-1, 5-dial (Glut-PC) in PBS was prepared as follows -

A. Glutaraldehyde solution contains 0.35% w/v glutaraldehyde and 154mM sodium chloride and has a pH in the range 7.0 to 7.5. B. Glut-PC solution contains 1.03% w/v Glut PC (equimolar with glutaraldehyde),

154mM sodium chloride and 5.26mM trisodium phosphate for adjusting the pH to be in the range 6.5 to 7.0.

Two sets of 9 strips of tissue were placed into vials containing the control fixation solution (45mls) or the test solution. Each vial was then stored inside a brown-glass bottle for 96 hours at 25 °C.

3.3 Method for testing the Cross-linked tissue:

A modified method of testing the shrinkage temperature of leather (BSI 3144: 1968 Sampling and Physical Testing of Leather) was used to test the tissue.

The three types of tissues were suspended in a beaker containing a solution of PB S with a thermometer placed at close proximity to the tissue. The solution of PB S was heated in water at a constant power to provide a temperature rise of approximately 2 °C/min from 40 °C to 90 °C. The change in physical appearances of the samples was recorded prior to testing and recorded in Table 1. Also a thermometer positioned close to the sample was used to measure the temperature rise with time. Inflexion points in the curve were recorded and are reported in Tables 2-4. The results of the DSC test is reported in Table 5. Results

Physical Appearance prior to testing:

Table 1

Experimental Results: Experiment 1

Table 2 Starting temperature - 40°C Final Temperature - 89°C

Experiment 2

Table 3

Starting temperature - 36.5°C Final temperature - 88.5°C

Experiment 3 :

Table 4 Starting temperature - 36°C Final Temperature - 86°C

DSC Experimentation- Conditions - Sealed pans containing samples.

Heating 25°C - 100°C at 10°C/minute. Table 5

The results show that the PC group containing compound performs differently to glutaraldehyde. The physical properties of the treated tissue appear to be more similar to those of the untreated tissue, which is believed to be desirable.

Example 4

4.1 Synthesis of 5-|^"(oxy)-( 1.1.1 trimethyl ammonium) phosphoranate methyl] -bicyclo f2.2.11 hept-2-ene.

To a solution of 5-norbornene-2-methanol (1.00 g, 0.0079 mol) and N,N,N\N'- tetramethylethylenediamine (0.46 g, 0.0038 mol) in acetonitrile (30 ml) at 0 °C, a solution of 2-chloro- 1 ,3 ,2-dioxaphospholane-2-oxide (1.13 g, 0.0079 mol) in MeCN (10 ml) was slowly added over 20 minutes under N₂. The solution was stirred for 4h. The solution was filtered and cooled to 0 °C. Trimethylamine (1.17 g, 0.0198 mol) was added and the solution heated at 50 °C overnight in a closed system. The solution was cooled, degassed, and the solution decanted. The solvent was evaporated and the residue partitioned between H₂O (20 ml) and Et₂O (20 ml). The aqueous layer was washed with Et₂O (20 ml), separated and evaporated. The yellow oil was identified as the product: Η NMR (400 MHz, D₂O) δ complex spectra.

¹³C NMR (50.1 MHz, D₂O) δ 27.0 (CH₂CH(CH₂O)), 38.0 (CHCH₂OP), 41.8 (C(CH₂)CH), 43.5 (C(CH₂)CH₂), 47.8 (CH₂ bridge), 54.0 (M&N), 58.1 (CH₂NMe₃), 64.8 (CH₂OP), 68.5 (CHCH₂OP), 131.1 (CHCH, trans), 135.4 (CHCH, cis), 136.2 (CHCH, cis) 136.7 (CHCH, trans).

4.2 Synthesis of 4-[Yoxy (l.l.l trimethyl ammonium) phosphoranate methyll- cyclopentan-1. 3-dicarbaldehvde. A 5% solution of 5-[(oxy)-(l , 1 , 1 trimethyl ammonium) phosphoranate methyl] -bicyclo [2.2.1] hept-2-ene (1.00 g, 0.0035 mol) in H₂O (20 ml) was subjected to a stream of O₃

(0.0017 mol/min, 2 min.) at 5 °C. Triphenyl phosphine (1.00 g, 0.0038 mol) was added and stirred for lh at room temperature. The solution was filtered, frozen and the water removed by freeze-drying.

Η NMR (400 MHz, D₂O) δ complex spectra.

¹³C NMR (50.1 MHz, D₂O) δ 29.3 (CH₂CH(CH₂O)), 41.2 (CHCH-OP), 43.5

(C(CH₂)CH), 44.7 (C(CH₂)CH₂), 51.1 (CH₂ bridge), 54.0 (M&N), 59.5 (CH₂NMe₃),

61.2 (CH₂OP), 66.0 (CHCH₂OP), 97.4 & 101.4 (CH(OH)₂).

DNPH test - yellow precipitate formed (positive result).

Proton and carbon nmr are consistent with the following proposed reaction scheme

Analogous methods to those of Example 4.1 may be used to make the following cycloalkene compounds

From these dialdehydes may be formed using techniques analogous to Example

4.2. Example 5

5.1 Synthesis of 1.6-heptadien-4-ol:

To a suspension of activated magnesium ribbon [activated overnight by stirring in an inert atmosphere] (19.6 g, 0.82 mol) and anhydrous diethyl ether [Et₂O] (307 ml), an initiating solution of allyl bromide (10%, 9.0 g, 0.074 mol) in Et₂O (13 ml) was added. Once the reaction had started to reflux gently, the remainder of the allyl bromide (81.0 g, 0.646mol) in Et₂O( 119 ml) was slowly added maintaining a gentle reflux. The reaction was left refluxing for 2h. The mixture was cooled in an ice bath and a solution of ethyl formate (24.77 g, 0.33 mol) in Et₂O (53 ml) was slowly added to the stirred mixture over lh. The reaction was left to warm to room temperature overnight. The reaction was quenched with saturated ammonium chloride solution (260 ml), and H₂O then added (250 ml). The aqueous layer was acidified with dilute (0.1 M) hydrochloric acid and the organic layer separated. The organic layer was washed with NaHCO₃ (300 ml), H₂O (200 ml), andNaCl (200 ml). The organic layer was dried with anhydrous sodium sulphate and the solvent removed. The resulting pale yellow oil was identified as a mixture of the alcohol and the formate ester (approximately 5:2 ratio):

Η NMR (400 MHz, CDC1₃) δ 1.90 (1H, m, CHOH), 2.20 (2H,m, CHCH₂), 2.35 (2H, m, CHCH₂), 3.70 (1H, m, CHOH), 5.15 (4H, m, 2xCHCH₂), 5.75 (2H, m, 2xCHCH₂), 8.05 (1H, s, OC(H)-O-CH). ¹³C NMR (50.1 MHz, CDC1₃) δ 37.8 (CH₂), 41.2 (CH₂), 69.7 (CHOH), 72.4 (CHO- COH), 118.1 (CHCH₂), 118.3 (CHCH₂), 133.0 (CHCH₂), 134.6 (CHCH₂), 160.7 (OC(H)-O-CH).

5.2 Synthesis of 4-[foxy)( 1,1.1 trimethyl ammonium) phosphoranate]- 1.6-heptadiene. To a solution of l,6-heptadien-4-ol (10.00 g, 0.089 mol) and N,N,N',N'- tetramethylethylenediamine (5.18 g, 0.045 mol) in acetonitrile [MeCN] (100 ml) at 0 °C, a solution of 2-chloro-l,3,2-dioxaphospholane-2-oxide (12.72 g, 0.089 mol) in MeCN (60 ml) was slowly added over 20 minutes under N₂. The solution was stirred for 4h. The solution was filtered and cooled to 0 °C. Trimethylamine (13.17 g, 0.223 mol) was added and the solution heated at 50 °C overnight in a closed system. The solution was cooled, degassed, and the solution decanted. The solvent was evaporated and the residue partitioned between H₂O (150 ml) and dichloromethane [DCM] (150 ml). The aqueous layer was washed with DCM (150 ml), separated and evaporated. The residue was washed with acetone (2x 150 ml) and stored in acetone (100 ml) overnight. The acetone layer was evaporated to leave a solid residue. The solid residue was identified as the product: Η NMR (400 MHz, D₂O) δ complex spectra.

¹³C NMR (50.1 MHz, D₂O) δ 43.5 (CH₂), 54.0 (M&NCH,), 55.5 (CH₂NMe₃), 59.8 (CH₂OPO), 65.7 (CHOP), 118.1 (CHCH₂), 134.2 (CHCH₂).

5.3 Synthesis of 3-|Yoxy)(l.l.l trimethyl ammonium) phosphoranate]- pentan-1.5- dial. A 10% solution of 4-[(oxy) (1,1,1 trimethyl ammonium) phosphoranate]- 1,6- heptadiene (2.00 g, 0.0073 mol) in H₂O (20 ml) was subjected to a stream of O₃ (0.0017 mol min, 10 min.) at 5 °C. Triphenyl phosphine (4.75 g, 0.018 mol) was added and stirred for lh at room temperature. The solution was filtered, frozen and the water and formaldehyde removed by freeze-drying. Η NMR (400 MHz, D₂O) δ complex spectra.

¹³C NMR (50.1 MHz, D₂O) δ 43.8 (CH₂), 54.0 (M&NCH,), 55.6 (CH₂NMe₃), 60.0

(CH₂OPO), 65.8 (CHOP), 91.8 & 92.7 (CH(OH)₂).

DNPH test - orange precipitate formed. [Formaldehyde produces a bright yellow precipitate] The reaction is believed to take place according to the following scheme:

Example 6

6.1 Synthesis of 3. 5 -dimethyl- 1.6-heptadien-4-ol:

To a suspension of activated magnesium ribbon [activated overnight by stirring in an inert atmosphere] (6.22 g, 0.26 mol) and anhydrous tetrahydrofuran [THF] (110 ml), an initiating solution of 3 chloro-1-butene (10%, 2.0 g, 0.02 mol) in THF (6 ml) and a crystal of iodine were added. Once the reaction had started to reflux gently, the remainder of the 3 chloro- 1 -butene ( 18.0 g, 0.199 mol) in THF (51 ml) was slowly added maintaining a gentle reflux. The reaction was left refluxing for 2h and heated further at 70 °C for 1 h. The mixture was cooled to room temperature and a solution of ethyl formate (7.38 g, 0.10 mol) in THF (20 ml) was slowly added to the stirred mixture over lh. The reaction was left overnight. The reaction was quenched with saturated ammonium chloride solution (270 ml). The organic layer was separated and washed with NaHCO3 (275 ml) and brine solution (250 ml). Ethyl acetate (300 ml) was added and organic layer separated. The organic layer was treated with charcoal, filtered with celite and the solvent removed. The resulting yellow oil was believed to be a mixture of the three types of alcohol and their ester derivatives:

Η NMR (400 MHz, CDC1₃) complex spectra, δ 0.9 - 1.10 (3H, m, CHMe), 1.60 (3H, d, MeCH=), 2.20 (2H,m, CHCH₂), 2.35 - 2.55 (2H, m, CHCH₂), 3.25 - 3.70 (1H, m, CHOH), 5.00 - 5.15 (4H, m, 2xCHCH₂), 5.65 - 5.85 (2H, m, 2xCHCH₂ and MeCHCH), 8.05 - 8.15 (1H, s, OC(H)-O-CH).

¹³C NMR (50.1 MHz, CDC1₃) complex spectra, δ 15 - 20 (CH₃), 38.0 - 41.5 (CH₂ and CHMe), 75 - 80 (CHOH and CHO-COH), 114 - 118 (CHCH₂ and CHCHMe), 138 - 141 (CHCH₂ and CHCHMe), 160.7 (OC(H)-O-CH).

6.2 Synthesis of 4-[(oxy)-( 1.1.1 trimethyl ammonium) phosphoranate] -3. 5-dimethyl- 1.6-heptadiene.

To a solution of 3, 5-dimethyl-l,6-heptadien-4-ol (2.00 g, 0.014 mol) and N,N,N',N' -tetramethylethylenediamine (0.83 g, 0.007 mol) in acetonitrile [MeCN] (20 ml) at 0 °C, a solution of 2-chloro-l,3,2-dioxaphospholane-2-oxide (2.04 g, 0.14 mol) in MeCN (25 ml) was slowly added over 20 minutes under N₂. The solution was stirred for 4h. The solution was filtered and cooled to 0 °C. Trimethylamine (3.10 g, 0.053 mol) was added and the solution heated at 50 °C overnight in a closed system. The solution was cooled, degassed and the solution decanted. The solvent was evaporated and the residue partitioned between H₂O (125 ml) and ether [Et₂O] (130 ml). The aqueous layer washed with Et₂O (130 ml), separated and evaporated. The residue was identified as a phosphorous species, but without the dialkene. Η NMR (400 MHz, D₂O) δ complex spectra. ^I3C NMR (50.1 MHz, D₂O) δ 54.0 (M&NCH,), 55.0 (CH₂NMe₃), 62.2 (CH₂OPO).

The product contained several isomers and was not further treated. The product could be separated into the individual isomers and be subjected to ozonolysis to form analogous dialdehydes to those of example 5, for instance having the formula

Analogous methods may be used to make the following PC dialdehyde compounds

Claims

1. A process in which a zwitterionic crosslinker of the formula I x. (R¹) (CHO)_n I in which X is a zwitterionic pendant group having an overall neutral charge, R¹ is an organic group having n+m functionality, m is at least 1 and n is at least 2, or a gem-diol, hemiacetal or acetal derivative thereof, is contacted in aqueous solution with a substrate having pendant primary amine groups, under conditions allowing reaction of the CHO groups of the compound of the formula I with the primary amine groups of the substrate.

2. A process according to claim 1 in which the substrate comprises protein, preferably comprising collagen.

3. A process according to claim 1 or claim 2 in which the zwitterionic group comprises a phosphate group as the anion and a quaternary ammonium group at the cation.

4. A process according to claim 1 or claim 2 in which X has the general formula N

in which the moieties X¹ and X², which are the same or different, are -O-, -S-, -

╬¥H- or a valence bond, preferably -O-, and W⁺ is a group comprising an ammonium, phosphomum or sulphonium cationic group having a group linking the anionic and cationic moieties which is a C ₂ alkylene group optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, optionally containing one or more fluorine substituents.

5. A process according to claim 4 in which W⁺ is a group of formula -W¹-

╬¥⁺R²³ ₃, -W^P⁺R²³³,, -W'-S⁺R²³", or -W'-Hef in which: either the groups R²³ are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms or aryl, or two of the groups R²³ together with the heteroatom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R²³ together with the heteroatom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R²³ is substituted by a hydrophilic functional group, and the groups R²³" are the same or different and each is R²³ or a group OR²³, where R²³ is as defined above; or

Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring.

6. A process according to claim 5 in which X is a group of formula VI:

where the groups R¹² are the same or different and each is hydrogen or C alkyl, and e is from 1 to 4, preferably in which e is 2 or 3 and each R¹² is methyl.

7. A process according to any preceding claim in which m is 1.

8. A process according to any preceding claim in which R¹ is a C₃.₂₄, preferably a C₃.₁₂-alkyl group.

9. A process according to any preceding claim in which the compound has the general formula VII

in which X is as defined in any of claims 1 and 3 to 6 R² is hydrogen or a C₁.₄-alkyl group, each of the groups R³ and R⁴ is independently selected from hydrogen, halogen, optionally substituted C₁.₂₄-alkyl, and hydroxyl groups, or two groups R³ or two groups R⁴ or one group R³ and one group R⁴ together represent an optionally substituted

alkylene, an optionally substituted C₂._g-alkenylene or an optionally substituted C₂.₈-alkynylene group, or two groups R³ or two groups R⁴ attached to adjacent carbon atoms may, together with the carbon atoms to which they are attached form a 1,2-arylene group, p is 1-4; q is 1-4; R⁵ and R⁶ are each independently selected from hydrogen and C,.₄-alkyl, each R⁷ is the same and is hydroxyl or C^-alkoxy, each R⁸ is the same and is selected from hydroxyl and C^ alkoxy, or the groups R⁷ and R⁸ attached to the same carbon atom together are =O or . ₄-oxaalkyleneoxy, and R⁹ is a bond or a C^ alkylene group.

10. A process according to claim 9 in which m and n are both 1 , R³ and R⁴ are selected from hydrogen and C_M alkyl and R⁵ and R⁶ are each hydrogen.

11. A compound of the general formula VII

X is a zwitterionic group having an overall neutral charge;

R² is hydrogen or a C^-alkyl group, each of the groups R³ and R⁴ is independently selected from hydrogen, halogen, optionally substituted C^-alkyl, and hydroxyl groups, or two groups R³ or two groups

R⁴ or one group R³ and one group R⁴ together represent an optionally substituted C₁.₈- alkylene, an optionally substituted C₂.₈-alkenylene or an optionally substituted

C₂._g-alkynylene group, or two groups R³ or two groups R⁴ attached to adjacent carbon atoms may, together with the carbon atoms to which they are attached form a 1 ,2-arylene group, p is 1-4; q is 1-4; R⁵ and R⁶ are each independently selected from hydrogen and C,.₄-alkyl, each R⁷ is the same and is hydroxyl or C^-alkoxy, each R⁸ is the same and is selected from hydroxyl and C_M alkoxy, or the groups R⁷ and R⁸ attached to the same carbon atom together are =O or C_╬╗.

₄-oxaalkyleneoxy, and R⁹ is a bond or a C^ alkylene group.

12. A compound according to claim 11 in which p and q are both 1, R³ and R⁴ are selected from hydrogen and C ₄ alkyl and R⁵ and R⁶ are each hydrogen.

13. A compound according to claim 11 or claim 12 in which the zwitterionic group comprises a phosphate group as the anion and a quaternary ammonium group as the cation.

14. A compound according to claim 11 or claim 12 in which X has the general formula V

in which the moieties X¹ and X², which are the same or different, are -O-, -S-, - NH- or a valence bond, preferably -O-, and W⁺ is a group comprising an ammonium, phosphonium or sulphonium cationic group having a group linking the anionic and cationic moieties which is a C^ alkylene group optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, optionally containing one or more fluorine substituents.

15. A compound according to claim 14 in which W⁺ is a group of formula -W'-N⁺R²³ ₃, -W'-P⁺R²³*,, -W'-S⁺R²³^ or -W^l-Het⁺ in which W¹ is C_2. ₆-alkylene and either the groups R²³ are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, or aryl, or two of the groups R²³ together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R²³ together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R²³ is substituted by a hydrophilic functional group, and the groups R^23a are the same or different and each is R²³ or a group OR²³, where R²³ is as defined above; or

16. A compound according to claim 15 in which X is a group of formula VI:

where the groups R¹² are the same or different and each is hydrogen or C^ alkyl, and e is from 1 to 4, preferably in which e is 2 or 3 and each R¹² is methyl.

17. A process in which a compound of the formula VIII

in which a) either R¹⁰ is CR¹² ₂ and R^u is CR⁵=CR¹² ₂ or b) R¹⁰ and R¹¹ together are CR⁵; and in which each R¹² is selected from hydrogen and C_M alkyl; X is a zwitterionic group having an overall neutral charge; each of the groups R³ and R⁴ is independently selected from hydrogen, halogen, optionally substituted C^-alkyl, and hydroxyl groups, or two groups R³ or two groups R⁴ or one group R³ and one group R⁴ together represent an optionally substituted C_{l g}- alkylene, an optionally substituted C₂.₈-alkenylene or an optionally substituted C₂.₈-alkynylene group, or two groups R³ or two groups R⁴ attached to adjacent carbon atoms may, together with the carbon atoms to which they are attached form a 1,2-arylene group, p is 1-4; q is 1-4;

R⁹ is a bond or a C^ alkylene group.

18. A process according to claim 17 in which R¹⁰ is CR¹² ₂ and R¹¹ is CR =CR¹² ₂ and each R¹² is hydrogen.

19. A process according to claim 17 in which R¹⁰ and R¹¹ together are CR⁵.

20. A process according to any of claims 17 to 19 in which p and q are both 1, and R³ and R⁴ are selected from hydrogen and C-.₄ alkyl.

21. A process according to any of claims 17 to 20 in which the zwitterionic group comprises a phosphate group as the cation and a quaternary ammonium group as the cation.

22. A process according to any of claims 17 to 20 in which X has the general formula V

NH- or a valence bond, preferably -O-, and W⁺ is a group comprising an ammonium, phosphonium or sulphonium cationic group having a group linking the anionic and cationic moieties which is a C ₂ alkylene group optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, optionally containing one or more fluorine substituents.

23. A process according to claim 22 in which W⁺ is a group of formula -W^l-N⁺R² , -W'-P⁺R²³";., -W'-S⁺R²³*, or -W'-Hef in which W¹ is C₂. ₆-alkylene and either the groups R²³ are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, or aryl, or two of the groups R²³ together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R²³ together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R²³ is substituted by a hydrophilic functional group, and the groups R^23a are the same or different and each is R²³ or a group OR²³, where R²³ is as defined above; or Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring.

24. A process according to claim 23 in which X is a group of formula VI:

^ΓÇó( (CCIH-M₂)e-.NNRR^1,2:3 VI ╧î╬ÿ

where the groups R¹² are the same or different and each is hydrogen or C alkyl, and e is from 1 to 4, preferably in which e is 2 or 3, and each R¹² is methyl.

25. A process according to any of claims 17 to 24 which is carried out using an oxidising agent selected from ozone in conjunction with hydrogen and a palladium- carbon catalyst, with zinc and acetic acid, with iodide and acetic acid, with dimethyl sulphide, with thiourea, with triphenylphosphine, with trimethylphosphite, or with pyridine or periodate, for instance in the presence of a catalyst such as osmium tetroxide, or potassium permanganate, sodium periodate with potassium permanganate catalyst, with ruthenium (III) chloride or ruthenium (VI) dioxide catalyst, (bi py)H₂CrOCl₅ and potassium permanganate and silica gel, preferably ozone with triphenylphosphine.

26. A process according to claim 24 including steps for the formation of the compound of the formula VIII by the reaction of a compound of the formula IX

in which the groups R³, R⁴, R⁶, R⁹, R¹⁰, R^╧Ç have the same meanings as in the compound of the formula VIII, X¹ and X² are as defined in the group of formula V is reacted of the compound with a phospholane reagent of the formula II

in which Hal is a halogen atom, preferably chlorine,

R¹³ is a bond or a group C(R¹⁴)R¹⁵, each group R¹⁴ is selected from hydrogen and C_M-alkyl groups; each group R¹⁵ is selected from hydrogen and C^-alkyl, or two groups R¹⁵ may form a Cj.j-alkylene group to produce a phospholane intermediate of the formula XI

in which all the groups have the same meanings as in the respective compounds of the general formulae IX and X, and in which the intermediate of the formula XI is subjected to a ring opening amination with an amine NR²³ ₃ reagent to produce the compound of the formula VIII.

27. Crosslinked proteinaceous materials produced by a process in which a zwitterionic crosslinker of the formula I x. (R¹) (CHO)_n I in which X is a zwitterionic pendant group having an overall neutral charge, R¹ is an organic group having n+m functionality, m is at least 1 and n is at least 2, or a gem-diol, hemiacetal or acetal derivative thereof, is contacted in aqueous solution with a substrate having pendant primary amine groups, under conditions allowing reaction of the CHO groups of the compound of the formula I with the primary amine groups of the substrate.

28. A product according to claim 27 in which the substrate comprises protein, preferably comprising collagen.