The invention relates to processes of making comb polymers from monomers comprising alkoxy polyethers, such as polyalkylene glycol such as poly (ethylene glycol), or polytetrahydrofuran (PTHF). Such methods may include the use of an initiator compound which comprises a moiety which, when attached to the comb polymer, is capable of binding to a protein or polypeptide. The initiator compounds and finished comb polymers, and their uses, are also included within the invention.

The modification of proteins with polymers such as poly (ethylene glycol), which is known by the abbreviation PEG, is well-known in the art. PEG-derivatives are manufactured, for example, by Shearwater Corporation, Huntsville, AL., USA, and Enzon, Inc., Bridgewater, NJ., USA. Uses of PEG are reviewed in catalogues from both of those companies, and indeed in the 2002 Enzon, Inc. Annual Report.

The attachment of PEG to proteins or polypeptides, known as PEGylation has been found to have a number of benefits. Firstly, this reduces the antigenicity and immunogenicity of a molecule to which PEG is attached. PEG also produces markably improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and/or through evasion of cellular clearance mechanisms. PEG can markably improve the solubility of proteins and polypeptides to which it is attached, for example PEG has been found to be soluble in many different solvents, ranging from water to many organic solvents such as toluene, methylene chloride, ethanol and acetone. An application of this has been to use PEG-modified antibodies, for example to phase partition target molecules or cells. PEGylation has also been found to enhance proteolytic resistance of the conjugated protein, and improve bioavailability via reduced losses at subcutaneous injection sites. PEGylation also has been observed to reduce the toxicity of the proteins or polypeptides to which it is attached, improve thermal and mechanical stability of the molecules and allow the improved formulation into materials used for some slow release administration strategies. These advantages are reviewed in, for example, the articles by Chapman A.P. (Advanced Drug Delivery Reviews, Vol. 54 (2002), pages 531-545). The chemistry of polypeptide and protein PEGylation is further reviewed in the article by Roberts, M.J., et al. (Advanced Drug Delivery Reviews, Vol. 54 (2002), pages 459-476), and the article by Kinstler, O., et al. (Advanced Drug Delivery Reviews, Vol. 54 (2002), pages 477-485).

A number of PEGylated drugs are on the market, For example, PEG-INTRON™ is an α-interferon product produced by Schering-Plough and Enzon, Inc. which is used to treat hepatitis C and cancer. Prothecan™ is a PEG-enhanced version of camptothecin, a topoisomerase I inhibitor that is effective against some cancers. PEGylated taxol and several enzyme-based products have also been produced which show, for example, better uptake in tumours and reduced side-effects compared to non-PEGylated compounds. As discussed in the review by Roberts (Supra), polymers such as PEG may be attached via a number of reactive amino acids on protein or polypeptide molecules, including lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, N-terminal amino groups and C-terminal carboxylic acid groups. In the case of glycoproteins, vicinal hydroxyl groups can be oxidised with periodate to form two reactive formyl moieties. A wide range of functional groups may be attached to compounds such as PEG to allow them to attach to lysine amine groups and N-terminal amine groups. These include succinimidyl succinate, hydroxysuccinamide and hydroxysuccinamide esters, aldehyde derivatives such as propionaldehyde and acetaldehyde, propionate and butanoate derivatives of succinimidyl, benzotriazole carbonate, p-nitrophenyl carbonate, trichlorophenyl carbonate and carbonylimidazole. Compounds such as tresylate are known to bind to proteins via nucleophilic attack. There are also a number of compounds which can react with cysteine residues on proteins or polypeptides. These include maleimides, vinylsulphones, pyridyl sulphides and iodoacetamides. Furthermore, succinimidyl carbonate can also be used as a functionalised group to attach PEG or other polymers to alanine or histidine amino acids within a protein or polypeptide. As already indicated, the reaction of such functionalised groups is already well-characterised as indicated in the articles by Roberts, Kinsler and Chapman, and indeed as shown in, for example, the Shearwater Catalogue (2001). The PEG currently on the market is usually in the form of long poly (ethylene glycol) polymers or branched or star-shaped poly (ethylene glycols).

The Applicants have now identified that it is possible to produce comb polymers which allow the size of the polymer attached to biological substances, for example, proteins and polypeptides, nucleic acids (DNA and RNA), carbohydrates and fats, to be varied and to be controlled. This allows the possibility of producing a wide variety of different polymers for attaching to proteins and polypeptides, which may be varied in their size and hydrodynamic volume to vary the properties of the compound to which the polymer is attached. For example, this may be used to vary the stability, solubility, toxicity and/or drug retention time of a drug which has been covalently attached to such co-polymers. Such co-polymers are capable of being produced in a controlled manner by so-called living radical polymerisation.

Living radical polymerisation is subject of International Patent Application No. WO 97/47661. Supported polymerisation catalysts and specific polymerisation initiators are also shown in WO 99/28352 and WO 01/94424. Basically, the system uses a compound complexed with a transition metal. This compound is preferably an organodiimine, although one of the nitrogens of the diimine is preferably not part of an aromatic ring (e.g. a l,4-diaza-l,3-butadiene, a 2-pyridinecarbaldehyde imine, an oxazolidone or a quinoline carbaldehyde).

Living free radical systems, which involve the use of free radical initiators are also known, see for example WO 96/30421 and WO 97/18247. This is reviewed in Kamigaito, et al, Chem. Rev. (2001), Vol. 12, pages 3689-3745.

A combination of the catalyst and the initiators has in the past been used to polymerise olefinically un-saturated monomers, such as vinylic monomers. The inventors have now realised that these systems may be used to produce comb polymers in a controlled manner. These comb polymers may have a functional group attached to them via conventional chemistry. However, the inventors have also realised that the initiators used in living radical polymerisation are attached to the comb polymer as a result of the reaction of the initiator with the monomers. This means that it is possible to functionalise the comb polymer at the same time as producing the co-polymer, by using a functionalised initiator.

Accordingly, the first aspect of the invention provides a method of producing a comb polymer comprising the steps of:

(a) Providing: (i) a plurality of monomers which are linear, branched or star-shaped, substituted or non-substituted, preferably containing 2, especially from 3 to 10, carbon atoms, and have an olefinically unsaturated moiety attached thereto, the olefinically unsaturated moiety being capable of undergoing addition polymerisation; (ii) an initiator compound; the initiator compound comprising a homolytically cleavable bond; (iii) a catalyst capable of catalysing the polymerisation of the monomer; and (b) Causing the catalyst to catalyse, in combination with the initiator, the polymerisation of a plurality of the monomers to produce the comb polymer;

wherein the initiator compound (ii) comprises a moiety which, when attached to the comb polymer, is capable of binding to a biological substance.

The monomers in (i) are preferably alkoxy polyethers such as poly (alkylene glycol) or polytetrahydrofuran.

The comb polymer may have a moiety which, when attached to the comb polymer, is capable of binding e.g. a protein or polypeptide, attached to it using conventional chemistry. However, as already indicated, it is possible to produce initiator compounds which have that moiety attached to them. Therefore, preferably the initiator compound comprises a moiety which, when attached to a comb polymer, is capable of binding to a biological substance, such as a protein or polypeptide, nucleic acid (DNA or RNA), carbohydrates or fats. Preferably, the poly (alkylene glycol) is a polymer of an alkylene glycol containing from 2-10, especially at least 3, carbon atoms, most preferably poly (ethylene glycol), poly (propylene glycol) or poly (butylene glycol). For example, poly (ethylene glycol) may be used.

In its most common form, this is a linear or branched polyether terminated with hydroxyl groups. This is synthesised by anionic ring opening polymerisation of ethylene oxide initiated by nucleophilic attack of a hydroxide ion on the epoxide ring. It is also possible to modify polyethylene glycol, for example by placing a monomethoxy group on one end to produce monomethoxy PEG (mPEG). This is synthesised by an ionic ring opening polymerisation initiated with methoxide ions and is commercially available. However, trace amounts of water present in the reaction mixture causes the production of significant quantities of PEG which is terminated at both ends by hydroxy groups. This is undesirable, as the moiety capable of binding to proteins or peptides will then attach to both ends of the polymer chain, which will cause unwanted cross-linking of proteins in the body.

A method intended to minimise the production of this impurity is to initiate the ring opening of ethylene oxide by nucleophilic attack of a benzoxy ion on the epoxide ring. In a similar manner to the above process, monobenzoxy PEG is produced, as well as the PEG chain terminated at both ends by hydroxy. This mixture is methylated, producing one chain terminated with BzO and OMe, and dimethoxy PEG. Hydrogenation of this mixture eliminates the benzoxy group to yield mPEG and dimethoxy PEG. Dimethoxy PEG remains present as an inert impurity. However, even using this process, the product obtained still contains 5-10% of the unwanted dihydroxy PEG according to its certificate of analysis.

The process of the present invention yields a product which is substantially 100% pure, eliminating substantially all of the dihydroxy PEG impurity, thus avoiding the disadvantages of the known processes, and removing the possibility of the cross-linking of proteins. Branched and star-shaped polymers such as PEG are available from a number of commercial sources, such as Enzon and Shearwater. Polytetrahydrofurans may also be obtained from commercial sources, such as Aldrich (Gillingham, Dorset, UK.).

Preferably, the molecular weight of the PEGmethacrylate is 475, 1100, 2080, 5000 or 20,000.

The polyalkylene glycol and polytetrahydrofuran comprises an olefinically unsaturated moiety, for example at the end of the polymer chain. This olefinically unsaturated moiety is capable of undergoing additional polymerisation.

The olefinically unsaturated monomer may be a methacrylate, an acrylate, a styrene, methacrylonitrile or a diene such as butadiene.

Examples of olefinically unsaturated moieties that may be used include methyl methacrylate, ethyl methacrylate, propyl methacrylate (all isomers), butyl methacrylate (all isomers), and other alkyl methacrylates; corresponding acrylates; also functionalised methacrylates and acrylates including glycidyl methacrylate, trimethoxysilyl propyl methacrylate, allyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, dialkylarninoalkyl methacrylates such as dimethylethylamino methacrylate; fiuoroalkyl (meth)acrylates; methacrylic acid, acrylic acid; fumaric acid (and esters), itaconic acid (and esters), maleic anhydride: styrene, α-methyl styrene; vinyl halides such as vinyl chloride and vinyl fluoride; acrylonitrile, methacrylonitrile; glycerol; vinylidene halides of formula CH2 = C(HaI)2 where each halogen is independently Cl or F; optionally substituted butadienes of the formula CH2 = C(R15) C(R15) = CH2 where R15 is independently H, Cl to ClO alkyl, Cl, or F; sulphonic acids or derivatives thereof of formula CH2 = CHSO2OM wherein M is Na, K, Li, N(R16)4 where each R16 is independently H or C, to C10 alkyl, COZ, ON, N(Rl6)2 or SO2OZ and Z is H, Li, Na, K or N(R16)4; acrylamide or derivatives thereof of formula CH2 = CHCON(R16)2 and methacrylamide or derivative thereof of formula CH2 = C(CH3)CON(R15)2.

Mixtures of such monomers may be used. Such unsaturated moieties may be attached, for example, at an end of the polymer, by conventional chemistry. Alternatively, such monomers may be obtained commercially. For example, PEGacrylate, diacrylate, methacrylate and dimethacrylate are commercially available from Aldrich (Gillingham, Dorset, UK.).

The unsaturated moiety may be attached to the polyalkylene glycol or polytetrahydrofuran by means of any suitable linkage groups, for example via a methyl ether linkage. Hence, it is possible to use poly (ethylene glycol) methyl ether methacrylate (available from Aldrich Chemicals). One advantage of using the living radical polymerisation technique is that commercially available compounds such as this, which have free-radical inhibitors, such as hydroquinones, may be used without further purification. With conventional free-radical-based systems the presence of a free-radical inhibitor will inhibit the addition polymerisation reaction. This is not the case with living radical polymerisation.

The initiator compound may comprise a homolytically cleavable bond with a halogen atom. This may contain a bond that breaks without integral charge formation on either atom by homolytic fission. As described in WO 97/01589, WO 99/28352 and WO 01/94424, it is believed that true free-radicals do not appear to be formed using some catalysts. It is believed that this occurs in a concerted fashion whereby the monomer is inserted into the bond without formation of a discrete free-radical species in the system. That is, during propagation this results in the formation of a new carbon-carbon bond and a new carbon-halgen bond without free-radical formation. A free-radical which is an atom or group of atoms having an unpaired valance electron and which is a separate entity without interactions, is not produced by the interaction of the initiator compound with the monomer with which it interacts. Suitable initiator compounds are described in, for example, WO 97/47661. However, it is preferable that the initiator compound also comprises a moiety which, when attached to the comb polymer, is capable of binding to a protein or polypeptide. These moieties are known in the art, as indeed described in Roberts, et al. (Supra), Chapman (Supra) and, for example, in the catalogues of Enzon and Shearwater.

The initiator may be a thioester or xanthate. These are used in so-called RAFT (Reversible Addition Fragmentation chain transfer and nitric oxide mediated polymerisation) and MADIX catalysation. The initiators and their reactions are described in WO 99/31144, WO 98/01478 and US 6,153,705.

Preferably, the initiator compound (ii) is selected from:

A-S-C(O)-R, A-S-C(S)-O-R, R-S-C(O)-A, R-S-C(S)-O-A, where R is C, to C20 substituted or non-substituted, straight chain, branched chain, cyclic, heterocyclic or aromatic alkyl;

A-B-X A

cozBA coaB A

Σ

where: X = a halide, especially Cl or Br, A = a moiety which, when attached to the comb polymer, is capable of binding to a protein or polypeptide, B is a linker and may or may not be present.

A is preferably selected from succinimidyl succinate, N-hydroxy succimimide, succinimidyl propionate, succinimidyl butanoate, propionaldehyde, acetaldehyde, tresylate, triazine, vinylsulfone, benzotriazole carbonate, maleimide, pyridyl sulfide, iodoacetamide and succinimidyl carbonate.

The linker is preferably selected from a Ci to C20 substituted or non-substituted, straight chain, branched chain cyclic, heterocyclic or aromatic alkyl group; -(CH2Z)3 CH2-, -CH2ZCH2-, -(CH2CH2Z)n-R, -(CH2CH(CH3)Z)n-R, -(CH2)b-C(O)-NH-(CH2)c-, -(CH2)a-NH-C(O)-(CH2)y-, -N(R)2-; -S-; -N-R; or -O-R; where R = C1 to C20 substituted or non-substituted, straight chain, branched chain cyclic, heterocyclic or aromatic alkyl, Z is O or S, and n, a, b and c are independently selectable integers between 1 and 10. Preferably, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Most preferably, the linker is methyl, ethyl, propyl, butyl or pentyl.

Preferably, the moiety which is capable of reacting with the protein or polypeptide has the formula:

o o -0-C-CHjCH1-C-O-N''

where n = integer of 0 to 10

integer of 0 to 10, Y is an aliphatic or

aromatic moiety

O Il -NHCCH1I

X' where R1 is H, methyl, ethyl, propyl or butyl, X is a halide, especially Cl or Br.

Most preferably, the initiator (ii) has a formula:

n is an integer of 0 to 10, and X is a halide, especially Cl or Br.

The initiator has a compound selected from:

The catalyst may be capable of catalysing the polymerisation reaction by living radical polymerisation (see e.g. WO 97/47661) or living free radical polymerisation (see e.g. WO 96/30421, WO 97/18247 and Kamagaito M., et al., Chem. Rev. (2001), Vol. 101 (12), pages 3689-3745).

Preferably the catalyst comprises a ligand which is any N-, O-, P- or S- containing compound which can coordinate in a δ-bond to a transition metal or any carbon-containing compound which can coordinate in a π-bond to the transition metal, such that direct bonds between the transition metal and growing polymer radicals are not formed.

The catalyst may comprise a first compound

MY

where: M is a transition metal having an oxidation state which is capable of being oxidised by one formal oxidation state,

Y is a mono, divalent or polyvalent counterion.

The catalyst may also be defined by the formula:

[MLm]"+ A""

where: M = a transition metal having an oxidation state which is capable of being oxidised by one formal oxidation state,

L = an organodiimine where at least one of the nitrogens of the diimine is not part of an aromatic ring,

A = anion, n = integer of 1 to 3,

m = an integer of 1 to 2.

The metal ion may be attached to a coordinating ligand, such as (CH3 CNV Y may be chosen from Cl, Br, F, I, NO3, PF6, BF4, SO4, CN, SPh, SCN, SePh or triflate (CF3 SO3). Copper (I) triflate may be used. This is available in the form of a commercially available benzene complex (CF3SO3CUhCoHe.

The especially preferred compound used is CuBr.

A may be F, Cl, Br, I, N, O3, SO4 or CuX2 (where X is a halogen).

The transition metal may be selected from Cu+, Cu2+, Fe2+, Fe3+, Ru2+, Ru3+, Cr2+, Cr3+, Mo2+, Mo3+, W2+, W3+, Mn3+ Mn4+, Rh3+, Rh4+, Re2+, Re3+, Co+, Co2+, V2+, V3+, Zn+, Zn2+, Au+, Au2+, Ag+ and Ag2+.

Preferably the organodiimine has a formula selected from:

a l.4-diaza-1.3-butadiene

R* an oxazolidone.

or a quinoline carbaldehvde

where Ri, R?, Rio, Ru, Rn and R13 may be varied independently and Ri, R2, Rio, Rn, Ru and R13 may be H, straight chain, branched chain or cyclic saturated alkyl, hydroxyalkyl, carboxyalkyl, aryl (such as phenyl or phenyl substituted where substitution is as described for Rt to Rg) CEbAr (where Ar = aryl or substituted aryl) or a halogen. Preferably Ri , R2, Rio, Rii, Rn and Ri3 may be a Ci to C20 alkyl, hydroxyalkyl or carboxyalkyl, in particular C] to C4 alkyl, especially methyl or ethyl, n-propylisopropyl, n-butyl, sec-butyl, tert butyl, cyclohexyl, 2-ethylhexyl, octyl decyl or lauryl.

Preferred ligands include:

Formula 28 Formula ?Q π,,. ..,T- in Fo Formula 32 Formula 33

Formuia 34 Foπnula 35 Formula 36

Forrnυ in 37 Foπnula 38 Foπrrøtø 39

Foπnula 40 Foπnula 42

Formula 4-6 Foπnula 47

Foπnula Foπnula Formula

Formula 51 *\ where: * indicates a chiral centre cocW R14 = Hydrogen. C. to C10 branched chain alkyi. carboxy- or hydroxy- C1 to C10 alkyl. Freferably the catalyst is

Preferably the organodiimine is N-(n-propyl)-2-pyridylmethanimine (NMPI), N-ethyl-2- pyridyl methanimine or N-(n-ethyl)-2-pyridylmethanimine. Other catalysts are described in WO 96/30421 and WO 97/18247. Preferably the catalyst comprises a bipyridine group, such as 4,4'-di(5-nonyl)-2.2'-bipyridyl (dNbpy).

A plurality of different monomers as defined in part (i) of the invention may be used. This allows the production of statistical co-polymers.

Alternatively, or additionally, a block co-polymer may be produced by additionally polymerising one or more different olefinically unsaturated monomers. For example, the olefinically unsaturated monomers may be selected from methyl methacrylate, ethyl methacrylate, propyl methacrylate (all isomers), butyl methacrylate (all isomers), and other alkyl methacrylates; corresponding acrylates; also functionalised methacrylates and acrylates including glycidyl methacrylate, trimethoxysilyl propyl methacrylate, allyl methacrylate, hydroxyethyl methacrylate, hydroxypropyl methacrylate, dialkylaminoalkyl methacrylates; fiuoroalkyl (meth)acrylates; methacrylic acid, acrylic acid; fumaric acid (and esters), itaconic acid (and esters), maleic anhydride: styrene, α-methyl styrene; vinyl halides such as vinyl chloride and vinyl fluoride; acrylonitrile, methacrylonitrile; vinylidene halides of formula CH2 = C(HaI)2 where each halogen is independently Cl or F; optionally substituted butadienes of the formula CH2 = C(R15) C(R15) = CH2 where R15 is independently H, Ci to C)0 alkyl, Cl, or F; sulphonic acids or derivatives thereof of formula CH2 = CHSO2OM wherein M is Na, K, Li, N(RI6)4 where each R16 is independently H or Cl to ClO alkyl, COZ, ON, N(Ri6)2 or SO2OZ and Z is H, Li, Na, K or N(R16)4; acrylamide or derivatives thereof of formula CH2 = CHCON(RI6)2 and methacrylamide or derivative thereof of formula CH2 - C(CH3)CON(R1 %.

The monomers may be polymerised prior to or after the polymerisation of the monomers as defined in part (I) of the invention.

The polymerisation reaction may be reactive in a number of different solvents, such as hydrophobic or hydrophilic solvents. These include water, propionitrile, hexane, heptane, dimethoxyethane, diethoxyethane, tetrahydrofuran, ethylacetate, diethylether, N,N-dimethylformamide, anisole, acetonitrile, diphenylether, methylisobutyrate, butan-2-one, toluene and xylene.

The reaction temperature may be carried out from -20 to greater than 2000C, especially +5 to 13O0C. WO 97/47661, for example, shows examples of living radical polymerisation and the typical conditions that may be used.

Preferably, the ratio of organodiimine : transition metal is 0.01 to 1000, preferably 0.1 to 10, and transition metal ion (as MY) : initiator is 0.0001 to 1000, preferably 0.1 to 10, where the degree of polymerisation is controlled by the ratio of monomer to initiator. AU ratios are given as weight : weight. Preferably the components are the catalyst of formula: [ML1n ]n+A"' (defined above) are at a ratio of catalyst : initiator of 3 : 1 to 1 : 100.

Preferably, the amount of diimine : metal used in the system is between 100 : 1 and 1 : 1, preferably 5 : 1 to 1 : 1, more preferably 3 : 1 to 1 : 1, by weight.

Preferably the concentration of monomer in a solvent used is 100% - 1%, preferably 100% - 5%, vol. : vol.

Preferred ratios of initiator to catalyst or 1 : 100 - 100: 1 „ typically 1:1.

Preferred ratios of monomer : initiator are 1:1 to 10,000:1, especially 5:1 to 100:1.

The reaction may be undertaken under an inert atmosphere such as nitrogen or argon, and may be carried out in suspension, emulsion, mini-emulsion or in a dispersion.

Preferably, the catalyst is a supported catalyst, that is, at least a part of the catalyst is attached to a support. Such supported catalysts are shown in, for example, WO 99/28352.

The support may be inorganic, such as silica, especially silica gel. Alternatively, the support may be organic, especially an organic polymer, such as a cross-linked organic polymer, including poly (styrene-w-divinylbenzone). The support may be in the form of beads. The advantage of using a supported catalyst is that it allows the catalyst to be removed from the system and recycled/reused.

The comb polymer may incorporate a fluorescently-labelled monomer. For example, the method may additionally comprise a step of copolymerising or block polymerising with at least one fluorescently-labelled monomer capable of undergoing addition polymerisation. This can be carried out simply by using a monomer which has a fluorescent moiety, such as fluorescein, or coumarin, attached to an olefinically unsaturated moiety. The olefinically unsaturated moiety may be selected from those unsaturated moieties defined above.

Preferably, the fluorescent label is coumarin, especially coumarin 343. Coumarin is particularly advantageous because it allows the comb polymer to be used to attach to proteins and the attachment of the proteins to be visualised using a confocal microscope. This allows, for example, the detection of individual proteins or indeed the visualisation of whole bacterial or other cells. Indeed, initial results have indicated that bacterial cells can be readily visualised, using a comb polymer according to the invention, to attach to E.coli and Streptomyces cells.

A further aspect of the invention provides initiator compounds capable of being used in a living radical polymerisation reaction comprising a moiety which, when attached to a polymer, is capable of binding to a protein or polypeptide. Initiators for use in a living radical polymerisation reaction having the following formulae are also provided:

A-S-C(O)-R, A-S-C(S)-O-R, R-S-C(O)-A, R-S-C(S)-O-A, where R is C1 to C20 substituted or non-substituted, straight chain, branched chain, cyclic, heterocyclic or aromatic alkyl;

A-B-X

NN/ cαzBA

X

where: X = a halide, especially Cl or Br, A = a moiety which, when attached to the comb polymer, is capable of binding to a protein or polypeptide, B is a linker and may or may not be present.

Preferably, A is selected from succinimidyl succinate, N-hydroxy succimimide, succinimidyl propionate, succinimidyl butanoate, propionaldehyde, acetaldehyde, tresylate, triazine, vinyl sulfone, benzotriazole carbonate, maleimide, pyridyl sulfide, iodoacetamide and succinimidyl carbonate.

Preferably, the linker is selected from a Ci to C2o substituted or non-substituted, straight chain, branched chain cyclic, heterocyclic or aromatic alkyl group; -(CH2Z)3 CH2-, -CH2ZCH2-, -(CH2CH2Z)n-R5 -(CH2CH(CH3)Z)n-R, -(CH2)b-C(O)-NH-(CH2)c-, -(CH2VNH-C(OHCH2V3 -N(R)2-; -S-; -N-R; or -O-R; where R = C1 to C20 substituted or non-substituted, straight chain, branched chain cyclic, heterocyclic or aromatic alkyl, Z is O or S, and n, a, b and c are independently selectable integers between 1 and 10.

Preferably the moiety capable of reacting with a protein or polypeptide has a formula:

integer of 0 to 10

where m = integer of 0 to 10, Y is an aliphatic or

aromatic moiety

ϋ Il -KHCCH1I

where R' is H, methyl, ethyl, propyl or butyl, X is a halide, especially Cl or Br.

Preferably the initiator has a formula of:

where n is an integer of 0 to 10, and X is a halide, especially Cl or Br.

The initiator especially has the formula:

Boc

The terminal amine group may be protected by any suitable protecting group, such as BOC. Deprotection is achieved by addition of acid, such as trifluoroacetic acid. Alternatively a furan intermediate may be produced which can then be converted to maleimide.

Under normal conditions, the aldehyde-based initiators will tend to react non-selectively with proteins, i.e. they will react substantially equally with both terminal nitrogen atoms and, for example, a lysine NH2 group, if the reaction conditions are not controlled. However, under the right reaction pKa for the particular aldehyde chosen, the aldehyde can be controlled to specifically target the terminal nitrogen.

A further aspect of the invention provides comb polymers capable of binding a protein or polypeptide obtainable by a method of the invention.

A further aspect provides a comb polymer having a general formula:

A-(D)d-(E)e-(F)f

where: A may or may not be present, and where present is a moiety capable of binding to a

protein or a polypeptide,

D, where present, is obtainable by additional polymerisation of one or more

olefinically unsaturated monomers which are not as defined in E.

E is obtainable by additional polymerisation of a plurality of monomers which are

linear, branched, or star-shaped, substituted or non-substituted, and have an

olefinically unsaturated moiety.

F, where present, is obtainable by additional polymerisation of one or more

olefinically unsaturated monomers which are not as defined in E.

d and f are an integer between 0 and 500, especially 0 to 300 or 0 to 100.

e is an integer of 0 to 1000, especially 0 to 10, 50, 100, 200, 300, 400, 500, 600,

700, 800 or 900

and wherein when A is present, at least one of D, E and F is present.

Preferred monomers used to obtain E are poly (alkylene glycol) or polytetrahydrofuran. This includes both functionalised comb polymer and non-functionalised comb polymer,

where the moiety capable of attaching to a protein or polypeptide may be attached later by

other chemistry.

Preferably the comb polymer has an average total molecular weight of 2,000-80,000,

especially 20,000-40,000.

Examples of preferred comb polymers, obtainable according to the process of the

invention, are:

11 These polymers can be used either directly to react with useful biomolecules or converted simply into new macromolecules that will react with useful biomolecules.

The comb polymer may be fluorescently labelled, especially with a coumarin. A still further aspect of the invention provides a method of attaching a polymer to a compound comprising reacting a comb polymer according to the invention with said compound. The compound may be a protein or polypeptide or may indeed be any compound having a suitable free thiol or free amine group, depending on the initiator used. Such compounds include amines, such as benzylamines and ethylenediamine, amino acids and carbohydrates such as sugars.

Preferably such compounds are biologically-active compounds, such as drugs. The combination of such compounds in combination with a pharmaceutically acceptable carrier are also provided. The compounds may include cancer chemotherapeutic agents, antibiotics, anti-fungal and/or immunosuppressants.

For example, Figures 23 and 24 show HPLC traces and SDS-PAGE for the reaction of lysozyme with a polymer prepared according to the invention. These figures clearly illustrate the progress of the reaction as the polymer selectively conjugates to only one of lysozyme's seven amino groups.

A still further aspect of the invention provides a method of fluorescently labelling a compound, virus, microorganism or cell comprising the step of reacting the compound, virus, microorganism or cell with a fluorescently labelled comb polymer according to the invention. The use of a comb polymer as a fluorescent label is also provided.

The fluorescently labelled comb polymer may be used to attach antibodies which in turn may be used to selectively bind to pre-defined antigens. This allows the selective labelling of the compounds to take place.

Methods of producing such antibodies are well-known in the art and indeed monoclonal antibodies may be produced by the well-known Kohler-Milstein method. Previously, when polymers have been used to bind to proteins, they have had to be of a low molecular weight, as a polymer with a molecular weight of e.g. 20,000 could not be excreted from the body by the liver. To combat this problem, four polymers of approximately 5,000 molecular weight each were bound to the protein, and eventually excreted without problem. An advantage that is provided by the comb polymers of the invention is that they can possess molecular weights of 20,000 and still be bound to the proteins without the problems of excretion found with conventional polymers. This is due to an ester linkage which is found in each "finger" of the comb polymer. Preliminary results show that this ester linkage is readily hydrolysed by enzymes, causing the fingers to gradually break off the main polymer backbone. This enables a 20,000 molecular weight polymeer to become smaller over time until it reaches a molecular weight which enables it to be excreted by the liver. Conventional chain polymers cannot offer this advantage but remain in the bloodstream without being excreted.

Initial results indicate that the comb polymers of the invention are stable over weeks in rat serum, but slowly break down in the manner detailed above.

The invention will now be described by way of example only with reference to the following examples:

Figure 1 shows the evolution of molecular weight distribution and polydispersity for the LRP (living radical polymerisation) of methyl methacrylate initiated by a N-hydroxysuccinimide (NHS) initiator.

Figure 2 shows SEC curves for NHS functionalised poly (MMA), solid curve, and the produce (N-benzylamide functionalised poly (MMA), dashed curve).

Figure 3. First order kinetic plot for the LRP of PEGMA initiated by NHS-Br, [PEGMA]0/[CuBr]o/[NHSBr]0/[L]o = 10/1/1/2.1 in toluene (33% v/v) at 3O0C. Figure 4. Evolution of the molecular weight distribution and polydispersity for the LRP of PEGMA initiated by NHS-Br, [PEGMA]o/[CuBr]0/[NHSBr]0/[L]0 = 10/1/1/2.1 in toluene (33% v/v) at 3O0C.

Figure 5. Evolution of the molecular weight distribution and polydispeersity for the LRP of MPEG(395)MA initiated using initiator 8, [MPEG(395)MA]0/[CuBr]0/[NHSBr]0 = 10/1/1/2 in tuluene (50% v/v) at 3O0C.

Figure 6. Selected region (2.7-4.3 ppm) of the 1H NMR spectrum of a NHS ester functionalised ρoly(MPEG(395)MA) prepared from initiator 8 (Mn = 6400 g.mol"1, Mw/Mn - 1.09).

Figure 7. First order kinetic plot for the LRP of MPEG(395)MA using initiator 7, [MPEG(395)MA]o/[CuBr]0/[NHSBr]o/[Propyl Ligand]0 = 10/1/1/2 ub toluene (50% v/v) at 3O0C.

Figure 8. Evolution of the molecular weight distribution and polydispeersity for the LRP of MPEG(395)MA using initiator 7, [MPEG(395)MA]0/[CuBr]0/[NHSBr]0/[Propyl Ligand]o = 10/1/1/2 in toluene at 3O0C.

Figure 9. Rate plot for TMM-LRP of MPEG(IOOO)MA initiator 12, [monomer]: [initiator]: [CuCl]: [L] = 5:1:2:2, T = 7O0C.

Figure 10. Dependence of Mn on conversion for MPEG(IOOO)MA initiator 12, [monomer]: [initiator]: [CuCl]: [L] = 5:1:1:2, T = 7O0C.

Figure 11. Rate plot for TMM-LRP of MPEG(IOOO)MA initiator 12, [monomer]: [initiator]: [CuBr]: [L] = 20:1:1 :2, T = 5O0C.

Figure 12. Dependence of Mn on conversion for TMM-LRP of MPEG(IOOO)MA initiator 12, [monomer]: [initiator]: [CuBr]: [L] = 20:1:1:2, T = 5O0C. Figure 13. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 6:1:1:2.

Figure 14. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 6:1 :1:2.

Figure 15. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14, [monomer] : [initiator] : [CuBr] : [L] = 28 : 1 : 1 :2. T = 4O0C.

Figure 16. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 6:1:1:2. T = 4O0C.

Figure 17. Rate plot for TMM-LRP of MPEG(395)MA using initiator 14, [monomer] : [initiator] : [CuBr] : [L] = 28 : 1 : 1 :2. T = 6O0C.

Figure 18. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 6:1:1:2. T = 6O0C.

Figure 19. Online 1H NMR experiment: Rate plot for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 10:1 :1:2. T = 4O0C.

Figure 20. Rate plot for TMM-LRP of MPEG(395)MA using initiator 15, [monomer] : [initiator] : [CuBr] : [L] = 8 : 1 : 1 :2. T = 3O0C.

Figure 21. Dependence of Mn on conversion for TMM-LRP of MPEG(395)MA using initiator 15, [monomer]: [initiator]: [CuBr]: [L] = 8:1:1:2. T = 3O0C.

Figure 22. Kinetic plot for the hydrolysis of N-succinimidyl terminated poly(MPEG(395)MA initiated by 8 in different buffers. Figure 23. HPLC traces for the reaction of succinimide terminated poly(MPEG(395)MA) prepared from initiator 8 (Mn = 6400 g.mol"1, Mw/Mn = 1.11) with Lysozyme ([polymer] / [lysozyme] 20:1).

Figure 24. SDS-PAGE for the conjugation of lysozyme with succinimide terminated poly(MPEG(395)MA) prepared from initiator 8 (Mn = 6400 g.mol"1, Mw/Mn = 1.11) (20 equivalents).

Figure 25. HPLC traces for the reaction of succinimide terminated poly(MPEG(395)MA) prepared from initiator 8 (Mn = 6400 g-mol"1, M91Mn = 1.11) with Lysozyme ([polymer] / [lysozyme] 5:1).

Figure 26. HPLC traces for the reaction of succinimide terminated poly(MPEG(395)MA) prepared from initiator 8 (Mn = 6400 gjϋol"1, Mw/Mn = 1.11) with Lysozyme ([polymer] / [lysozyme] 2:1).

Figure 27. Comparison of the HPLC traces of various conjugates of lysozyme obtained with different ratios of polymer/lysozyme using succinimide terminated poly(MPEG(395)MA) prepared from initiator 8.

Figure 28. Kinetic plot for the hydrolysis of the succinimide end group of poly(MPEG(395)MA) polymer initiated by 7 in different buffers.

Figure 29. 1H NMR spectrum of a NHS ester functionalised (initiator 7) poly(MPEG(395)MA) (Mn = 2700 g.mol"1, Mw/Mn = 1.12).

Figure 30. 1H NMR spectrum of a N-benzylamide functionalised poly(MPEG(395)MA) (Mn = 2800 g.mol"1, Mw/Mn = 1.15).

Figure 31. HPLC traces for the reaction of poly(MPEG(395)MA) prepared from initiator 7 (Mn = 2700 g.mol"1, Mw/Mn = 1.12) with lysozyme ([polymer] / [lysozyme] 30:1). Figure 32. SDS-PAGE for the conjugation of ρoly(MPEG(395)MA) prepared from initiator 7 with lysozyme at different reaction time and different ratio polymer / protein (a) 5/1, (b) 10/1 and (c) 30/1.

Figure 33. SEC-HPLC chromatography of the conjugation reaction of Lysozyme with the aldehyde-terminated polymer (Mπ~22,000, PDi 1.09).

Figure 34. Retro-Diels-Alder reaction: (■ = "initiator" and ▲ = maleimido signals) a) t - 0; b) t = 3.5 h; c) t = 7 h.

Synthesis ofiV-[2-(2'-bromo-2'-methylpropionyloxy)-ethyl]phthalimide, 6.

N-(2-hydroxyethyl)phthalimide (Aldrich, 99%) (19.12 g, O.lmol) was dissolved in anhydrous THF (250 niL) with triethylamine (28.1 mL, 0.2 mol) under nitrogen in a 500 mL round-bottomed flask equipped with a magnetic stirrer. The flask was cooled to 0°C with an ice bath before the dropwise addition of 2-bromoisobutyryl bromide (13.9 mL, 0.11 mol). The mixture was stirred for 45 minutes and allowed to reach room temperature. Subsequently the reaction mixture was poured into an excess of cold water and extracted with diethyl ether (3 x 50 mL). The organic layer was washed with a saturated aqueous solution of Νa2Cθ3 (3 x 50 mL), acidified water (pH = 4.5, 3 x 50 mL), and again the saturated aqueous solution of Na2CO3 (3 x 50 mL). The organic layer was dried over anhydrous MgSO4 and filtered. Finally the solvent was removed under reduced pressure by using the rotary evaporator in order to isolate the title compound (30.6 g, yield 90 %) as a yellowish solid.

m.p. 63-65°C, IR (solid, ATR cell) v (cm"1) 1774 (Ccy0l=O), 1705 (C=O); 1H NMR (CDCl3, 298 K, 300MHz) δ 1.81 (s, 6H, C(CHB)2Br), 3.95 (t, 2H, J= 5.3 Hz, CH2N), 4.35 (t, 2H, J= 5.4 Hz, CH2O), 7.67 (m, 2H, CH Ar), 7.78 (m, 2H, CH Ar). 13C NMR (CDCl3, 298 K, 75 MHz) δ 31.00 (2C, C(CH3)2Br), 37.12 (1C, CH2N), 55.92 (1C, C(CH3)2Br), 63.42 (1C, CH2O), 123.78 (2C, CH Ar), 132.35 (1C, C Ar), 133.54 (2C, CH Ar), 168.40 (2C, Coycl=0), 171.87 (1C, C=O).

Synthesis of iV-(2-bromo-2-methylpropionyloxy) succinimide, 7.

This was prepared from iV-hydroxysuccinimide (NHS) using a similar procedure to that given above for the synthesis of compound 6. The solvent used in this case was anhydrous dichloromethane as NHS is insoluble in THF. The title compound was obtained in 85 % yield as a white solid.

m.p. 72-74°C; IR (solid, ATR cell) v (cm"1) 1772 (Ccycp0), 1728 (C=O); 1H NMR (CDCl3, 298 K, 300 MHz) δ 2.08 (s, 6H, C(CHa)2Br), 2.87 (s, 4Η, CH2). 13C NMR (CDCl3, 298 K, 75 MHz) δ 26.03 (2C, CH2), 31.09 (2C, C(CH3)2Br), 51.60 (1C, C(CH3)2Br), 167.89 (1C, C=O), 169.02 (2C, Ccyd=O); MS (+EI), (m/z) 266, 265, 156, 151, 149, 123, 121, 116, 115, 91, 87, 70, 69. Anal. Calcd for C8H10NO4Br: C = 36.39; H = 3.82; N = 5.30, Br = 30.26. Found: C = 36.35; H = 3.82; N = 5.03; Br = 30.17.

4-[(4-chloro-6-methoxy -l,3>5-triaziii-2-yI)amino]phenol, 4.

A solution of 2,4-dichloro-6-methoxy-l,3,5-triazine29 (9.00 g, 50.0 mmol) in 100 mL of acetone was cooled to 0 °C and, under stirring, solid 4-aminophenol (5.46 g, 50.0 mmol) was added in small portions over ca. 2 min. The white suspension was then left to warm to ambient temperature and stirred for further 1 h, whilst being neutralized with a 2 M aqueous solution OfNa2CO3. The mixture was then poured into 500 mL of ice/water and the resulting white precipitate was filtered and dried, to give 9.60 g (38.0 mmol, yield 76%) of 4-[(4-chloro-6-methoxy-l,3,5-triazin-2-yl)amino]phenol that can be used for the next step without further purifications. An analytical sample was obtained by flash chromatography (CC, SiO2, petroleum ether/ Et2O 1 :1, Rf = 0.14). The NMR analysis (Cfi-DMSO) revealed the presence, in solution, of 2 rotational isomers (molar ratio 7:3). m.p. 172 °C dec; IR v(NH)3476 cm'1. V(OH) 3269 cm"1. Major isomer 1H NMR (J6-DMSO, 298K, 300 MHz) δ 3.94 (s, 3H, OCH3); 6.79 (d, J = 8.8 Hz, 2H, CH Ar), 7.48 (d, J - 8.8 Hz, 2H, CH Ar), 9.40 (s, IH, OH), 10.46 (s, IH, NH); 13C(1H) NMR tø-DMSO, 298K, 75 MHz) δ 55.52 (1C, OCH3); 115.46 (2C, CH Ar), 123.91 (2C, CH Ar), 129.49 (1C, C Ar), 154.44 (1C, C Ar), 164.81 (1C, C Ar), 169.57 (IC5 C Ar), ,171.23 (1C, C Ar). Minor isomer 1H NMR tø-DMSO, 298K, 300 MHz) δ 3.96 (s, 3H, OCH3); 6.79 (d, J = 8.9 Hz, 2H, CH Ar), 7.39 (d, J= 8.9 Hz, 2H, CH Ar), 9.42 (bs, IH, OH), 10.10.32 (s, IH, NH); 13C(1H) NMR tø-DMSO, 298 K, 75 MHz) δ 55.10 (1C, OCH3); 115.46 (2C, CH Ar), 123.03 (2C, CH Ar), 129.26 (1C, C Ar), 154.76 (1C, C Ar), 165.20 (1C, C Ar), 170.48 (IC3 C Ar), 170.64 (1C, C Ar); Anal. Calcd for C10H9ClN4O2: C = 47.54, H = 3.59, N - 22.18, Cl = 14.03, Found: C = 47.57, H = 3.55, N = 22.10, Cl = 14.8.

't'-K'i-chloro-β-methoxy-ljSjS-triazin-Z-y^aminolpheny^- bromo-l-methylpropionate, 5

A solution of 2-bromoisobutyryl bromide (1.0 rαL, 7.90 mmol) in 20 mL of THF was added dropwise to a solution of 4-[(4-chloro-6-methoxy-l,3,5-triazin-2-yl)amino]phenol (1.9 g, 7.52 mmol) and triethylamine in 100 mL of THF, at -10 0C. During the addition (ca. 15 min) precipitation of triethylammonium bromide was observed. The reaction was monitored by TLC (SiO2, petroleum ether/ Et2O 1:1, 4-[(4-chloro-6-methoxy -l,3,5-triazin-2-yl)amino]phenol (starting material) Rf = 0.14; 4-[(4-chloro-6-methoxy- 1 ,3 ,5-triazin-2-yl)amino]phenyl 2-bromo-2-methylpropionate (final product) Rf = 0.26). After 1.5 h the white suspension was poured into a conical flask containing 150 mL OfEt2O and the ammonium salt removed by filtration on a sintered glass frit. The solvent was then evaporated at reduced pressure to give a white crude residue that was suspended in 10 mL of pentane and filtered. We obtained 2.56 g (6.37 mmol, yield 85%) of 4-[(4-chloro-6-methoxy-l,3,5-triazin-2-yl)amino]phenyl 2-bromo-2-methylpropionate as a white solid. The 1H NMR analysis (^6-DMSO) revealed the presence, in solution, of 2 rotational isomers (molar ratio 7:3). m.p. 107-108 °C; IR vm) 3365 cm4. v(O-o) 1747 cm"1. Major isomer. 1H NMR (J6-DMSO, 298 K, 400 MHz) δ 2.05 (s, 6H, C(CH3)2Br), 3.96 (s, 3H, OCH3), 7.17 (d, J = 8.9 Hz, 2H, CH Ar) , 7.77 (d, J = 8.9 Hz, 2H, CH Ar), 10.78 (s, IH, NH); 13C(1H) NMR (J6-DMSO5 298 K, 100.6 MHz) δ 30.42 (2C, CH3), 55.75 (bs, 1C, OCH3), 57.29 (1C, C(CH3)2Br), 121.96 (2C3 CH Ar), 122.12 (2C, CH Ar), 136.29 (1C, C Ar), 146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar), , 170.16 (1C, C=O), 171.33 (bs, IC3 C Ar). Minor isomer: 1H NMR (J6-DMSO, 298 K, 400 MHz) δ 2.05 (s, 6H, C(CH3)2Br), 3.96 (s, 3H, OCH3), 7.17 (d, J= 8.9 Hz, 2H, CH Ar) , 7.69 (d, J= 8.9 Hz, 2H, CH Ar), 10.66 (s, IH, NH); 13C(1H) NMR (J6-DMSO, 298 K, 100.6 MHz) δ 30.42 (2C, CH3), 55.75 (bs, 1C, OCH3), 57.29 (1C, C(CH3)2Br), 121.96 (2C, CH Ar), 122.73 (2C, CH Ar), 136.29 (1C, C Ar), 146.61 (bs, 1C, C Ar)5 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar), , 170.16 (1C, C=O)5 171.33 (bs, IC3 C Ar).

Typical Polymerisation of MMA.

CuBr (0.134 g, 0.934 mmol) was placed in an oven-dried Schlenk tube. The tube was fitted with a rubber septum, evacuated and flushed with dry N2 three times. Methyl methacrylate (10 mL, 93.4 mmol) and xylene (20 mL) were transferred to the tube via degassed syringe. The mixture was stirred rapidly under nitrogen and N-(n-propyl)-2-pyridylmethanimine (NMPI) (0.408 g, 1.86 mmol) was added which imparted a deep red/brown colour to the solution. Appropriate initiator (0.934 mmol) was added and the resulting solution was degassed by three freeze-pump-thaw cycles. The resulting mixture was placed in a thermostatically controlled oil bath at 90 0C. Samples were taken periodically for conversion and molecular weight analysis. Conversion was measured by gravimetry by drying to constant weight in a vacuum oven at 70 0C. The catalyst was removed from the samples by passing through a column of activated basic alumina prior to SEC. (see Figure I)- Table 1 Polymerisation of MMA in Xylene Solution (33% v/v) at 90 0C

Initiator [MMA]/(Cu( I)Br) [CuI ll)Br:/[NMPI)/[lnmaιor] Time \U PDt Coπv. MPoIT Mm Ϊ mol ' % • IOV 5 ιoo/i/o/2/r 4S0 8200 1.14 66 6 100/1/0/1/2.1 600 . S900 1.20 75 0.047 19000") 7 37/1/0/1/2.1 138 5800 1.05 89 0.32 1580011) 7 60/0.95/0.05/11. 28S0 3200 1.04 37 0.22 13100") EiBr 100/1/0/2/111 2SSO 2500 1 16 71 (2400")

° kp[Pol*] = rate constant of propagation x [active propagating polymer chains] from first order kinetic plot. b determined by the 1H NMR peak intensity ratio on a BrUker DPX 300 MHz cΛKn-octyl)-2-pyridylmethanimine used as the ligand Λ 10 mole % HEMA/90 mole % MMA

Typical Polymerisation of Styrene.

CyBr (0.055 g, 0.38 mmol) was placed in an oven dried Schlenk tube. The tube was fitted

with a rubber septum, evacuated and flushed three times with dry N-. Styrene (10 mL, 96

mmol) was transferred to the tube via degassed syringe. The mixture was stirred rapidly

under nitrogen and 4,4'-di(5-nonyl)-2.2'-bipyridyl (dNbpy) (0.314 g, 0.768 mmol) was

added, imparting a deep red/brown colour to the solution. Initiator 7 (0.035 g, 0.048 mmol,

0.192 mmol of initiating sites) was added and the resulting solution was degassed by three

freeze-pump-thaw cycles. The resulting mixture was placed in a thermostatically controlled

oil bath at 110 0C for 4.5 hours. The catalyst was removed from the samples by passing

through a column of activated basic alumina prior to SEC.

Kinetic studies for initiators 6 and 7.

Samples were removed periodically using degassed syringes and quenched in liquid

nitrogen for conversion and molecular weight analysis. Conversion was determined by

NMR on a Bruker DPX 300. For Living Radical Polymerisation initiated by 6, samples

were passed over a basic alumina column and then filtered in a syringe equipped with a

0.22 μm hydrophobic filter prior to molecular weight studies. In the case of LRP initiated by 7, molecular weight was determined by diluting the sample with THF and letting it settle overnight to precipitate the catalyst residues. The upper liquid was then filtered with a 0.22 μm hydrophobic filter. This method was chosen for N-hydroxysuccinimide-functionalised polymers as these polymers could not be passed over basic alumina.

Synthesis of a N-benzylamide fimctionalised poly(MMA).

Benzylamine was added to a solution of N-hydroxysuccinimide terminated poly(methyl methacrylate) in anhydrous THF. N-hydroxysuccinimide-terminated poly(methyl methacrylate) (Mn = 3200 g mol"1, PDI = 1.06) (1.00 g, 0.313 mmol) and three equivalents of benzylamine (0.100 mL, 0.938 mmol) were dissolved in 10 mL of dry THF in a dry Schlenk and stirred at 50°C for 3 days under nitrogen. After reaction, the polymer was precipitated in cold petroleum ether (see Figure 2).

This shows that N-benzylamide functional groups may be added and can be used to reach with free amide groups of the sort found in proteins.

day! Scheme Coupling of a N-hydroxysuccinimide terminated poly(MMA) with benzylamine.

(7) N-hydroxysuccinimide initiator (7) (NHS-Br)

Reagents.

Poly(ethylene glycol) methyl ether methacrylate (Mn = ca 475, Aldrich, 99%) and anhydrous toluene was degassed by bubbling with dry nitrogen for 30 minutes before use. The ligandN-(«-propyl)-2-pyridylmethanimine was prepared as described previously1. Copper(I) bromide (Avocado, 98%) was purified as necessary by a method based on that of Keller and Wycoff2. Other reagents were all commercial products and used without further purification.

Typical procedure.

Polymerizations were carried out at 30°C mediated by copper(I) bromide / N-(«-propyl)-2-pyridylmethanimine. A typical polymerization recipe is based on 33% v/v monomer in toluene. The ratio of initiator/Cu(I)Br/ligand is 1/1/2.1 on a molar basis. A dry Schlenk tube was charged with Cu(I)Br (0.3099 g, 2.16xlO"3 mol), NHS-Br (7) (0.5704 g, 2.16xlO'3 mol) and a magnetic bar prior to being deoxygenated by cycling between nitrogen and vacuum three times. To the flask was then added PEGMA (10 ml, 2.27x10"2 mol) and toluene (20 ml). The mixture was immediately subjected to three freeze-pump-thaw degassing cycles. Finally N-(«-propyl)-2-pyridylmethanimine (0.707 ml, 4.54x10"3 mol) was added and the flask was placed in an oil bath thermostatted at 30°C.

Kinetic studies.

Samples were removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular weight analysis. Conversion was determined by NMR on a Briiker DPX 300 MHz. Molecular weight was determined by diluting the sample with toluene and allowing it to settle down overnight to remove the copper complexes. The upper liquid was then filtered with a 0.22μm hydrophobic filter. This method was chosen because of the difficulty encountered to pass the polymer over a basic alumina column. Number average molecular weights (Mn) were determined by Size Exclusion Chromatography (SEC) in a system fitted with a 5 mm guard column, two Polymer Labs mixed E columns, a differential refractive index detector, and an auto sampler. The system was eluted with THF at a rate of 1 mL/min. Toluene was used as the flow marker.

Purification. N-hydroxysuccinimide functionalised poly(PEGMA) were purified by two consecutive

purifications from a Toluene solution in diethyl ether.

Table 1. Kinetic data for the polymerisation of PEGMA initiated by NHS-Br in toluene solution (33% v/v) at 300C ([PEGMA]o/[CuBr]o/[NHSBr]o/[L]o= 10/1/1/2.1).

Time Conversion Mn eXp Mw/Mn a Mn ,h=ob (h) (%) (g-mol-1) femol-1) 1 8.9 2350 1.10 450 2 18.4 2860 1.26 920 3 27.1 3100 1.20 1360 4 34.7 3600 1.13 1730 17 80.8 5670 1.06 4040

1 determined by SEC analysis calibrated with PoIy(MMA) standards - THF h M1, Ulu> = ([M]0 / [I]11 x M W MMΛ x Conv ) / 100

Table 2. Characterisation of PoIy(PEGMA) prepared by LRP

Kp[PoI*]" Mn exp b MJMn Mn theo b (IvO (g-mol-1) (g-rnol-1) NHS-PoIy(PEGMA) 0.096 6200 L05 4040

" Kp[PoI*] = rate constant of propagation b determined by SEC calibrated with PoIy(MMA) standards - THF (stabilised with topanol) Mn ,,,u, = ([M],) / [I]1, x M W MMΛ X Conv ) / 100

References

(a) D. M. Haddleton, M. C. Grossman, B. H. Dana, D. J. Duncalf, A. M. Henning, D.

Kukulj and A. J. Shooter, Macromolecules, 1999, 32, 2110.

(b) R. N. Keller and W. D. Wycoff, Inorg. Synth, 1947, 2, 1.

Polymerisation of methoxypolyethyleneglycol methacrylate (2080) using the initiator

derived from N-hydroxy succinimide

[PEG]/[I]/[Cu]/[L] = 19.2/1/1/2 in 80% toluene solution (AJ U2-27a) @30"C

N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1

eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 2080, 7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 30°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (400 mL). The resulting white powder was filtered, dissolved in toluene (2OmL) and precipitated in diethyl ether (400 mL). This procedure was repeated three times.

Table 1 : Data for the polymerization of methoxypolyethyleneglycol methacrylate (2080) with an initiator derived from N-hydroxy succinimide at 300C in 80% toluene solution.

Sample Time Conversion" Mn'' PDi* / minutes / % 89 4 3380 1.04 291 9 9820 1.09 901 17 10030 1.07 1369 23 11080 1.07 2760 26 12610 1.07 3965 28 14830 1.04

" Conversion was determined using IH NMR. b Molecular mass determined by SEC using PMMA standards.

Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate) was freeze dried prior to use to remove all water.

[PEG]/[ϊ]/[Cu]/[L] = 19.2/1/1/2 in 80% toluene solution (AJ U2-27b) @50°C N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 2080, 7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (28 niL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (400 mL). The resulting white powder was filtered, dissolved in toluene (2OmL) and precipitated in diethyl ether (400 mL). This procedure was repeated three times.

Table 2: Data for the polymerization of methoxypolyethyleneglycol methacrylate (2080) with an initiator derived from N-hydroxy succinimide at 50 0C in 80% toluene solution.

SampleTime Conversion" Mn'' PDi* /minutes /% 86 7 8700 1.06 289 12 10920 1.07 899 24 14450 1.05 1367 33 15810 1.04 2758 45 20220 1.07 3962 53 23180 1.07

" Conversion was determined using IH NMR. h Molecular mass determined by SEC using PMMA standards. Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate) was freeze dried prior to use to remove all water.

[PEG]/[I]/[Cu]/[L] = 19.2/1/1/2 in 80% toluene solution (AJ U2-27c) @90°C

N-hydroxy succinimide initiator, (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 2080, 7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.05 g, 0.38 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 90°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (400 mL). The resulting white powder was filtered, dissolved in toluene (2OmL) and precipitated in diethyl ether (400 mL). This procedure was repeated three times.

Table 3: Data for the polymerization of methoxypolyethyleneglycol methacrylate (2080) with an initiator derived from N-hydroxy succinimide at 90 0C in 80% toluene solution.

Sample Time Conversion" Mn* PDiΛ / minutes / % 86 18 11100 1.08 289 26 14870 1.08 899 31 17900 1.08 1367 35 18110 1.09 2758 38 18110 1.09 3962 39 18240 1.08 "Conversion was determined using IH NMR. * Molecular mass determined by SEC using PMMA standards.

Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate) was freeze dried prior to use to remove all water.

[PEG]/[I]/[Cu]/[L] = 23.9/1/1/2 in 66% toluene solution (AJ U2-11) @90"C N-hydroxy succinimide initiator, (2.5 g, 9.47 mmol), Cu(I)Br (1.35 g, 9.47 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 628, 142.0 g, 0.226 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (261 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-propyl-2-pyridylmethanimine (2.80 g, 0.019 mol) was added. The reaction was placed in a thermostatically controlled oil bath at 90°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (1000 mL). The resulting oil was washed with diethyl ether (3 x 1000 mL) and then dried in vacuo.

Table 4: Data for the polymerization of methoxypolyethyleneglycol methacrylate (628) with an initiator derived from N-hydroxy succinimide at 90 0C in 66% toluene solution.

Sample Time Conversion" Mn* PDi'' / minutes / % 48 21 4449 1.11 132 40 7198 1.08 185 44 7779 1.07 245 46 8105 1.09 300 48 8331 1.09 a Conversion was determined using IH NMR h Molecular mass determined by SEC using PMMA standards.

Bisomer MPEG550MA was used as provided.

Polymerisation of methoxypolyethyleneglycol methacrylate (1080) using the N-hydroxy succinimide derived initiator

[PEG]/[I]/[Cu]/[L] = 13.9/1/1/2 in 66% toluene solution (AJ U2-13) @90°C

N-hydroxy succinimide initiator, (0.526 g, 1.99 mmol), Cu(I)Br (0.29 g, 2.02 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 1080, 29.62 g, 0.027 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (60 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridylmethanimine (0.51 g, 3.96 mol) was added. The reaction was placed in a thermostatically controlled oil bath at 900C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (1000 mL). The resulting oil was washed with diethyl ether (3 x 1000 mL) and then dried in vacuo.

Table 5: Data for the polymerization of methoxypolyethyleneglycol methacrylate 1080) with an initiator derived from N-hydroxy succinimide at 90 0C in 66% toluene solution. Sample Time Conversion" Mn* PDi* / minutes / % 1250 47.3 12180 1.16 2460 50.4 12460 1.16 3890 52.8 12540 1.20 " Conversion was determined using IH NMR. b Molecular mass determined by SEC using PMMA standards.

[PEG]/[I]/[Cu]/[L] = 9.3/1/1/2 in 66% toluene solution (AJ U2-15) @90°C

N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 1080, 18.90 g, 0.018 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (35 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 90°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

Table 6: Data for the polymerization of methoxypolyethyleneglycol methacrylate (1080) with an initiator derived from N-hydroxy succinimide at 90 0C in 66% toluene solution.

Sample Time Conversion" Mn6 PDiή /minutes /_% 4160 88.7 9870 1.22

" Conversion was dertimined using IH NMR. * Molecular mass determined by SEC using PMMA standards.

Bisomer SlOW (50% aqueous solution of methoxypolyethyleneglycol methacrylate) was freeze dried prior to use to remove all water.

Polymerisation of methoxypolyethyleneglycol methacrylate (628) using the N-hydroxy succinimide derived initiator

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 66% toluene solution (AJ U2-31a) @30"C N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 628, 7.57 g, 0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (14 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 30°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

Table 7: Data for the polymerization of methoxypolyethyleneglycol methacrylate (628) with an initiator derived from N-hydroxy succinimide at 30 0C in 66% toluene solution. _ Sample Time Conversion" Mn* / minutes / % 60 19 2850 1.04 131 32 3230 1.10 199 45 3560 1.12 250 53 3760 1.12 298 56 3980 1.12

α Conversion was determined using IH NMR. h Molecular mass determined by SEC using PMMA standards.

Bisomer MPEG550MA was used as provided.

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 66% toluene solution (AJ U2-31B) @50"C N-hydroxy succinimide initiator, (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and methoxypolyethyleneglycol methacrylate (PEG) (average molecular weight = 628, 7.57 g, 0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (14 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50°C (t=0) and samples were removed periodically for conversion and molecular weight analysis. Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

Table 8: Data for the polymerization of methoxypolyethyleneglycol methacrylate (628) with an initiator derived from N-hydroxy suceinimide at 50 0C in 66% toluene solution. __ Sample Time Conversion" MnΛ / minutes / % 59 39 3212 Ϊ09 126 56 3958 1.11 195 69 4375 1.13 246 75 4649 1.13 295 82 4874 1.13

"Conversion was determined using IH NMR. ^Molecular mass determined by SEC using PMMA standards. Bisomer MPEG550MA was used as provided. Experimental

General experimental

For all following polymerisations conversion data was obtained by 1H NMR spectroscopy and molecular weight data (Mn and PDi) by SEC using PMMA standards.

Methoxypolyethyleneglycol methacrylates were obtained from Sigma- Aldrich or Laporte Performance Chemicals and used either as received (MPEG(395)MA: Mn 475 g mol'1 and BISOMER MPEG(550)MA: Mn 628 g mol"1) or freeze dried prior to use to remove all water (BISOMER SlOW MPEG(IOOO)MA: Mn 1080 g mol"1 and BISOMER S20W MPEG(2000)MA: Mn = 2080 g mol"1).

The ligands N-(«-alkyl)-2-pyridylmethanimine were prepared as described previously.1 Copper(I) bromide was purified as necessary by a method based on that of Keller and Wycoff.2

All other reagents were obtained from either Sigma- Aldrich, Romil, Fisher or Acros and used as received. Functional initiators The following table lists the functional initiators used to polymerise the methoxypolyethyleneglycol methacrylates. Table 1 : Functional initiators. Initiator code Initiator structure

Functional polymers

The following table lists the functional polymers prepared using methoxypolyethyleneglycol methacrylates and the initiators shown in Table 1. These polymers can be used either directly to react with useful biomolecules or converted simply into new macromolecules that will react with useful biomolecules.

Table 2: Functional polymers.

Initiator used Polymer structure

Preparation of initiators and intermediates

Preparation of initiator 8

N-hydroxysuccinimide^-bromopropionate

N-hydroxysuccinimide (4.51 g, 39.22 mmol) and 2-bromopropionic acid (2.9 mL, 32.68 mmol) were dissolved in anhydrous DCM (1000 ml) in a 2000 mL round- bottomed flask under nitrogen equipped with a magnetic stirrer. The flask was then cooled to 00C with an ice bath before the dropwise addition of a solution of N5N1- Dicyclohexylcarbodiimide (6.70 g, 32.68 mmol) in 50 mL of anhydrous DCM. After addition, the mixture was stirred at room temperature overnight. The reaction mixture was then filtered and the solvent evaporated to give a yellow solid that was purified by flash chromatography (CC, SiO2, Et2O, Rf (e8ter) = 0.31). Obtained 7.2 g (28.91 mmol, 74%) of product as a white solid. Melting point: 69-7O0C. 1H NMR (CDCl3) δ (ppm) 1.96 (d, 3H, CH(CH3)Br, J = 6.78 Hz), 2.86 (s, 4H, Hcycl), 4.61 (q, IH, CH(CH3)Br, J= 7.03 Hz). 13C NMR (CDCl3) δ (ppm) 21.67 (1C, CH(CH3)Br) 25.74 (2C, Ccyci), 34.97 (1C, CH(CH3)Br), 166.17 (1C, C=O), 168.69 (2C, Ccycl=O). IR (solid, ATR cell) v (cm'1) 1808, 1781 (Ccyci=O), 1729 (C=O). Mass spectroscopy (+EI, m/z) 248.964. Elem. Anal. Theoretical for C7H8NO4Br: C, 33.62; H, 3.22; N, 5.60. Found: C, 33.47; H, 3.16; N, 5.46.

Preparation of initiator 7

N-hydroxysuccinimide-l-brotno-l-methylpropionate

7

N-Hydroxysuccinimide (11.51 g, 0.1 mol) was dissolved in anhydrous dichloromethane (100ml) with triethylamine (28.ImL, 0.2 mol) under nitrogen in a 250ml round-bottomed flask equipped with a magnetic stirrer. The flask was cooled to 00C with an ice bath before the dropwise addition of 2-bromo-2-methylpropionyl bromide (13.9 mL, 0.11 mol). Next the mixture was stirred for 45 minutes and allowed to reach room temperature. After this the reaction mixture was poured into an excess of cold water and extracted with diethyl ether (3 x 50 mL). The organic layer was subsequently washed with a saturated aqueous solution of sodium carbonate (3 x 50 mL), acidified water (pH= 4.5, 3 x 50 mLl), and again the saturated aqueous solution of sodium carbonate (3 x 50 mL). The organic layer was dried over anhydrous magnesium sulphate and filtered. Finally the solvent was removed under reduced pressure by using the rotary evaporator in order to isolate the title compound in quantitative yield as a white solid. 1H NMR (CDCl3) δ (ppm) 2.08 (s, 6H, C(CHs)2Br), 2.87 (s, 4H, Hcycl). 13C NMR (CDCl3) δ (ppm) 26.03 (2C, Ccycl), 31.09 (2C, C(CH3)2Br), 51.60 (1C, C(CH3)2Br), 167.89 (1C, C=O), 169.02 (2C, Ccyci=O). IR (solid, ATR cell) v (cm 1) 1803, 1772 (Ccycf=O), 1728 (C=O), 1394, 1359, 1197, 1121, 1071, 996, 924, 856, 811, 731, 648. Mass spectroscopy (+EI, m/z) 266, 265, 156, 151, 149, 123, 121, 116, 115, 91, 87, 70, 69. Elem. Anal. Theoretical for C8H10NO4Br: C, 36.39; H, 3.82; N, 5.30; Br, 30.26. Found: C, 36.35; H, 3.82; N, 5.03; Br, 30.17. Melting point 72-74°C.

Preparation of initiator 5

2,4-Dichloro-6-methoxy-l,3,5-triazine3

To 200 ml of methanol and 25 ml of water were added 33.6 g. (0.4 mole) of sodium bicarbonate and 36.8 g. (0.2 mole) of cyanuric chloride. This mixture was stirred at 30 0C for 30 minutes until the evolution of carbon dioxide had nearly ceased, and water was then added. The crystalline solid which separated was filtered, washed with water, and dried in a vacuum desiccator. The yield of crude 2,4-dichloro-6-methoxy- triazine was 10.5 g. (58%), m.p. 87-890C. After recrystallization from heptane the m.p. was 88-9O0C. Elem. Anal. Calcd. for C4H3N3OCl2: C, 26.67; H, 1.67; N,23.35; Cl, 39.44. Found: C, 26.96; H, L84; N, 23.25; Cl, 39.19.

4-[(4-chloro-6-methoxy -1,3, 5-triazin-2-yl)amino]phenol

A solution of 2,4-dichIoro-6-methoxy-l,3,5-triazine (9.00 g, 50.0 mmol) in 100 mL of acetone was cooled to 0 0C and, under stirring, solid 4-amino phenol (5.46 g, 50.0 mmol) was added in small portion, over ca. 2 rain. The white suspension was then let to warm to room temperature and stirred for further 1 h, while being neutralized with a 2 M aqueous solution Of Na2CO3 during the reaction. The mixture was then poured into 500 mL of ice/water and the resulting white precipitate was filtered and dried, to give 9.6 g (38.0 mmol, yield 76%) of 4-[(4-chloro-6-methoxy -l,3,5-triazin-2- yl)amino]phenol that can be used without further purifications. An analytical sample can be obtained by flash chromatography (CC, SiO2, petroleum ether/ Et2O 1:1, Rf = 0.14). The NMR analysis (DMSO d6) reveals the presence, in solution, of 2 rotational isomers (molar ratio 7:3). M.p was 172 °C. IR v( NH) 3476 cm"1. v( 0H) 3269 cm'1. Major isomer: 1H NMR (DMSO d6) δ = 3.94 (s, 3H, OCH3); 6.79 (d, J = 8.8 Hz, 2H, CH Ar), 7.48 (d, J = 8.8 Hz, 2H, CH Ar), 9.40 (s, IH, OH), 10.46 (s, IH, NH). 13C NMR (DMSO d6) δ = 55.52 (1C, OCH3); 115.46 (2C, CH Ar), 123.91 (2C, CH Ar), 129.49 (1C, C Ar), 154.44 (1C, C Ar), 164.81 (1C, C Ar), 169.57 (1C, C Ar), ,171.23 (1C, C Ar). Minor isomer: 1H NMR (DMSO d6) δ = 3.96 (s, 3H, OCH3); 6.79 (d, J = 8.9 Hz, 2H, CH Ar), 7.39 (d, J= 8.9 Hz, 2H, CH Ar), 9.42 (bs, IH, OH), 10.10.32 (s, IH, NH). 13C NMR (DMSO d6) δ = 55.10 (1C, OCH3); 115.46 (2C, CH Ar), 123.03 (2C, CH Ar), 129.26 (1C, C Ar), 154.76 (1C, C Ar), 165.20 (1C, C Ar), 170.48 (1C, C Ar), 170.64 (1C, C Ar).

4-[(4-chloro-6-methoxy-l,3,5-triazin-2-yl)amino]phenyl-2- bromo-2- wethylpropionate

A solution of 2-bromoisobutryl bromide (1.0 mL, 7.90 mmol) in 20 mL of THF was added dropwise to a solution of 4-[(4-chloro-6-methoxy-l,3,5-triazin-2- yl)amino]phenol BIW009 (1.9 g, 7.52 mmol) and triethylamine (1.1 mL, 8.0 mmol) in 100 mL of THF, at - 10 'C. During the dropping {pa. 15 min) the precipitation of triethylammonium bromide was observed. The reaction was monitored by TLC (SiO2 ,ρetroleum ether/ Et2O 1:1, BIW009 (starting material) Rf = 0.14; BIWOlO (final product) Rf = 0.26). After 1.5 h the white suspension was poured into a conical flask containing 150 mL OfEt2O and the the ammonium salt removed by filtration on a sintered glass frit. The solvent was then evaporated at reduced pressure to give a white crude residue that was suspended in 10 ml of pentane and filtered. Obtained 2.56 g (6.37 mmol, yield 85%) of BIWOlO as white solid. The NMR analysis (DMSO d6) revealed the presence, in solution, of 2 rotational isomers (molar ratio 7:3). M.p. 107-108 0C. IR v( NH) 3365 cm'1. v( O=0) 1747 cm-1. Major isomer: 1H NMR (DMSO d6) δ = 2.05 (s, 6H, C(CH3)2Br), 3.96 (s, 3H5 OCH3), 7.17 (d, J = 8.9 Hz, 2H, CH Ar) , 7.77 (d, J = 8.9 Hz5 2H, CH Ar), 10.78 (s, IH, NH). 13C{1H} NMR (DMSO d6) δ = 30.42 (2C, CH3), 55.75 (bs, 1C, OCH3), 57.29 (1C, C(CH3)2Br), 121.96 (2C, CH Ar), 122.12 (2C, CH Ar), 136.29 (1C, C Ar), 146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar), , 170.16 (1C, OC(O)C(CH3)2Br), 171.33 (bs, 1C, C Ar). Minor isomer: 1H NMR (DMSO d6) δ = 2.05 (s, 6H, C(CH3)2Br), 3.96 (s, 3H, OCH3), 7.17 (d, J = 8.9 Hz, 2H, CH Ar) , 7.69 (d, J = 8.9 Hz, 2H, CH Ar), 10.66 (s, IH, NH). 13C NMR (DMSO d6) δ = 30.42 (2C, CH3), 55.75 (bs, 1C, OCH3), 57.29 (1C, C(CHa)2Br), 121.96 (2C, CH Ar), 122.73 (2C, CH Ar), 136.29 (1C, C Ar), 146.61 (bs, 1C, C Ar), 165.10 (bs, 1C, C Ar), 169.89 (bs, 1C, C Ar), , 170.16 (1C, OC(O)C(CH3)2Br), 171.33 (bs, 1C, C Ar).

Preparation of initiator 9

2~Hydroxyethyl-2-bromo-2-methylpropionate

Ethylene glycol (279 g, 4500 mmol) and Et3N (3.34 g, 33.0 mmol) were poured in a 2-necked round bottom flask. To this was added dropwise and a solution of 2- bromoisobutryl bromide (6.90 g, 30.0 mmol) in anhydrous THF (50 mL) at room temperature over ca. 1 h. The colourless solution was stirred overnight, then diluted in 500 mL of water and extracted with 3x200 mL of a mixture of Et2O/CH2Cl2 (4:1). The organic layers, reunited, were washed with 2x200 mL of water and dried over MgSO4. Evaporation of the solvent at reduced pressure (rotavapor, without heating) gave a pale yellow liquid. The latter was dissolved in ca. 30 mL Of CH2Cl2 then 1O g of SiO2 were added and the solvent evaporated again until a white powder was obtained. This was poured in a column packed with SiO2, (ca 15 cm depth) previously eluted with petroleum ether/Et2O 5:1 and purified by column chromatography (elute with petroleum ether/Et2θ 5:1) to eliminate the impurities. The desired product, using this solvent mixture, has an Rf -O (stays on the bottom of the TLC plate). When the impurities have been eliminated, the column was eluted with 100% Et2O, to give a colourless liquid. Yield 82%. IR v^ 3388 cm"1 (broad); v(c=0) 1731 cm"1. 1H NMR (CDCl3) δ = 1.97 (s, 6H, CH3); 3.89 (t, J = 4.6 Hz; 2H, OCH2CH2OH); 4.33 (t, J = 4.6 Hz; 2H, OCH2CH2OH). 13C NMR (CDCl3) δ = 30.45 (2C, CH3), 55.55 (1C, C(CH3)2Br); 60.66 (1C, OCH2CH2OH); 65.90 (1C, OCH2CH2OH); 171.69 (1C, C=O).

Preparation of initiator 6

2-Phthalimidoethyl-2-bromo-2-methylpropionate

6

N-(2-hydroxyethyl)phthalimide (19.12 g, 0.1 mol) was dissolved in anhydrous THF (25OmL) with triethylamine (28.1 mL, 0.2 mol) under nitrogen in a 500 mL round bottom flask equipped with a magnetic stirrer and dropping funnel. The flask was cooled to 0 0C with an ice/salt bath before the dropwise addition of 2-bromo-2- methylpropionyl bromide (13.9 mL, 0.11 mol). The mixture was stirred for 45 minutes and allowed to reach room temperature before the mixture was poured into an excess of cold water and the product extracted with diethyl ether (3x100 mL). The organic layer was subsequently washed with a saturated aqueous solution of sodium carbonate (3x100 mL), acidified water (pH 4.6, 3x100 mL) and again with saturated aqueous solution of sodium carbonate (3x100 mL). The organic layer was dried over anhydrous magnesium sulphate and filtered. The product was isolated via reduction under reduced pressure to obtain a white solid (25.79 g, 75.8 % yield). 1H NMR (CDCl3) δ (ppm) 1.81 (s, 6H, C(CHj)2Br), 3.95 (t, 2H, J = 5.3 Hz, CH2-N), 4.35 (t, 2H, J = 5.4 Hz, CHj-O), 7.67 (m, 2H, Haro), 7.78 (m, 2H, Har0). 13C NMR (CDCl3) δ (ppm) 31.00 (2C, C(CH3)2Br), 37.12 (1C, CH2-N), 55.92 (1C, C(CH3)2Br), 63.42 (1C, CH2-O), 123.78 (2C, Caro), 132.35 (1C, Civaro), 133.54 (2C, Caro), 168.40 (2C, CcycpO), 171.87 (1C, C=O). IR (solid, ATR cell) v (cm"1) 2975, 1774 (Ccyci=O), 1705 (C=O), 1417, 1392, 1321, 1276, 1158, 1105, 1063, 985, 763, 716, 632. Melting point 63-65°C.

Preparation of initiator 10

THtylthiolether propanol

Sodium hydride (10.95 g, 0.273 mol, 60% in oil) was suspended in THF (750 mL) at O0C. Triphenylmethanethiol (75.5 g, 0.273 mol) in THF (600ml) was added to the suspension and stirred at O0C for 10 minutes. 3-Bromo-l-propanol (24.75 mL, 0.273 mol) in THF (300 mLl) was added and the mixture stirred at 0 0C for 20 minutes. After this time TLC showed mostly one major product (Rf approx 0.3 ethyl acetate/hexane 1:9). Water was added and the product extracted into ethyl acetate (2x1000 mL) (an aqueous NaCl solution was used to break up the emulsion), dried with sodium sulfate, filtered and concentrated. The product was purified by column chromatography using ethyl acetate/hexane 1:9 to 1:4 as the eluent (SiO2) to give a white solid, which was triturated with hexane and filtered to give 65.2 g of product. 1H NMR (CDCl3) δ ppm 7.6-7.1 (m, 15H Haro), 3.58 (t, 2H, CH2OH), 2.29 (t, 2H, SCH2), 1.65 (q, 2H, CH2CH2CH2), 1.45 (OH).

3-Tritylthioletherpropyl-2-bromo-2-methylpropionate 10

Tritylthiolether propanol (50 g, 0.150 mol), triethylamine (31.3 mL, 0.225 mol) and anhydrous tetrahydrofuran (125 mL) were placed in a three-necked round bottom flask containing a magnetic follower and fitted with a pressure equalizing dropping funnel. The flask was cooled with the use of an ice bath and 2-bromoisobutyrl bromide (27.8 mL, 0.225 mol) was added to the dropping funnel. Whilst stirring the 2-bromoisobutyrl bromide was added drop-wise to the cooled solution and the solution left stirring over night. The mixture is then filtered to remove the triethylamine hydrochloride salt before the addition of dichloromethane (500 mL) and subsequently washed with dilute hydrochloric acid (2x300 mL), dilute sodium hydroxide (2x300 mL) and finally distilled water (3x300 mL). The Organic layer was separated and the product isolated by flash evaporation of solvent, the product was then triturated with hexane, filtered and the product collected in quantitative yield. 1H NMR (CDCl3) δ ppm 7.4-7.1 (m, 15H, Har0), 4.02 (t, 2H, CH2CO2), 2.15 (t, 2H, SCH2), 1.78 (s, 3H5 C(CH3)2Br), 1.63 (q, 2H, CH2CH2CH2).

Preparation of initiator 11

4-(2-bromo-2-methylpropionate) benzaldehyde 4-Hydroxybenzaldehyde 12.21 g (0.1 moles), triethylamine, 15.3 mL (0.11 moles) and anhydrous THF 400 mL were placed in a 3 neck round bottomed flask. Bromoisobutyryl bromide 13.6 mL (0.11 moles) was added slowly with stirring. A white precipitate of triethylammonium bromide was formed. The mixture was left to react for 6 hours with stirring. On completion of the reaction triethylammonium bromide was removed by filtration and the THF was removed by rotary evaporation. The resulting orange liquid was dissolved in dichloromethane and subsequently washed with 2 x 200 mL portions of saturated Na2CO3 (aq), dil. HCl (aq) and distilled water. The dichloromethane was dried using MgSO4 and the solvent removed by rotary evaporation to give a yellow oily liquid which crystallised upon standing. This was recrystalised from diethyl ether x 2.Yield = 18.95g (69.9 %). 1H NMR (CDCl3) δ (ppm) 10.00 (s, IH, CHO), 7.94 (d, J = 4.6 Hz, 2H, H3T0), 7.31 (d, J = 4.8 Hz 2H, Haro), 2.06 (s, 6H, C(CHs)2Br). 13C NMR (CDCl3) δ (ppm) 190.59, 169.33, 155.08, 134.07, 131.02, 121.71, 54.94, 30.25. IR (Solid, ATR Cell) 2984, 2820, 2730 (O=C- H), 1746 (C=O), 1693 (H-C=O), 1590, 1500, 1374, 1262, 1207, 1153, 1132, 1099, 1009, 932, 881, 808, 658: +EI MS (m/z) 273, 271 (mass peaks), 210, 193, 163, 151, 149, 140, 123, 121, 102. Elem. Anal. Theoretical for Hl 103Br: C = 48.73 , H = 4.09; Found: C = 48.63 , H = 4.03.

Preparation of initiator 12

2-(2,2-Dimethoxy-ethoxy)-ethanol

— O O OH

Potassium hydroxide (30 g, 0.51 mol) was suspended in ethylene glycol (100 ml) and the mixture was heated to 115 0C with stirring. After the KOH dissolved completely, 2-chloro-l,l-dimethoxy-ethane (30.0 mL, 0.263 mol) was added dropwise (ca. 30 minutes) and the solution was stirred at 115 0C for 72 h. The resulting suspension was cooled to room temperature and 150 mL water was added. The solution was extracted with dichloromethane (3x10OmL) and the organic layers combined was washed with brine (2x100 mL) and dried with MgSO4. After filtration the solvent was removed under reduced pressure to give the product as a yellow oil (Yield, 17.7 g, 44.9%). 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 2.20 (s, IH, OH), 3.40 (s, 6H, OCH3), 3.55 (d, J= 5.3 Hz, 2H5 CHCH2), 3.63-3.61 (m, J= 4.0 Hz, OCH2), 3.74-3.72 (m, J= 4.0 Hz, 2H, CH2OH), 4.52 (t, IH, CH(OCH3)2. 13C(1H) NMR (100.59 MHz, CDCl3, 298 K) δ = 54.12 (2C, CH3), 61.82 (1C, CH2OH), 70.78 (1C, CHCH2O), 73.00 (1C, OCH2CH2), 102.73 (1C, CH). Anal. Calcd. for C5HJ4O4: C, 47.99; H, 9.40; Found C, 45.02; H, 8.74

2-Bromo-2-methyl-propionic acid 2-(2i2~dimeihoxy-eihoxy)-ethyl ester

Yl A solution of 2-(2,2-dimethoxy-ethoxy)-ethanol (Hg, 0.073 mol) and triethylamine (12 mL, 0.088 mol) in dichloromethane (150 mL) was cooled to O0C and a solution of 2-bromo-2-methyl propionyl bromide (8.5 mL, 0.069 mol) in 50 mL of dichloromethane was added dropwise in ca. 30 min. After stirring overnight at room temperature the resulting suspension was filtered and the yellow solution was washed with saturated NaHCO3 aqueous solution (2 x 100 mL) and dried with MgSO4. After filtration the solvent was removed under reduced pressure and the yellow oily residue was purified by distillation (b.p. 70°C/0.02mbar) to give 14.0 g (0.061mol, yield: 89%) of product as a colourless oil. 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 1.94 (s, 6H, (CHj)2CBr), 3.39 (s, 6H, OCH3), 3.56 (d, J= 5.3 Hz, 2H, CHCH2), 3.76 (t, J= 4.8 Hz, CH2OCH2), 4.33 (t, J = 4.8 Hz, 2H, CH2OCO), 4.50 (t, IH, CH(OCH3)2v 13C(1H) NMR (100.59 MHz, CDCl3, 298 K) δ = 30.90 (2C, C(CH3)2), 54.13 (2C, CH3O), 55.79 (1C, BrC(CH3)2), 65.24 (1C, CH2OC(O)), 69.21 (1C, CHCH2O), 71.18 (1C, OCH2CH2), 102.83 (1C, CH).

Preparation of initiator 13

3-(2,5-Dioxo-2,5-dihydro-pyrml-l-yl)-propionic acid A solution of maleic anhydride (5.00 g, 0.0561 mol) in acetic acid (70 ml) was added dropwise to a solution of β-alanine (5.50 g, 0.0561 mol) in acetic acid (25 ml) and the mixture was stirred at room temperature for three hours. 50 mL of acetic acid was added to the white suspension and the mixture heated up to 115°C. After 1 h a limpid colourless solution was observed. The mixture was then stirred at this temperature overnight and the colour turned to orange. The solvent was then removed under reduced pressure and 30 mL of toluene was then added to the resulting orange oil. This was then evaporated under reduced pressure and this operation was repeated 3 times. The orange residue was then purified by flash chromatography (CC, SiO2, CH2Cl2/ethyl acetate 9:1) to give the product as a white solid (3.86 g, 0.0228 mol, 41%). m.p. 105-1070C IR (neat): 3092, 2883, 2537, 1695, 1445, 1411, 1373, 1337, 1305, 1230, 1151, 1081, 1043, 924, 830, 773, 694, 618 cm"1. 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 2.69 (t, J = 7.3 Hz, 2H, CH2), 3.82 (t, / = 7.3 Hz, 2H, CH2), 6.71 (s, 2H, CHvinyi), 10.07 (bs, IH, COOH). 13C{1H} NMR (100.59 MHz, CDCl3, 298 K) δ = 32.62 (1C, CH2), 33.36 (1C, CH2), 134.38 (2C, CHvinyl), 170.48 (1C, C), 176.64 (2C, C). Anal. Calcd. for C7H7NO4: C, 49.71; H, 4.17; N, 8.28. Found C, 49.35; H, 4.19; N, 7.95.

3-(2,5-Dioxo-2,5-dihydro-pyrrol-l-yl)-propionyl chloride (3-maleimidopropionyl chloride) 3-(2,5-Dioxo-2,5-dihydro-pyrrol-l-yl)-propionic acid (2.20 g, 0.0130 mol) was dissolved in CH2Cl2 (100 mL). Oxalyl chloride (1.1 mL, 0.0130 mol) was then added, at room temperature. 50 μL of DMF were added dropwise and an intense evolution of gas was observed. The solution was colourless before the addition of DMF and remained colourless for Ih, then slowly turned to very pale yellow (but still very limpid). After 3 h the solvent was removed under reduced pressure to give an off- white solid that became pale brown after standing under vacuum at room temperature for 1 h. The acid chloride product so obtained was used directly without further purifications. IR (neat): 3095, 1803 1698, 1446, 1410, 1387, 1360, 1307, 1230, 1148, 1131, 1083, 1011,948, 922, 833, 719, 689, cm"1. 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 3.25 (t, J= 6.9 Hz, 2H, CH2), 3.86 (t, J= 6.9 Hz, 2H, CH2), 6.73 (s, 2H, CHvinyi). 13C(1H) NMR (100.59 MHz, CDCl3, 298 K) δ = 33.21 (1C, CH2), 44.97 (1C, CH2), 134.47 (2C, CHvinyO, 170.08 (2C, C), 171.50 (1C, C).

2-Bromo-2-methyl-propionic acid 2-[3-(2, 5-dioxo-2, 5-dihydro-pyrrol-l-yl)- propionyloxyj-ethyl ester

2-Hydroxyethyl-2-bromo-2-methylpropionate (initiator 9) (0.187 g, 0.887 mmol) and 3-(2,5-Dioxo-2,5-dihydro-pyrrol-l-yl)-propionic acid (0.300 g, 1.77 mmol) were dissolved in dichloromethane (10 ml) under nitrogen in a 25 ml round-bottomed flask. ThenN,N'-Dicyclohexylcarbodiimide (DCC) (0.366 g, 1.77 mmol) was added to the solution. After one day at room temperature, very low conversion was observed and 0.5 ml (9.5OxIO"3 mmol) of a solution of 4-dimethylaminopyridine (DMAP) in dichloromethane (Conc.DMAP = 19 mmol/1) was added. Total conversion was then achieved in 12 hours and the solvent was removed under reduced pressure. The solid residue was extracted with 3 x 50 ml of petroleum ether and the petroleum ether was removed by evaporation under vacuum in order to isolate a colourless oil (0.27Og, 0.745 mmol, 84%). The pale pink residue was extracted with 3 x 50 ml of diethylether, but TLC (CH2Cl2 / AcOEt 9:1) revealed that only traces of the ester were present. An analytical pure sample was obtained by flash chromatography (cc, SiO2, Pet. Ether/ Et2O 3:1). 1H NMR (CDCl3) δ (ppm) 1.88 (s, 6H, C(CHa)2Br)5 2.62 (t, 2H, CH2-COO(CH2)2-O, Jab=7.02 Hz), 3.78 (t, 2H, (CO)2N-CH2, Jab= 7.07 Hz), 4.27-4.35 (m, 4H5O-(CH2)Z-O), 6.68 (s, 2H, OC-CH=CH-CO). 13C NMR (CDCl3) δ (ppm) 30.61 (2C5 C(CH3)2Br), 32.75 (1C, CH2-COO(CH2)2-O), 33.46 (1C, (CO)2N-CH2), 55.45 (1C, C(CH3)2Br), 62.04 (IC5 CH2-COO-CH2), 63.35 (IC5 CH2-OOC-C(CH3)2Br), 134.25 (2C5 OC- CH=CH-CO), 170.29 (IC5 C=O ester), 170.40 (1C, C=O ester), 171.39 (2C, O=C- N(CH2)-C=O). IR (solid, ATR cell) v (cm"1) 1769 (v (C=0, ωde)), 1736 (v (c=o, acid)), 1707 (F (C=O, imide)).

Preparation of initiator 14

2-Bromo-2-methyl-propionic acid 3-tert-butoxycarbonylamino-propyl ester

14-

A solution of 3-amino propanol (3.00 mL, 0.0392 mol) in 100 mL of THF was cooled to 0 0C and Boc2O (8.56 g, 0.0322 mol) in THF (50 mL) was added dropwise (ca. 20 min.). The solution was then warmed up to room temperature and stirred for 3 h. TLC analysis (SiO2, 100% Et2O) revealed the complete disappearance of the amino alcohol starting material (Rf=O) and the presence of the expected N-Boc-protected amino alcohol intermediate (Rf=0.25). The mixture was then cooled to 0 0C and Et3N (6.0 mL, 0.0431 mol) was added via syringe. A solution of 2-bromo isobutyryl bromide (4.85 mL, 0.0392 mol) in THF (50 mL) was added dropwise in ca. 30 min. and the resulting white suspension stirred at 0 0C for 1 h and at room temperature for further 2 h. The mixture was then diluted with Et2O (200 mL) and the ammonium salt was filtered off and washed with 3x50 mL of Et2O. The colourless solution was washed with 3x100 mL of water and dried over MgSO4. Removal of the solvent under reduced pressure gave the product a colourless oil that was purified by flash chromatography (CC, SiO2, Petroleum ether/Et2O 8:1). Obtained 10.42 g (0.0321 mol, 82%) of (1) as colourless oil. IR (neat): 3295, 2976, 1734, 1713, 1695, 1517, 1463, 1391, 1366 1273, 1163, 1109, 1013, 633 cm'1.1H NMR (400.03 MHz, CDCl3, 298 K) δ = 1.43 (s, 9H, CH3), 1.88 (quint., /= 6.3 Hz, 2H, CH2), 1.93 (s, 6H, CH3), 3.23 (q, / = 6.0 Hz, 2H, CH2), 4.24 (q, / = 6.0 Hz, 2H, CH2), 4.77 (bs, IH, NH). 13C{1H} NMR (100.59 MHz, CDCl3, 298 K) δ = 28.54 (3C, CH3), 28.99 (1C, CH2), 30.88 (2C, CH3), 37.57 (1C, CH2), 55.95 (1C, C), 63.86 (1C, CH2), 156.03 (1C, C), 171.89 (1C, C). Anal. Calcd. for C12H22BrNO4: C, 44.46; H, 6.84; N, 4.32; Br, 24.65. Found C, 44.48; H, 6.91; N, 4.33. Br, 24.91.

Preparation of initiator 15

^lO-Dioxa-tricyclofS^.l.^JdecS-eneSJ-dione

Maleic anhydride (30.00 g, 0.306 mol) was suspended in 150 mL of toluene and the mixture warmed to 80°C. Furan (33.4 mL, 0.459 mol) was added via syringe and the turbid solution stirred for 6 h. The mixture was then cooled to room temperature and the stirring was stopped. After 1 h the resulting white crystals were filtered off and the solid washed with 2 x 30 mL of petroleum ether. Obtained 44.40 g (0.267 mol, 87% yield) of the desired product as small white needless, m.p. 124-127 °C (dec.) IR (neat): 1857, 1780, 1309, 1282, 1211, 1145, 1083, 1019, 947, 920, 902, 877, 847, 800, 732, 690, 674, 633, 575 cm 1. 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 3.17 (s, 2H, CH), 5.45 (t, /= 1.0 Hz, 2H, CHO); 6.57 (t, J= 1.0 Hz, 2H, CHvinyl). 13C(1HJ NMR (100.59 MHz, CDCl3, 298 K) δ = 48.85 (2C, CH), 82.35 (2H, CHO), 137.12 (2C, CHvinyi), 170.04 (2C, CO). Anal. Calcd. for C8H6O4: C, 57.84; H, 3.64;. Found C, 57.74; H, 3.68.

4-(2-Hydroxy-ethyl)-10-oxa-4-aza-tricyclo[5.2.L02'6]dec-8 -ene-3,5-dwne

The anhydride, 4,10-dioxa-tricyclo[5.2.1.02>6]dec-8-ene-3,5-dione, (2.00 g, 12.0 x 10'3 mol) was suspended in 50 mL of MeOH and the mixture cooled to 0°C. A solution of ethanolamine (0.72 mL, 12.0 x 10"3 mol) in 20 mL of MeOH was added dropwise (10 min) and the resulting solution was stirred for 5 min at 0°C, then 30 min at room temperature and finally refluxed for 4 h. After cooling to room temperature the solvent was removed under reduced pressure, the white residue was dissolved in 150 mL OfCH2CI2 and washed with 3 x 100 mL of water. The organic layer was dried over MgSθ4 and filtered. Removal of the solvent under reduced pressure furnished the desired product (1.04 g 5.0 x 10'3 mol, 42 %yield) as white solid that was used for the next step without further purifications. An analytical sample was obtained by flash chromatography (CC, SiO2, 100% ethyl acetate, Rf(6)=0.26). m.p. 139-141 "C (dec). IR (neat): 3472, 1681, 1435, 1405, 1335, 1269, 1168, 1100, 1053, 1013, 959, 916, 875, 850, 807, 722, 705, 654 cm"1. 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 1.90 (bs, IH, OH); 2.90 (s, 2H, CH), 3.69-3.72 (m, 2H, CH2), 3.76-3.78 (m, 2H, CH2), 5.28 (t, J= 0.9 Hz, 2H, CH), 6.52 (t, J= 0.9 Hz, 2H, CHvinyi). 13C(1H) NMR (100.59 MHz, CDCl3, 298 K) δ = 41.77 (2C, NCH2), 60.18 (2C, OCH2), 47.50 (2C, CH), 81.04 (2C, CH), 136.60 (2C, CH^i), 176.97 (2C, C). Anal. Calcd. for C10Hi 1NO4: C, 57.41; H, 5.30; N, 6.70. Found C, 57.16; H, 5.37; N, 6.62.

2-Bromo-2-methyl-proρionic acid 2-(3,5-dioxo-10-oxa-4-aza-tricyclo[5.2.1. Φ6]dec- 8-en-4-yl)-ethyl ester A solution of the alcohol, 4-(2-hydroxy-ethyl)-10-oxa-4-aza-tricyclo[5.2.1.02>6]dec- 8- ene-3,5-dione, (2.22 g, 10.6 x 10~3mol) and Et3N (1.6 mL, 11.7 x 10~3 mol) in 120 mL of THF (the solution remains slightly turbid) was cooled to 0°C and a solution of 2- bromo isobutiryl bromide (1.4 mL, 11.1 x 10"3 mol) in 40 mL of THF was added dropwise (30 min). The white suspension was stirred for 3 h at 0°C, then at room temperature overnight. The ammonium salt was filtered off and the solvent removed under reduced pressure to give a pale-yellow residue that was purified by flash chromatography (CC, SiO2, petroleum ether/ethyl acetate 1:1, Rf(7)=0.23). Obtained 3.54 g (9.88 x 10'3 mol, 93% yield) of initiator 15 as a white solid, m.p. 83-85°C IR (neat): 1733, 1695, 1419, 1395, 1336, 1278, 1157, 1106, 1015, 874, 852, 824, 724, 706, 654, 603 cm"1. 1H NMR (400.03 MHz, CDCl3, 298 K) δ = 1.86 (s, 6H, CH3), 2.84 (s, 2H, CH), 3.78 (t, J= 5.3 Hz, 2H, NCH2); 4.30 (t, J= 5.3 Hz, 2H, OCH2); 5.23 (t, J= 1.0 Hz, 2H, CHO); 6.49 (t, J= 1.0 Hz, 2H, CHviπyi). 13C(1H) NMR (100.59 MHz, CDCl3, 298 K) δ = 30.65 (2C, CH2), 37.65 (2C, NCH2), 47.56 (2C, CH), 55.80 (1C, C(CHa)2Br)5 62.26 (OCH2), 80.91 (2H, CHO), 136.62 (2C, CHvinyO, 171.46 (1C, COester), 175.95 (2C, COimide). Anal. Calcd. for C14H16NO5: C, 46.95; H,4.50; N.3.91; Br, 22.31. Found C, 46.88; H, 4.55; N, 3.79; Br, 22.22 Preparation of PoIyPEG polymers

Polymerisations using Initiator 8

Polymerisation of MPEG(395)MA

[PEGJ/flJ/fCuJ/fLJ = 10/1/1/2 in 50 v/v% toluene solution at 300C A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 x 10"3 mol), initiator 8 (0.569 g, 2.27 x 10"3 mol) and a magnetic follower prior to being deoxygenated by reeling between nitrogen and vacuum three times. To a second Schlenk tube was added MPEG(395)MA (10 mL, 22.74 x 10'3 mol), N-(«-ethyl)-2-ρyridylmethanimine (0.64 mL, 4.54 x 10"3 mol) and toluene (10 mL). The mixture was immediately subjected to five freeze-pump-thaw degassing cycles. This solution was then transferred to the Schlenk tube containing the initiator and Cu(I)Br via a cannula. The resulting brown solution was stirred at 30 °C. Samples were removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular weight analysis.

Table 3: Kinetic data for the polymerisation of MPEG(395)MA initiated by 8 in toluene solution (50% v/v) at 300C ([MPEG(395)MA]o/[CuBr]o/[NHSBry[ligand]o = 10/1/1/2).

Experiment Time Conversion ln([M]0/[M]) Mn, theo Mn, SEC Mw/Mn GO (%) (g-mor1) (g-mor1) Toluene 2 4.5 0.046 230 3040 1.07 Ethyl Ligand 4 17.3 0.190 870 3710 1.30 6 30.2 0.359 1510 4480 1.13 8 40.2 0.514 2010 4950 1.11 10 49.3 0.679 2470 5530 1.10 21 92.3 2.564 4620 7980 1.09

Polymerisation of MPEG(550)MA

[PEGJ/[I]/[Cu]/[L] = 6.37/1/1/2 in 73 w/v% toluene solution at 50/70 "C Initiator 8 (0.10 g, 0.400 mmol), Cu(I)Br (0.057 g, 0.400 mmol, 1 eq) and MPEG(550)MA, (1.60 g, 2.55 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.90 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-propyl-2-pyridylmetharώnine (0.114 g, 0.797 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The temperature was increased to 700C after 5 hours 32 minutes.

Table 4: Data for the polymerization of MPEG(550)MA with initiator 8 at 50/70 0C in 73 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 385 15 4730 1.05 1357 42 10440 1.10 1663 45 10280 1.12

[PEG]/p]/[Cu]/[L] = 6.37/1/1/2 in 73 w/v% toluene solution at 900C Initiator 8 (0.10 g, 0.400 mmol), Cu(I)Br (0.057 g, 0.400 mmol, 1 eq) and MPEG(550)MA (1.60 g, 2.341 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.90 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed iV-propyl-2-pyridylmethanimine (0.114 g, 0.797 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 90 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 5: Data for the polymerization of MPEG(550)MA with initiator 8 at 90 0C in 73 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 138 46 6950 1.09 240 46 7220 1.12 314 48 7370 1.12 1293 59 7690 1.14

[PEG]/[I]/[Cu]/[L] = 31.9/1/1/2 in 67 v/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(550)MA (8.0 g, 12.7 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (14.7 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product.

Table 6: Data for the polymerization of MPEG(550)MA with initiator 8 at 500C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 120 7 7189 1.089 240 15 8976 1.074 360 20 10477 1.074 1320 87 23051 1.147 [PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 67 v/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridyhnethanimine (0.107 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 7: Data for the polymerization of MPEG(550)MA with initiator 8 at 500C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 180 13 4984 1.064 360 31 6628 1.110 1320 86 11282 1.104

[PEG]/[I]/[Cu(I)]/[Cu(II)]/[L] = 6.4/1/0.95/0.05/2 in 67 v/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0545 g, 0.38 mmol, 0.95 eq), Cu(II)Br (0.0045 g, 0.02 mmol, 0.05 eq), and MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 8: Data for the polymerization of MPEG(550)MA with initiator 8 at 500C in 67 v/v% toluene solution. Sample Time Conversion Mn PDi / minutes / % 180 5 4743 4743 360 16 5904 5904 1320 65 11202 11202 1800 78 12245 12245

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 67 v/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(55O)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (3.0 niL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-propyl-2-pyridyhnethanimine (0.119 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 9: Data for the polymerization of MPEG(550)MA with initiator 8 at 50 0C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 180 13 4875 1.041 360 22 5601 1.087 1320 66 9897 1.091

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 67 v/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassedN-octyl-2-pyridyhnethanimine (0.175 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 10: Data for the polymerization of MPEG(550)MA with initiator 8 at 50 0C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 180 19 5034 1.075 360 33 6636 1.101 1320 85 11294 1.097

fPEGJ/flJ/fCuJ/fLJ = 6.4/1/1/2 in 67 v/v% toluene solution at 700C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(550)MA (1.60 g, 2.55 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (3.0 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridyhnethanimine (0.107 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 70 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 11 : Data for the polymerization of MPEG(550)MA with initiator 8 at 700C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 60 20 5455 1.096 120 52 7898 1.114 180 73 9544 1.086 240 80 10207 1.095 [PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 67 v/v% toluene solution at 500C Initiator 8 (6.0 g, 24 mmol), Cu(I)Br (3.44 g, 24 mmol, 1 eq) and MPEG(550)MA (96 g, 0.153 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (176 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed JV- ethyl-2-pyridylmethanimine (6.44 g, 48 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product.

Table 12: Data for the polymerization of MPEG(550)MA with initiator 8 at 500C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 1200 66 8207 1.118 1500 75 9276 1.082 1680 81 9342 1.096

Polymerisation ofMPEG(1000)MA

[PEG]/[I]/[Cu]/[L] = 23.2/1/1/2 in 80 w/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(IOOO)MA (10.0 g, 9.3 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (40 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product. Table 13: Data for the polymerization ofMPEG(1000)MA with initiator 8 at 50 0C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 300 3 9760 1.056 1260 41 19436 1.087 3000 79 29013 1.132 7020 87 30046 1.149

[PEG]/[I]/[Cu]/[L] = 46.3/1/1/2 in 80 w/v% toluene solution at 500C Initiator 8 (0.10 g, 0.40 mmol), Cu(I)Br (0.0574 g, 0.40 mmol, 1 eq) and MPEG(IOOO)MA (20.0 g, 18.5 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (80 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (0.107 g, 0.80 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product.

Table 14: Data for the polymerization ofMPEG(1000)MA with initiator 8 at 50 0C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 300 4 11014 1.070 1260 15 14388 1.080 3000 31 26378 1.096 7020 53 33388 1.154 [PEG]/[I]/[Cu]/[L] = 23.2/1/1/2 in 80 w/v% toluene solution at 500C Initiator 8 (1.0 g, 4.0 mmol), Cu(I)Br (0.574 g, 4.0 mmol, 1 eq) and MPEG(IOOO)MA (100 g, 93.0 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (200 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N- ethyl-2-pyridyhnethanimine (1.07 g, 8.0 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product.

Table 15: Data for the polymerization of MPEG(IOOO)MA with initiator 8 at 500C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 4320 89" 37676 LΪ43 Polymerisations using Initiator 7

Polymerisation of MPEG(395)MA

[PEG]/[I]/[Cu]/[L] = 10/1/1/2 in 50 v/v% toluene solution at 300C A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), initiator 7 (0.601 g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling between nitrogen and vacuum three times. To a second Schlenk tube was added MPEG(395)MA (10 mL, 22.74 mmol), N-(n-ρropyl)-2-ρyridyhnethanimine (0.71 mL, 4.54 mmol) and toluene (10 mL). The mixture was immediately subjected to five freeze-pump-thaw degassing cycles. This solution was then transferred to the Schlenk tube containing the initiator and Cu(I)Br via a cannula. The resulting brown solution was stirred at 30 °C. Samples were removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular weight analysis.

Table 16: Kinetic data for the polymerisation of MPEG(395)MA initiated by 7 in toluene solution (50% v/v) at 30°C ([MPEG(395)MA]o/[CuBr]o/|>IHSBr]o/[ligand]o = 10/1/1/2). Solvent / Time Conversion ln([M]o/[M]) Mn, theo Mn, SEC Mw/Mn Ligand (h) (%) (g-mor1) (g-mor1) Toluene 1 8.9 0.0933 450 2350 1.10 Propyl ligand 2 18.4 0.204 920 2860 1.26 3 27.1 0.316 1360 3100 1.20 4 34.7 0.4259 1740 3600 1.13 17 80.8 1.6510 4050 5670 1.06 [PEG]/[I]/[Cu]/[L] = 10/1/1/2 in 50 v/v% anisole solution at 300C A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 rnmol), initiator 7 (0.601 g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling between nitrogen and vacuum three times. To a second Schlenk tube was added MPEG(395)MA (10 mL, 22.74 mmol), N-(n-ethyl)-2-pyridylmethanimine (0.64 mL, 4.54 mmol) and anisole (10 mL). The mixture was immediately subjected to five freeze-pump-thaw degassing cycles. This solution was then transferred to the Schlenk tube containing the initiator and Cu(I)Br via a cannula. The resulting brown solution was stirred at 30 0C. Samples were removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular weight analysis.

Table 17: Kinetic data for the polymerisation of MPEG(395)MA initiated by 7 in anisole solution (50% v/v) at 30°C ([MPEG(395)MA]o/[CuBr]o/[NHSBr]o/[ligand]o = 10/1/1/2). Solvent / Time Conversion ln([M]0/[M]) Mn, theo Mn, SEC Mw/Mn Ligand (h) (%) (g-nior1) (g-nior1) Anisole 2 17.0 0.1861 850 2670 1.11 Ethyl ligand 4 26.8 0.3116 1340 3460 1.12 6 34.8 0.4277 1740 4260 1.11 22 81.4 1.6809 4080 6350 1.06

[PEG]/[I]/[Cu]/[L] = 10/1/1/2 in 50 v/v% anisole solution at 30 °C A dry Schlenk tube was charged with Cu(I)Br (0.326 g, 2.27 mmol), initiator 7 (0.601 g, 2.27 mmol) and a magnetic follower prior to being deoxygenated by cycling between nitrogen and vacuum three times. To a second Schlenk tube was added MPEG(395)MA (10 mL, 22.74 mmol), N-(«-propyl)-2-pyridylmetharrimine (0.71 mL, 4.54 mmol) and anisole (10 mL). The mixture was immediately subjected to five freeze-pump-thaw degassing cycles. This solution was then transferred to the Schlenk kibe containing the initiator and Cu(I)Br via a cannula. The resulting brown solution was stirred at 30 0C. Samples were removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular weight analysis. Table 18: Kinetic data for the polymerisation of MPEG(395)MA initiated by 7 in anisole solution (50% v/v) at 30°C ([MPEG(395)MA]0/[CuBr]0/[NHSBr]0/[ligand]o = 10/1/1/2). Solvent / Time Conversion ln([M]0/[M]) Mn, theo Mn, SEC Mw/Mn Ligand (h) (%) (g-rnol4) (g-mor1) Anisole 2 9.8 0.1030 490 2070 1.10 Propyl ligand 4 16.8 0.1837 842 2480 1.12 6 28.7 0.3378 1440 2870 1.13 27 83.4 1.7985 4180 6280 1.06

Polymerisation of MPEG(550)MA

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 66 w/v% toluene solution at 300C Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and MPEG(550)MA (7.57 g, 0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (14 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2- pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 30 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 19: Data for the polymerization of MPEG(550)MA with initiator 7 at 30 0C in 66 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 60 19 2850 1.04 131 32 3230 1.10 199 45 3560 1.12 250 53 3760 1.12 298 56 3980 1.12 [PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 66 w/v% toluene solution at 50 0C Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and MPEG(550)MA) (7.57 g, 0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (15 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridyhnethanimine (0.51 g, 3.79 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 20: Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0C in 66 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 59 39 3212 1.09 126 56 3958 1.11 195 69 4375 1.13 246 75 4649 1.13 295 82 4874 1.13

[PEGJ/fIJ/fCu]/[LJ = 23.9/1/1/2 in 66 w/v% toluene solution at 900C Initiator 7 (2.5 g, 9.47 mmol), Cu(I)Br (1.35 g, 9.47 mmol, 1 eq) and MPEG(550)MA (142.0 g, 0.226 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (261 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N- propyl-2-pyridylmethanimine (2.80 g, 0.019 mol) was added. The reaction was placed in a thermostatically controlled oil bath at 90 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (1000 mL). The resulting oil was washed with diethyl ether (3 x 1000 mL) and then dried in vacuo. Table 21: Data for the polymerization of MPEG(550)MA with initiator 7 at 90 0C in 66 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 48 21 4449 1.11 132 40 7198 1.08 185 44 7779 1.07 245 46 8105 1.09 300 48 8331 1.09

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 66 w/v% toluene solution at 500C Initiator 7 (10.0 g, 0.038 mol), Cu(I)Br (5.41 g, 0.038 mol, 1 eq) and MPEG(550)MA (151.0 g, 0.240 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (302 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N- ethyl-2-pyridylmethanimine (10.2 g, 0.0761 mol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0). Conversion was followed by 1H NMR spectrometry and molecular weight analysis by SEC.

Table 22: Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0C in 66 w/v% toluene solution.

Sample Time Conversion Mn PD / minutes / % 235 86A 5Ϊ40 ΪΪ3

[PEG]/[I]/[Cu]/[L] = 6.46/1/1/2 in 62 w/v% toluene at 500C Initiator 7 (2.95 g, 1.119 xlO"2 mol), Cu(I)Br (1.60 g, 1.119 xlO"2 mol) and MPEG(550)MA (45.42 g, 7.23 xlO'2 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (73 mL) was then added to the Schlenk tube and the mixture degassed via three consecutive freeze, pump, thaw cycles. On completion deoxygenated N-ethyl-2-pyridylmethanimine (3.16 mL, 2.24 xlO'2 mol) was added and the Schlenk placed in a thermostatically controlled oil bath at 500C (t=0) and sampled for conversion and molecular weight analysis. The polymer was isolated by washing with diethyl ether and subsequently dialysed in acidified water (pH ~4)

Table 23: Data for the polymerization of MPEG(550)MA with initiator 7 at 500C in 62 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 300 849 45% L22

[PEG]/[I]/[Cu]/[L] = 23.9/1/1/2 in 67 w/v% toluene solution at 500C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), 2,2'-biρyridyl (0.059 g, 0.378 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.68 mL) was added to the Schlenk tube and the resulting solution was deoxygenated via three freeze pump thaw cycles. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 24: Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0C in 67 w/v% toluene solution using 2,2'-bipyridyl ligand. Sample Time Conversion Mn PDi / minutes / % 240 85 15443 LΪΪ

[PEG]/[I]/[Cu]/[LJ = 23.9/1/1/2 in 67 w/v% toluene solution at 500C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), 4,4'-dinonyl- 2,2'-dipyridyl (0.1545 g, 0.378 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.68 mL) was added to the Schlenk tube and the resulting solution was deoxygenated via three freeze pump thaw cycles. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 25: Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0C in 67 w/v% toluene solution using 4,4'-dinonyl-2,2'-dipyridyl ligand. Sample Time Conversion Mn PDi / minutes / % 240 79 15936 LΪ6

[PEG]/[I]/[Cu]/[L] = 23.9/1/1/1 in 67 w/v% toluene solution at 500C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), 1,1,4,7,10,10- hexamethyltriethylenetetramine (0.0435 g, 0.189 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.68 mL) was added to the Schlenk tube and the resulting solution was deoxygenated via three freeze pump thaw cycles. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 26: Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0C in 67 w/v% toluene solution using 1,1,4,7,10,10-hexamethyltriethylenetetramine ligand. Sample Time Conversion Mn PDi / minutes / % 240 8(5 19060 LΪ6

[PEGJ/flJ/fCuJ/fLJ = 23.9/1/1/1 in 67 w/v% toluene solution at 500C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), N,N,N',N",N"- pentamethyldiethylenetriamine (0.0328 g, 0.189 mmol), MPEG(550)MA, (2.84 g, 4.52 mmol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.68 mL) was added to the Schlenk tube and the resulting solution was deoxygenated via three freeze pump thaw cycles. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 27: Data for the polymerization of MPEG(55O)MA with initiator 7 at 50 0C in 67 w/v% toluene solution using N,N,N',N",N"-pentamethyldiethylenetriamine ligand. Sample Time Conversion Mn PDi / minutes / % 240 95 19019 L20

Polymerisation of MPEG(1000)MA

[PEG]/[I]/[Cu]/[L] = 13.9/1/1/2 in 66 w/v% toluene solution at 900C Initiator 7 (0.526 g, 1.99 mmol), Cu(I)Br (0.29 g, 2.02 mmol, 1 eq) and MPEG(IOOO)MA (29.62 g, 0.027 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (60 niL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridyhnethanimine (0.51 g, 3.96 mol) was added. The reaction was placed in a thermostatically controlled oil bath at 90 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (1000 mL). The resulting oil was washed with diethyl ether (3 x 1000 mL) and then dried in vacuo.

Table 28: Data for the polymerization of MPEG(IOOO)MA with initiator 7 at 90 0C in 66 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 1250 47.3 12180 1.16 2460 50.4 12460 1.16 3890 52.8 12540 1.20 [PEG]/[I]/[Cu]/[L] = 9.0/1/0.24/0.24 in 75 w/v% toluene solution at 500C. Initiator 7 (5.0 g, 0.019 mol), Cu(I)Br (0.66 g, 4.61 mmol, 0.24 eq) and MPEG(IOOO)MA (185.0 g, 0.171 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (740 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridyhnethanimine (1.24 g, 9.24 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 29: Data for the polymerization of MPEG(IOOO)MA with initiator 7 at 50 0C in 75 w/v% toluene solution.

Sample Time Conversion Mn PDi

/ minutes / %

60 7 5650 0.93 120 11 5595 0.97 285 20 6315 1.02 1340 43 7993 1.08 7476 63 9543 1.06

[PEG]/[I]/[Cu]/[L] = 18.5/1/1/2 in 75 w/v% toluene solution at 500C Initiator 7 (1.0 g, 3.79 mmol), Cu(I)Br (0.54 g, 3.79 mmol, 1 eq) and MPEG(IOOO)MA (151.4 g, 0.140 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (608 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (1.02 g, 7.57 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 30: Data for the polymerization of MPEG(IOOO)MA with initiator 7 at 50 0C in 75 w/v% toluene solution. Sample Time Conversion Mn PDi

/ minutes / %

4005 100 8607 1.32

[PEG]/[I]/[Cu]/[L] = 18.5/1/1/2 in 75 w/v% toluene solution at 50 °C Initiator 7 (2.0 g, 7.57 mmol), Cu(I)Br (1.08 g, 7.57 mmol, 1 eq) MPEG(IOOO)MA (151.47 g, 0.140 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (606 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N- ethyl-2-pyridylmethanimine (2.03 g, 0.015 mol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 31: Data for the polymerization of MPEG(IOOO)MA with initiator 7 at 50 0C in 75 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 195 20 7270 1.04 1380 53 11964 1.08 2735 73 13945 1.08

Polymerisation of MPEG(2000)MA

[PEG]/[I]/[Cu]/[L] = 19.2/1/1/2 in 80 w/v% toluene solution at 300C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq) and (MPEG(2000)MA) (7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethammine (0.05 g, 0.38 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 30 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (400 mL). The resulting white powder was filtered, dissolved in toluene (2OmL) and precipitated in diethyl ether (400 mL). This procedure was repeated three times.

Table 32: Data for the polymerization of MPEG(2000)MA with initiator 7 at 30 0C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 89 4 3380 1.04 291 9 9820 1.09 901 17 10030 1.07 1369 23 11080 1.07 2760 26 12610 1.07 3965 28 14830 1.04

[PEG]/[I]/[Cu]/[L] = 19.2/1/1/2 in 80 w/v% toluene solution at 500C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq) and MPEG(2000)MA (7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridyhnethanimine (0.05 g, 0.38 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (400 mL). The resulting white powder was filtered, dissolved in toluene (2OmL) and precipitated in diethyl ether (400 mL). This procedure was repeated three times.

Table 33: Data for the polymerization of MPEG(2000)MA with initiator 7 at 50 0C in 80 w/v% toluene solution. Sample Time Conversion Mn PDi / minutes / %

289 12 10920 1.07 899 24 14450 1.05 1367 33 15810 1.04 2758 45 20220 1.07 3962 53 23180 1.07

[PEG]/p]/[Cu]/[L] = 19.2/1/1/2 in 80 w/v% toluene solution at 900C Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq) and MPEG(2000)MA (7.55 g, 3.63 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (28 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridyhnethanimine (0.05 g, 0.38 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 90 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by the dropwise addition of the reaction solution to a vigorously stirred solution of diethyl ether (400 mL). The resulting white powder was filtered, dissolved in toluene (2OmL) and precipitated in diethyl ether (400 mL). this procedure was repeated three times.

Table 34: Data for the polymerization of MPEG(2000)MA with initiator 7 at 90 0C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 86 18 11100 1.08 289 26 14870 1.08 899 31 17900 1.08 1367 35 18110 1.09 2758 38 18110 1.09 3962 39 18240 1.08 [PEG]/[I]/[Cu]/[L] = 9.3/1/1/2 in 66 w/v% toluene solution at 900C Initiator 7 (0.5 g, 1.89 mmol), Cu(I)Br (0.27 g, 1.89 mmol, 1 eq) and MPEG(IOOO)MA (18.90 g, 0.018 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (35 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.51 g, 3.79 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 90 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 35: Data for the polymerization ofMPEG(1000)MA with initiator 7 at 900C in 66 w/v% toluene solution.

Sample Time Conversion Mn PDi /minutes / % 4160 88J 9870 L22

[PEG]/[I]/[Cu]/[L] = 19.2/1/1/2 in 80 w/v% toluene solution at 50/700C Initiator 7 (0.67 g, 2.53 mmol), Cu(I)Br (0.36 g, 2.53 mmol, 1 eq) and MPEG(2000)MA (101.24 g, 0.049 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (405 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridyhnethanimine (0.68 g, 5.07 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The temperature was increased to 70 0C after 45 hours 15 minutes.

Table 36: Data for the polymerization of MPEG(2000)MA with initiator 7 at 50/70 0C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 4030 47 24,600 L06

[PEG]/[I]/[Cu]/[L] = 19.2/1/1/2 in 75 w/v% toluene at 50/70 °C Initiator 7 (0.66 g, 2.5 xlO"3 mol), Cu(I)Br (0.36 g, 2.5 xlO"3 mol) and MPEG(2000)MA (100.0 g, 4.81 xlO'2 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (300 mL) was then added to the Schlenk tube and the mixture deoxygenated by purging with nitrogen for 1 hour. Deoxygenated N-ethyl-2- pyridylmethanimine (0.706 mL, 5.0 xlO"3 mol) was then added and the Schlenk placed in a thermostatically controlled oil bath at 50 0C (t=0). The temperature was increased to 7O0C after 24 hours. The polymer was isolated by washing with diethyl ether and subsequently dialysed in acidified water (pH ~4).

Table 37: Data for the polymerization of MPEG(2000)MA with initiator 7 at 50/700C in 75 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 2880 942 21900 L2Ϊ

[PEG]/[I]/[Cu]/[L] = 28.8/1/1/2 in 75 w/v% toluene at 50/700C. Initiator 7 (0.44 g, 1.67 xW3 mol), Cu(I)Br (0.24 g, 1.67 xlO"3 mol) and MPEG(2000)MA (100.0 g, 4.81 xlO'2 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (300 mL) was then added to the Schlenk tube and the mixture deoxygenated by purging with nitrogen for 1 hour. Deoxygenated N-ethyl-2- pyridylmethanimine (0.47 mL, 3.33 xlO"3 mol) was then added and the Schlenk placed in a thermostatically controlled oil bath at 50 0C C (t=0). The temperature was increased to 7O0C after 24 hours. The polymer was isolated by washing with diethyl ether and subsequently dialysed in acidified water (pH ~4).

Table 38: Data for the polymerization of MPEG(2000)MA with initiator 7 at 50/70 0C in 75 w/v% toluene solution. Sample Time Conversion Mn PDi / minutes / % 2880 92^8 26370 L26 Polymerisations using Initiator 5

Polymerisation of MPEG(550)MA

[PEG]/[I]/[Cu]/[L] = 64/1/1/2 in 75 w/v% toluene solution at 50/9IfC Initiator 5 (0.25 g, 0.622 mmol), Cu(I)Br (0.089 g, 0.622 mmol, 1 eq) and MPEG(550)MA (24.90 g, 0.040 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (100 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (0.167 g, 1.245 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The temperature was increased to 9O0C after 3 hours 15 minutes.

Table 39: Data for the polymerization of MPEG(550)MA with initiator 5 at 50/90 0C in 75 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 150 3 5376 1.07 353 9 10390 1.09 1750 27 13890 1.16

Polymerisation of MPEG(1000)MA [PEG]/[I]/[Cu]/[L] = 37/1/1/2 in 75 w/v% toluene solution at 50 "C Initiator 5 (0.125 g, 0.031 mmol), Cu(I)Br (0.044 g, 0.031 mmol, 1 eq) and MPEG(IOOO)MA (12.45 g, 0.012 mol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (50 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (0.083 g, 0.062 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 40: Data for the polymerization ofMPEG(1000)MA with initiator 5 at 50 0C in 75 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / %

352 10 9713 1.06 1716 15 10924 1.08 2725 16 11240 1.08 4142 15 12800 1.11 Polymerisations using Initiator 9

Polymerisation of MPEG(2000)MA

[PEG]/[I]/[Cu]/[L] = 28.8:1/1/2 in 67 w/v% acetone at 500C initiator 9 (0.035 g, 1.667 xlO"4 mol), Cu(I)Br (0.024 g, 1.667 xlO^4 mol) and MPEG(2000)MA (10 g, 4.81 xlO'3 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Acetone (20 mL) was then added to the Schlenk tube and the mixture degassed via three consecutive freeze, pump, thaw cycles. On completion deoxygenated N-ethyl-2-pyridylmethanimine (0.05 mL, 3.54 xlO"4 mol) was added and the Schlenk placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples removed periodically for conversion and molecular weight analysis.

Table 41 : Data for the polymerization of MPEG(2000)MA with initiator 9 at 500C in 67 w/v% acetone solution.

Sample Time Conversion Mn PDi / minutes / % 60 3 25270 1.06 360 19 22690 1.08 3480 74 32080 1.14 Polymerisations using Initiator 6

Polymerisation of MPEG(2000)MA

[PEG]/[I]/[Cu]/[L] = 14.4/1/1/2 in 67 w/v% toluene at 300C Initiator 6 (0.119 g, 3.333 Xl(T4 mol), Cu(I)Br (0.048 g, 3.333 xlO"4 mol) and MPEG(2000)MA (10 g, 4.81 *10"3 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (20 mL) was then added to the Schlenk tube and the mixture degassed via three consecutive freeze, pump, thaw cycles. On completion deoxygenated N-n-propyl-2-pyridylmethanimine (0.10 mL, 6.667 xlO"4 mol) was added and the Schlenk placed in a thermostatically controlled oil bath at 30 0C (t=0) and sampled for conversion and molecular weight analysis.

Table 42: Data for the polymerization of MPEG(2000)MA with initiator 6 at 30 0C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 3900 29J 24120 L08

[PEG]/[I]/[Cu]/[L] = 21.63/1/1/2 in 67 w/v% toluene at 300C Initiator 6 (0.079 g, 2.222 xlO"4 mol), Cu(I)Br (0.031 g, 2.222 xlO"4 mol) and PEG(2000)MA (10 g, 4.81 xlO"3 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (20 mL) was then added to the Schlenk tube and the mixture degassed via three consecutive freeze, pump, thaw cycles. On completion deoxygenatedN-«-propyl-2-pyridylmethanimine (0.066 mL, 4.444 xlO"4 mol) was added and the Schlenk placed in a thermostatically controlled oil bath at 300C (t=0) and sampled for conversion and molecular weight analysis.

Table 43: Data for the polymerization of MPEG(2000)MA with initiator 6 at 300C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 6600 27 27480 ΪTΪ0

[PEG]/[I]/[Cu]/[L] = 28.8/1/1/2 in 67 w/v% toluene at 30 0C Initiator 6 (0.059 g, 1.667 xlO"4 mol), Cu(I)Br (0.024 g, 1.667 XlO-4 mol) and MPEG(2000)MA (10 g, 4.81 xlO"3 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (20 mL) was then added to the Schlenk tube and the mixture degassed via three consecutive freeze, pump, thaw cycles. On completion deoxygenatedN-n-propyl-2-pyridylmethanimine (0.049 mL, 1.667 xlO"4 mol) was added and the Schlenk placed in a thermostatically controlled oil bath at 300C (t=0) and sampled for conversion and molecular weight analysis.

Table 44: Data for the polymerization of MPEG(2000)MA with initiator 6 at 300C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 6600 183 25290 • U)9 Polymerisations using Initiator 10

IO

Polymerisation of MPEG(395)MA

[PEGl/ffl/fCuJ/IL] = 25/1/1/2 in 67 w/v% toluene at 500C Initiator 10 (0.81 g, 1.68 χlθ"3 mol), Cu(I)Br (0.24 g, 1.68 xlO"3 mol) and MPEG(395)MA (20.0 g, 4.21 xlO"2 mol) and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Toluene (41 mL) was then added to the Schlenk tube and the mixture deoxygenated by purging with nitrogen for 1 hour. Deoxygenated N-«- propyl-2-pyridylmethanimine (0.53 mL, 3.37 xlO"3 mol) was then added and the Schlenk placed in a thermostatically controlled oil bath at 500C (t=0) and sampled for conversion and molecular weight analysis.

Table 45: Data for the polymerization of MPEG(395) with initiator 10 at 500C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 9(XJ 52^9 63(X) UΪ Polymerisations using Initiator 11

W Polymerisation of MPEG(550)MA

[PEGJ/[IJ/[Cu]/[L] = 6.4/1/1/2 in 67v/v% toluene solution at 500C Initiator 11 (0.103 g, 0.380 mmol), Cu(I)Br (0.054 g, 0.380 mmol, 1 eq) and MPEG(550)MA (1.51 g, 2.41 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (2.78 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 46: Data for the polymerization of MPEG(550)MA with initiator 11 at 500C in 61 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 60 24 3545 1.080 120 41 4002 1.102 180 53 4243 1.104 240 64 4506 1.109 300 70 4677 1.108

[PEG]/[I]/[Cu]/[L] = 6.4/1/1/2 in 67 v/v% toluene solution at 500C Initiator 11 (3.0 g, 11.1 mmol), Cu(I)Br (1.584 g, 11.1 mmol, 1 eq) and MPEG(550)MA (44.27 g, 70.5 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (81.3 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed iV-ethyl-2-pyridyhnethanimine (2.97 g, 22.2 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product.

Table 47: Data for the polymerization of MPEG(550)MA with initiator 11 at 50 0C in 67 v/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 60 27 4800 1.096 120 45 5427 1.115 180 60 5895 1.127 240 67 6215 1.133 300 72 6343 1.125

Polymerisation of MPEG(2000)MA

[PEG]/[I]/[Cu]/[L] = 12/1/1/2 in 80 w/v% toluene solution at 50/700C Initiator 11 (0.1 g, 0.369 mmol), Cu(I)Br (0.053 g, 0.369 mmol, 1 eq) and MPEG(2000)MA (9.24 g, 4.44 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (37 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed iV-ethyl-2-pyridyhnethanimine (0.10 g, 0.758 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The temperature was increased to 70 0C after 113 hours. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product. Table 48: Data for the polymerization of MPEG(2000)MA with initiator 11 at 50/70 0C in 80 w/v% toluene solution. — _ SampleTime Conversion /minutes /% 1020 21 1440 23 6780 40 9660 62 20333 1.117

[PEGJ/flJ/fCuJ/fLJ = 24/1/1/2 in 80 w/v% toluene solution at 50/700C Initiator 11 (0.1 g, 0.369 mmol), Cu(I)Br (0.053 g, 0.369 mmol, 1 eq) and MPEG(2000)MA (18.48 g, 8.88 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (74 mL) was added to the Schlenk tube. The resulting solution was deoxygenated by bubbling with nitrogen for 1 hour and then degassed N-ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The temperature was increased to 70 0C after 113 hours. The polymer was purified by removing the solvent in vacuo and dialysising the residue using acidic water (pH ~4). Subsequent freeze drying isolated the product.

Table 49: Data for the polymerization of MPEG(2000)MA with initiator 11 at 50/70 0C in 80 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 1020 16 2580 19 6780 22 8220 53 9660 0.57 22262 1.140 Polymerisations using Initiator 12

12

Polymerisation Of MPEG(IOOO)MA

[PEG]/[I]/[Cu]/[L] = 5/1/1/2 in 66 v/v% toluene solution at 70 0C N-(ethyl)-2-pyridy]methanimine ligand (1.41 mL, 10.92 mmol), initiator 12 (1.633 g, 5.46 mmol), and MPEG(IOOO)MA (27.3 mL, 30 g, 27.3 mmol) were charged to a dry Schlenk tube along with toluene (60 mL) as the solvent and mesitylene (1 mL) as an internal standard. The tube was sealed with a rubber septum and subjected to three freeze pump thaw cycles. This solution was then cannulated under nitrogen into another Schlenk tube, previously evacuated and filled with nitrogen, containing Cu(I)Cl (0.543 g, 5.46 mmol) and a magnetic follower. The brown solution was subsequently heated to 70 0C with constant stirring (t = 0). Samples were removed periodically using a degassed syringe for molecular weight and conversion analysis. After 48 h the mixture was diluted with 50 mL of toluene, air was bubbled for 6 h and the green suspension was kept at 0° C overnight. After filtration through a short neutral alumina column to remove the copper salt, the polymer was precipitated from diethyl ether. The polymer was collected by filtration and dried in vacuum oven (4O0C) overnight.

Table 50: Data for the polymerization of MPEG(IOOO)MA with initiator 12 at 700C in 66 v/v% toluene solution.

Sample Time Conversion Mn PDi / hours / % 1 17.8 1370 1.23 2.5 56.6 10700 1.09 4 73.5 11600 1.15 5 78.9 11600 1.11 6 81.3 11600 1.19 7 85.9 12600 1.15

[PEGJ/flJ/fCuJ/fLJ - 20/1/1/2 in 66 v/v% toluene solution at SO °C N-(ethyl)-2-pyridylmethanimine ligand (0.35 mL, 2.73 mmol), initiator 12 (0.41 g, 1.37 mmol), PEG(IOOO)MA (27.3 mL, 30 g, 27.3 mmol) were charged to a dry Schlenk tube along with toluene (60 mL) as the solvent and mesitylene (1 mL) as internal standard. The tube was sealed with a rubber septum and subjected to three freeze pump thaw cycles. This solution was then cannulated under nitrogen into another Schlenk tube, previously evacuated and filled with nitrogen, containing Cu(I)Br (0.197 g, 1.37 mmol) and a magnetic follower. The brown solution was subsequently heated to 50 °C with constant stirring (t = 0). Samples were removed periodically using a degassed syringe for molecular weight and conversion analysis. Half the reaction solution was removed with a dry cannula when conversion was at 66%, bubbled for 6 h with air, and passed over a short neutral alumina column to removed copper salt. The solvent was removed under vacuum and the unreacted monomer was removed by dialysis to give the polymer as a white powder. After 48 h the remaining reaction mixture was diluted with 50 mL of toluene, air was bubbled for 6 h and the green suspension was kept at 0° C overnight. After filtration through a short neutral alumina column to remove the copper salt, the polymer was precipitated from diethyl ether. The polymer was collected by filtration and dried in vacuum oven (400C) overnight.

Table 51: Data for the polymerization of MPEG(IOOO)MA with initiator 12 at 50 0C in 66 v/v% toluene solution.

Sample Time Conversion Mn PDi / hours / % 1 5 1566 1.14 3 11 11400 1.06 6 26 13000 1.07 8 31 12700 1.09 21 61 13600 1.13 24 66 13800 1.14 28 73 14500 1.2 31 76 14900 1.21 46 87 14600 1.18 50.5 89 16000 1.2 54 0.9 17600 1.19 Polymerisations using Initiator 13

Polymerisation of MPEG(550)MA

[PEG]/[I]/[Cu]/[L] = 15.9/1/1/2 in 67 w/v% toluene solution at 300C Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) and MPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridyhnethanimine (0.074 g, 0.56 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 30 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 52: Data for the polymerization of MPEG(550)MA with initiator 13 at 300C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % Ϊ440" ϊl 5626 L08 4260 14.0 6153 1.11

[PEG]/[I]/[Cu]/[L] = 8/1/1/2 in 67 w/v% toluene solution at 500C Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) and MPEG(550)MA (1.38 g, 2.20 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (2.75 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.074 g, 0.56 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 53: Data for the polymerization of MPEG(550)MA with initiator 13 at 500C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 7200 448 7880 LΪ6

[PEG]/[I]/[Cu]/[L] = 15.9/1/1/2 in 67 w/v% toluene solution at 500C Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) and MPEG(55O)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridylmethanimine (0.074 g, 0.56 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 54: Data for the polymerization of MPEG(550)MA with initiator 13 at 50 0C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % Ϊ440 Ϊ6A 797Ϊ LΪ2 8640 27.2 8378 1.14

[PEGJ/flJ/fCuJ/fLJ = 31.8/1/1/2 in 67 w/v% toluene solution at 500C Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) and MPEG(55O)MA (5.51 g, 8.77 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (11.0 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridyhnethanimine (0.074 g, 0.56 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 55: Data for the polymerization of MPEG(550)MA with initiator 13 at 500C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 1440 9.6 8504 1.11 5760 16.8 9999 1.14 8640 17.8 10208 1.13

[PEG]/[I]/[Cu]/[L] = 15.9/1/1/2 in 67 w/v% toluene solution at 700C Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Br (0.039 g, 0.28 mmol, 1 eq) and MPEG(55O)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed N-ethyl-2-pyridyhnethanimine (0.074 g, 0.56 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 70 °C (t=0) and samples were removed periodically for conversion and molecular weight analysis.

Table 56: Data for the polymerization of MPEG(550)MA with initiator 13 at 700C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 120 13.6 5768 1.09 300 21.3 6814 1.10 4260 41.0 8444 1.15 [PEG]/[I]/[Cu]/[L] = 15.9/1/1/2 in 67 w/v% toluene solution at 50/90 °C Initiator 13 (0.10 g, 0.28 mmol), Cu(I)Cl (0.0273 g, 0.28 mmol, 1 eq) and MPEG(550)MA (2.76 g, 4.39 mmol), and a magnetic follower were placed in an oven dried Schlenk tube. The Schlenk tube was evacuated and flushed with dry nitrogen three times. Deoxygenated toluene (5.5 mL) was added to the Schlenk tube. The resulting solution was deoxygenated via three freeze pump thaw cycles and then degassed iV-ethyl-2-pyridyhnethanimine (0.074 g, 0.56 mmol) was added. The reaction was placed in a thermostatically controlled oil bath at 50 0C (t=0) and samples were removed periodically for conversion and molecular weight analysis. The temperature was increased to 900C after 163 hours

Table 57: Data for the polymerization of MPEG(550)MA with initiator 13 at 50/90 0C in 67 w/v% toluene solution.

Sample Time Conversion Mn PDi / minutes / % 9780 Ϊ8 4091) U)9 12660 81.0 16504 1.31 Polymerisations using Initiator 14

Boc

14-

Polymerisation of MPEG(395)MA

[PEG]/[I]/[Cu]/[L] = 6/1/1/2 in 50 v/v% toluene solution at 400C N-(ethyl)-2-pyridylmethanimine ligand (1.07 mL, 1.017 g, 7.58 x 10"3 mol), initiator 14 (1.229 g, 3.79 x 10'3 mol) and MPEG(395)MA (10.80 g, 22.70 x 10"3 mol) were charged to a dry Schlenk tube along with toluene (10 mL) as the solvent (50% v/v). The tube was sealed with a rubber septum and subjected to three rreeze-pump-thaw cycles. This solution was then cannulated under nitrogen into another Schlenk tube, previously evacuated and filled with nitrogen, containing Cu(I)Br (0.544 g, 3.79 x 10" J mol) and a magnetic follower. The brown solution was subsequently heated to 40 0C with constant stirring (t = 0). Samples were removed periodically using a degassed syringe for molecular weight and conversion analysis. After 48 h the mixture was diluted with 50 mL of toluene, air was bubbled for 6 h and the green suspension was kept at 0 ° C overnight. After filtration through a celite® pad, the solvent was removed under reduced pressure to give a yellow-brown oil which was dissolved in water (250 mL) and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m2) to give the expected polymer as a pale yellow oil.

Table 58: Polymerisation data for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 6:1:1:2. Monomer/ Time Conv. ln([M]0/[M]) Mn PDI Initiator (mins) (%) 6:1 (40 0C) 60 34.76 0.478 5876 1.03 120 49.43 0.667 7250 1.08 180 62.05 0.889 8089 1.08 240 71.54 1.118 9013 1.10 300 76.33 1.359 9262 1.10 360 80.18 1.556 9561 1.07 420 86.93 1.904 9932 1.10 480 88.72 2.216 10195 1.10

[PEG]/[I]/[Cu]/[L] = 28/1/1/2 in 50 v/v% toluene solution at 40 °C N-(ethyl)-2-pyridylmethanimine ligand (1.07 mL, 1.017 g, 7.58 x 10'3 mol), initiator 14 (0.263 g, 0.812 x 1O-3 mol) and MPEG(395)MA (10.80 g, 22.70 x 10"3 mol) were charged to a dry Schlenk tube along with toluene (10 mL) as the solvent (50% v/v). The tube was sealed with a rubber septum and subjected to three freeze-pump-thaw cycles. This solution was then cannulated under nitrogen into another Schlenk tube, previously evacuated and filled with nitrogen, containing Cu(I)Br (0.116 g, 0.812 x 10'3 mol) and a magnetic follower. The brown solution was subsequently heated to 40 0C with constant stirring (t = 0). Samples were removed periodically using a degassed syringe for molecular weight and conversion analysis. After 48 h the mixture was diluted with 50 mL of toluene, air was bubbled for 6 h and the green suspension was kept at 0° C overnight. After filtration through a celite® pad, the solvent was removed under reduced pressure to give a yellow-brown oil which was dissolved in water (250 mL) and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m2) to give the expected polymer as a pale yellow oil.

Table 59: Polymerisation data for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]:[initiator]:[CuBr]:[L] = 28:1:1:2. T= 40 "C. Monomer/ Time Conv. ln([M]0/[M]) Mn PDI Initiator (mins) (%) 28:1 (40 0C) 60 21.8 0.246 5500 1.05 120 24.7 0.284 5798 1.06 180 37.4 0.468 7001 1.09 240 41.5 0.536 7202 1.08 300 46.2 0.620 7633 1.07 360 49.3 0.680 7733 1.09 420 50.7 0.707 7899 1.09 480 55.4 0.808 8099 1.08

[PEG]/[I]/[Cu]/[L] = 28/1/1/2 in 50 v/v% toluene solution at 600C N-(ethyl)-2-pyridylmethanimine ligand (1.07 mL, 1.02 g, 7.58 x 10"3 mol), initiator 14 (0.263 g, 0.812 x 10"3 mol) and MPEG(395)MA (10.80 g, 22.70 x IO^3 mol) were charged to a dry Schlenk tube along with toluene (10 mL) as the solvent (50% v/v). The tube was sealed with a rubber septum and subjected to three freeze-pump-thaw cycles. This solution was then cannulated under nitrogen into another Schlenk tube, previously evacuated and filled with nitrogen, containing Cu(I)Br (0.116 g, 0.812 x 10"3 mol) and a magnetic follower. The brown solution was subsequently heated to 60 0C with constant stirring (t = 0). Samples were removed periodically using a degassed syringe for molecular weight and conversion analysis. After 48 h the mixture was diluted with 50 mL of toluene, air was bubbled for 6 h and the green suspension was kept at 0° C overnight. After filtration through a celite® pad, the solvent was removed under reduced pressure to give a yellow-brown oil which was dissolved in water (250 mL) and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m2) to give the expected polymer as a pale yellow oil.

Table 60: Polymerisation data for TMM-LRP of MPEG(395)MA using initiator 14, [monomer]: [initiator]: [CuBr]: [L] = 28:1:1:2. T = 60 0C. Monomer/ Time Conv. ln([M]0/[M]) Mn PDI Initiator (mins) (%) 28:1 (60 0C) 60 38.0 0.427 5233 1.06 120 48.7 0.682 5656 1.07 180 58.9 0.969 6116 1.06 240 67.3 1.257 6185 1.08 300 74.3 1.441 6416 1.07 360 78.9 1.618 6284 1.08 420 85.1 2.035 6291 1.08 480 89.1 2.182 6610 1.08 [PEG]/[I]/[Cu]/[L] = 10/1/1/2 in 50 v/v% da-toluene solution at 400C N-(n-octyl)-2-pyridylmethanimine ligand (0.052 mL, 0.050 g, 0.228 x 10"3 mol), initiator 14 (0.037 g, 0.114 x 10"3 mol) and MPEG(395)MA (0.050 mL, 0.540 g, 1.14 x 10"3 mol) were charged to a dry Schlenk tube along with d8-toluene (0.50 mL) as the solvent (50% v/v). The tube was sealed with a rubber septum and subjected to three freeze-pump-thaw cycles. This solution was then cannulated under nitrogen into an NMR tube, previously evacuated and filled with nitrogen, containing Cu(I)Br (0.016 g, 0.114 x 10*3 mol). The tube was then heated to 40 0C and 1H NMR spectra were recorded every 15 minutes. Polymerisations using Initiator IS

Polymerisation of MPEG(395)MA

[PEGJ/[I]/[Cu]/[L] = 8/1/1/2 in 50 v/v% toluene solution at 300C N-(ethyl)-2-pyridylmethanimine ligand (0.80 mL, 0.76 g, 5.68 x 10'3 mol), initiator 15 (2.03 g, 5.68 x 10-3 mol) MPEG(395)MA (20.0 mL, 21.6 g, 45.50 x 10"3 mol) were charged to a dry Schlenk tube along with toluene (20 mL) as the solvent (50% v/v). The tube was sealed with a rubber septum and subjected to three freeze-pump-thaw cycles. This solution was then cannulated under nitrogen into another Schlenk tube, previously evacuated and filled with nitrogen, containing Cu(I)Br (0.41 g, 2.84 x 10"3 mol) and a magnetic follower (t = 0). The brown solution was subsequently stirred at 300C. Samples were removed periodically using a degassed syringe for molecular weight and conversion analysis. After 7 h the mixture was diluted with 50 mL of toluene, air was bubbled for 6 h and the green suspension was kept at 0° C overnight. After filtration through a celite® pad, the solvent was removed under reduced pressure to give a yellow-brown oil which was dissolved in water (250 mL) and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m2) to give the expected polymer as a pale yellow oil.

Table 61: Polymerisation data for the TMM-LRP of MPEG(395)MA using initiator 15, [monomer]: [initiator]: [CuBr]: [L] = 8:1:1:2. T = 30 "C.

Monomer/ Time Conv. ln([M]0/[M]) Mn PDΪ Initiator (mins) (%) 8:1 (30 0C) 60 22.90 0.478 4532" L07 120 34.64 0.667 5008 1.11 180 45.66 0.889 5379 1.11 246 50.97 1.118 5662 1.09 420 67.14 1.359 6165 1.08 Reactions of PoIyPEG Polymers

Reactions of PoIyPEG Polymers prepared from initiator 8

Hydrolytic stability of the succinimide end group of PoIyPEG polymer initiated by 8

On-line 1H NMR experiments was carried out, each using a different buffer. N-

succinimidyl (initiator 8) terminated poly(MPEG(395)MA (Mn= 6400g/mol, PDI=

1.09) (50mg, 0.00781 x 10'3 mol) were introduced in an NMR tube and dissolved in

0.5 mL of the appropriate phosphate buffer (pH = 8, C=IOO mM or 20OmM). NMR

spectra were recorded regularly.

Table 62: Kinetic data for the hydrolysis of N-succinimidyl terminated

poly(MPEG(395)MA initiated by 8 in different buffers.

13 45.1 13 56.9 13.5 45.0 13.5 53.7 14.5 48.9 15 48.4 15.5 50.1 16 49.6 16.5 50.1 17 52.6 17.5 50.2 18 53.2 18.5 52.0 19 54.9 19.5 55.1 20 53.9 20.5 55.5 21 55.9 21.5 53.0 22 54.5 22.5 55.9 23 55.3 23.5 57.6 24 58.9

Bioconjuction of succinimide teminated PoIyPEG polymer initiated by 8

A set of three experiments was carried out, each containing a different ratio polymer /

lysozyme. Low molecular weight succinimidyl ester terminated

poly(MPEG(395)MA) prepared from initiator 8 (Mn = 6400 g/mol, PDI= 1.11) (8.9

mg, 1.39xlO'6 mol) for a ratio 2/1, (22.6 mg, 3.50xl0"6 mol) for a ratio 5/1 and (89.5

mg, 13.99xlO'6 mol) for a ratio 20/1 and lysozyme (10 mg, 0.699xl0"6 mol) was

dissolved in 10 ml of anhydrous DMSO and 0.5 mL of anhydrous TEA and stirred at

room temperature under nitrogen. Samples were taken periodically and analyzed by

HPLC. The HPLC system was fitted with a guard column, a BioSep-SEC-S3000

column and a UV detector continuously measuring the relative absorbance of the

mobile phase at 215 nm. The system was eluted with 0.1% v/v trifluoroacetic acid

solution in water and acetonitrile (69/31 v/v) at a rate of 0.5 mL/min. In the case of a

ratio 30:1, the crude was analysed by SDS-PAGE (polyacrylamide resolving gel

cross-linking: 15%, running buffer: 25 mM TRIS base, 250 mM glycine, 0.1% SDS,

pH 8.7). Reactions of PoIyPEG Polymers prepared from initiator 7

Hydrolytic stability of the succinimide end group of PoIyPEG polymer initiated by 7

On-line IH NMR experiments was carried out, each using a different buffer. N-

succinimidyl (initiator 7) terminated Poly(MPEG(395)MA (Mn= 2700g/mol, PDI=

1.12) (50mg, 0.0185 x 10-3 mol) were introduced in an NMR tube and dissolved in

0.5 mL of the appropriate buffer (200 mM phosphate buffer (pH = 6 and pH = 8), 100

mM phosphate buffer (pH = 8) or 200 mM borate buffer (pH = 9.2)). NMR spectra

were recorded regularly.

Table 63: Kinetic data for the hydrolysis of the succinimide end group of

Poly(MPEG(395)MA polymer initiated by 7 in different buffers.

Conversion of succinimide end group of PoIyPEG to amine group

Ethylenediamine (22.4 mL, 0.333 mol) and a magnetic follower were placed into a

three necked round bottom flask fitted with a pressure equalising dropping funnel.

The system was flushed with nitrogen and placed under positive pressure. A solution

of succinimide terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (3.0 g, 9.38

xlO"4 mol) dissolved in anhydrous dichloromethane (12 mL) was added to the

dropping funnel and the solution added drop-wise to the ethylenediamine. The

solution was left stirring for 16 hours before dialysing and subsequently freeze drying

to isolate the product. 1H NMR spectra shows the reduction of the succinimide O=C-

CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group

Ethylenediamine (14.85 mL, 0.222 mol) and a magnetic follower were placed into a

three necked round bottom flask fitted with a pressure equalising dropping funnel.

The system was flushed with nitrogen and placed under positive pressure. A solution

of succinimide terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25

xlO"4 mol) dissolved in water (20 mL) was added to the dropping funnel and the

solution added drop-wise to the ethylenediamine. The solution was left stirring for 16

hours before dialysing and subsequently freeze drying to isolate the product. 1H NMR

spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75

ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (20.0 mL, 0.299 mol), water (20 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated ρoly(MPEG(550)MA) [Mn 4590 PDi 1.22] (5.0 g, 1.56 xlO"3 mol) dissolved in water (50 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was left stirring for 24 hours before dialysing and subsequently freeze drying to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (1.35 mL, 0.02 mol), water (5 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (1.0 g, 3.13 xlO"4 mol) dissolved in water (25 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was left stirring for 3 hours before adding the solution to 2 L of water and dialysing. Subsequently the dialysed solution was freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (1.35 mL, 0.02 mol), anhydrous dichloromethane (5 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated ρoly(MPEG(550)MA) [Mn 4590 PDi 1.22] (1.0 g, 3.13 xlO"4 mol) dissolved in anhydrous dichloromethane (25 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was left stirring for 3 hours before adding the solution to 2 L of water and dialysing. Subsequently the dialysed solution was freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm. Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (2.68 niL, 0.04 mol), water (10 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25 xlO"4 mol) dissolved in water (50 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was left stirring for 5.5 hours before adding the solution to 2 L of water and dialysing. Subsequently the dialysed solution was freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (2.67 mL, 0.04 mol), water (10 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated poly(MPEG(550)MA) [Mn 4590 PDi 1.22] (2.0 g, 6.25 xlO"4 mol) dissolved in water (50 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was stirred for 4 hours before neutralising the solution with 2M HCl and the water subsequently removed using high vacuum. The polymer was dialysed and then freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (0.836 mL, 0.013 mol), water (1 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated ρoly(MPEG(2000)MA) [Mn 24600 PDi 1.06] (5.0 g, 2.03 xlO"4 mol) dissolved in water (100 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was stirred for 4 hours before neutralising the solution with 2M HCl and the water subsequently removed using high vacuum. The polymer was dialysed and then freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (2.5 niL, 0.038 mol), water (10 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated poly(MPEG(2000)MA) [Mn 24600 PDi 1.06] (15.0 g, 7.5 χlθ'4 mol) dissolved in water (400 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was stirred for 4 hours before neutralising the solution with 2M HCl then adding NaCl (140 g) before extracting into dichloromethane (4 x 150 mL). The organic layers were combined and dried over Na2SO4 filtered and then evaporated to dryness before being washed with diethyl ether. The polymer was dialysed and then freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm.

Conversion of succinimide end group of PoIyPEG to amine group Ethylenediamine (5.60 mL, 0.08 mol), water (10 mL) and a magnetic follower were placed into a three necked round bottom flask fitted with a pressure equalising dropping funnel and the solution cooled by placing in an ice bath. The system was flushed with nitrogen and placed under positive pressure. A solution of succinimide terminated poly(MPEG(2000)MA) [Mn 21900 PDi 1.21] (33.5 g, 1.53 χlθ"3 mol) dissolved in water (500 mL) was added to the dropping funnel and the solution added drop-wise to the ethylenediamine. The solution was stirred for 4 hours before neutralising the solution with 2M HCl then adding NaCl (140 g) before extracting into dichloromethane (4 x 150 mL). The organic layers were combined and dried over Na2SO4 filtered and then evaporated to dryness before being washed with diethyl ether. The polymer was dialysed and then freeze dried to isolate the product. 1H NMR spectra shows reduction of the succinimide O=C-CH2-CH2-C=O resonance at 2.75 ppm. Conversion of amine end group of PoIyPEG to maleimide group Amine terminated poly(MPEG(550)MA) [3VIn 3200] (0.5 g, 1.56 xlO"4 mol), saturated sodium hydrogen carbonate (2.5 mL) and a magnetic follower were placed into a three necked round bottom flask and cooled by placing in an ice bath. The system was flushed with nitrogen and placed under an inert atmosphere. To this solution N-methoxycarbonylmaleimide (0.1 g, 6.45 xlO"4 mol) was added with vigorous stirring. After ten minutes water (5 mL) was added and the reaction left stirring for a further 45 minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl (0.15 g) was added. The polymer was then extracted in to dichloromethane (3 x 50 mL), the extracts were combined and dried over Na2SO4 before being filtered and evaporated to dryness. The polymer was then washed with diethyl ether and dried under vacuum at room temperature. 1H NMR spectra shows appearance of the maleimide resonances at ~5.9-6.4 and -6.7 ppm.

Conversion of amine end group of PoIyPEG to maleimide group Amine terminated ρoly(MPEG(55 O)M A) [Mn 3200] (1.0 g, 3.13 xlO"4 mol), saturated sodium hydrogen carbonate (5 mL) and a magnetic follower were placed into a three necked round bottom flask and cooled by placing in an ice bath. The system was flushed with nitrogen and placed under an inert atmosphere. To this solution N-methoxycarbonylmaleimide (0.2 g, 1.29 xlO'3 mol) was added with vigorous stirring. After ten minutes water (10 mL) was added and the reaction left stirring for a further 45 minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl (0.30 g) was added. The polymer was then extracted in to dichloromethane (3 x 50 mL), the extracts were combined and dried over Na2SO4 before being filtered and evaporated to dryness. The polymer was then washed with diethyl ether and dried under vacuum at room temperature. 1H NMR spectra shows appearance of the maleimide resonances at ~5.9-6.4 and -6.7 ppm.

Conversion of amine end group of PoIyPEG to maleimide group Amine terminated ρoly(MPEG(55 O)MA) [Mn 3200] (1.0 g, 3.13 xlO'4 mol), saturated sodium hydrogen carbonate (5 mL) and a magnetic follower were placed into a three necked round bottom flask and cooled by placing in an ice bath. The system was flushed with nitrogen and placed under an inert atmosphere. To this solution N-methoxycarbonylmaleimide (0.20 g, 1.29 *10"3 mol) was added with vigorous stirring. After ten minutes water (10 mL) was added and the reaction left stirring for a further 45 minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl (3.75 g) was added. The polymer was then extracted in to dichloromethane (3 x 50 mL), the extracts were combined and dried over Na2SO4 before being filtered and evaporated to dryness. The polymer was then washed with diethyl ether and dried under vacuum at room temperature. 1H NMR spectra shows appearance of the maleimide resonances at -5.9-6.4 and -6.7 ppm.

Conversion of amine end group of PoIyPEG to maleimide group Amine terminated poly(MPEG(2000)MA) [Mn 24600] (5.0 g, 2.03 *10'4 mol), saturated sodium hydrogen carbonate (15 mL) and a magnetic follower were placed into a three necked round bottom flask and cooled by placing in an ice bath. The system was flushed with nitrogen and placed under an inert atmosphere. To this solution N-methoxycarbonylmaleimide (0.13 g, 8.13 χlθ"4 mol) was added with vigorous stirring. After ten minutes water (15 mL) was added and the reaction left stirring for a further 45 minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl (7.5 g) was added. The polymer was then extracted in to dichloromethane (4 x 50 mL), the extracts were combined and dried over Na2SO4 before being filtered and evaporated to dryness. The polymer was then washed with diethyl ether and dried under vacuum at room temperature. 1H NMR spectra shows appearance of the maleimide resonances at ~5.9-6.4 and ~6.7 ppm.

Conversion of amine end group of PoIyPEG to maleimide group Amine terminated poly(MPEG(2000)MA) [Mn 24600 PDi 1.06] (15.0 g, 6.1 xlO"4 mol), saturated sodium hydrogen carbonate (45 mL) and a magnetic follower were placed into a three necked round bottom flask and cooled by placing in an ice bath. The system was flushed with nitrogen and placed under an inert atmosphere. To this ;:v»!ution N-methoxycarbonylmaleimide (0.38 g, 2.44 χlθ"3 mol) was added with vigorous stirring. After ten minutes water (15 mL) was added and the reaction left stirring for a further 45 minutes. The pH was then adjusted to 3 with 0.5N sulfuric acid and NaCl (7.5 g) was added. The polymer was then extracted in to dichloromethane (3 x 50 mL), the extracts were combined and dried over Na2S 04 before being filtered and evaporated to dryness. The polymer was then washed with diethyl ether and dried under vacuum at room temperature before final purification through dialysis and isolation by freeze drying. 1H NMR spectra shows appearance of the maleimide resonances at ~5.9-6.4 and -6.7 ppm.

Coupling of succinimide teminated PoIyPEG to benzylamine

CH2Cl2 or water

m ~ 6

Two different experiments were carried out, each using a different solvent. Low molecular weight poly(MPEG(395)MA) (Mn= 2700g/mol, PDI= 1.12) (Ig, 0.37OxI(T 3 mol) prepared from initiator 7 (i.e. succinimide terminated) and benzylamine (0.40 ml, 3.7xlO"3 mol) was dissolved in 10 ml of dry chloroform or distilled water and stirred at room temperature for 20 hours under nitrogen. After reaction, the solvent was removed under vacuum by using a rotary evaporator. The crude was purified by preparative GPC before being precipitated of the polymer in cold Petroleum Ether (40-60°C Fraction).

Bioconjuction of succinimide teminated PoIyPEG polymer A set of three experiments was carried out, each containing a different ratio polymer / lysozyme. Moreover each set of experiments was left to react for either 4 hours or 20 hours. Low molecular weight poly(MPEG(395)MA) prepared from initiator 7 (i.e. succinimide terminated) (Mn= 2700 g/mol, PDI= 1.12) (41.6 mg, 15.4xlO"3 mol) for a ratio 5/1, (83.2 mg, 30.8xl0"3 mol) for a ratio 10/1 and (249.5 mg, 92.4xlO"3 mol) for a ratio 30/1 and lysozyme (50 mg, 3.O8xlO"3 mol) was dissolved in 10 ml of 200 mM phosphate buffer (pH = 8) and stirred at 4 0C for 4 hours or 20 hours under nitrogen. The reaction was followed by HPLC in the case of a ratio polymer / lysozyme 30/1. The HPLC system was fitted with a guard column, a BioSep-SEC-S3000 column and a UV detector continuously measuring the relative absorbance of the mobile phase at 215 nm. The system was eluted with 0.1% v/v trifluoroacetic acid solution in water and acetonitrile (69/31 v/v) at a rate of 0.5 mL/min. In each case, the crude was purified in dialysis bag (Spectra/Porl, MWCO = 6-8000 g/mol) and analysed by SDS- PAGE (polyacrylamide resolving gel cross-linking: 15%, running buffer: 25 mM TRIS base, 250 mM glycine, 0.1% SDS, pH 8.7). Reactions of PoIyPEG Polymers prepared from initiator 12

Conversion ofacetal end group of PoIyPEG to aldehyde group Acetal-tertninated polymer (Mn 11000, PDi 1.15, 3.O g, 0.27 mmol) was dissolved in a 1:1 trifluoroacetic acid (TFA)/H2O solution (100 mL) and the solution was stirred at room temperature for 48 hours. Most of the acid was removed under reduced pressure and the crude was dissolved in water and purified by dialysis. The aqueous solution was then freeze-dried to give the desired aldehyde terminal polymer (2.8 g, 0.25 mmol, 93 %) as an off-white solid. (Mn-Il5OOO, PDi 1.13)

Conversion ofacetal end group of PoIyPEG to aldehyde group Acetal-terminated polymer (Mn 22,000, PDi 1.09, 3.0 g, 0.14 mmol) was dissolved in a 1:1 trifluoroacetic acid (TFA)/H2O solution (100 mL) and the solution was stirred at room temperature for 48 hours. Most of the acid was removed under reduced pressure and the crude was dissolved in water and purified by dialysis. The aqueous solution was then freeze-dried to give the desired aldehyde terminal polymer (2.8 g, 1.3 mmol, 93 %) as an off-white solid. (Mn~22,000, PDi 1.09)

Conversion ofacetal end group of PoIyPEG to aldehyde group Acetal-terminated polymer (Mn = 32,000, PDi = 1.09, 3.0 g, 0.094 mmol) was dissolved in a 1:1 trifluoroacetic acid (TFA)/H2O solution (100 mL) and the solution was stirred at room temperature for 48 hours. Most of the acid was removed under reduced pressure and the crude was dissolved in water and purified by dialysis. The aqueous solution was then freeze-dried to give the desired aldehyde terminal polymer (2.7 g, 0.084 mmol, 90 %) as an off-white solid. (Mn~32,000, PDi 1.11)

Bioconjugation of deprotected PoIyPEG polymers prepared from initiator 12

Bioconjuction of aldehyde-terminated PoIyPEG polymer Lysozyme (6 mg, 4.2x10"4 mmol) and aldehyde-terminated polymer (Mn~22,000, PDi 1.09, 110 mg, 0.01 mmol) was dissolved in 5 mL of acetate/acetic acid buffer (pH = 5) and 0.15 mL Of NaCNBH3 (0.25 mM in water) was added dropwise. The solution was stirred at room temperature and samples were taken at regular intervals. The reaction was monitored by HPLC fitted with a guard column, a bioSep-SEC-S3000 column and an UV detector.

Bioconjuction of aldehyde-terminated PoIyPEG polymer Lysozyme (6 mg, 4.2XlO"4 mmol) and aldehyde terminated polymer (Mn~22,000, PDi 1.09, 110 mg, 0.01 mmol) was dissolved in 5 mL of phosphate buffer (pH = 6) and 0.15 mL OfNaCNBH3 (0.25 mM solution in water) was added dropwise. The solution was stirred at room temperature and samples were taken at regular intervals. The reaction was monitored by HPLC fitted with a guard column, a bioSep-SEC-S3000 column and an UV detector. Reactions of PoIyPEG Polymers prepared from initiator 14

Conversion of BOC end group of PoIyPEG to amine group

BOC terminated polymer (Mn=6400 g mol'1, 3.2 g, 1.0 mmol) was dissolved in CH2Cl2 (25 mL), trifluoroacetic acid (3.9 mL, 50 mmol) was added via syringe and the resulting solution was stirred at room temperature for 16 h. The solvent was then removed under reduced pressure and the resulting orange-brown oil was dissolved in deionized water and dialyzed. The polymer solution was freeze-dried. Toluene (50 mL) was then added and the solvent was removed under reduced pressure. This procedure was repeated three times and the expected amine terminated polymer as the trifluoroacetic acid salt (2.5 g, 0.81 mmol, 81 % yield) was obtained as a yellow- orange oil. 1H NMR revealed the complete disappearance of the singlet relative to the Boc group at 1.4 ppm. Mw/Mn (GPC)= 1.11

Conversion of amine end group of PoIyPEG to maleimde group

3-Maleimidopropionyl chloride (13.0 mmol) was dissolved in 100 mL of CH2Gb, diisopropylethylamine (DIPEA, 2.3 mL, 13.0 mmol) was added via syringe and the solution was cooled to 0°C. A solution of amine terminated polymer as the trifluoracetic acid salt (1.5 g, 0.47 mmol) in 30 mL of CH2Cl2 was added dropwise (ca. 15 min) and the mixture was stirred at 00C for Ih, then at room temperature for 2 days. The solvent was then removed under reduced pressure and 200 mL of water were added to the brown residue. The suspension was centrifugate and purified by dialysis (Millipore, regenerated cellulose, MWCO 1 kDa, filtration area 0.23 m2). The polymer solution was freeze-dried. Toluene (50 mL) was then added and the solvent was removed under reduced pressure. This procedure was repeated three times and the expected maleimde terminated polymer was obtained as a pale yellow oil. A conversion of 80 % can be calculated by 1H NMR, comparing the integration of the vinylic protons of the maleimide moiety and that of the terminal OCH3 of the PEG side-chains. Mw/Mn (GPC) = 1.06. Reactions of PoIyPEG Polymers prepared from initiator 15

Conversion offuran end group of PoIyPEG to maleimde group

A solution of polymer prepared from initiator 15 (3.Og, 0.36 mmol) in toluene (25 mL) was warmed to reflux and the reaction was monitored by 1H NMR analysis on samples taken at regular intervals of time. After 7 h the solvent was removed under reduced pressure to give the maleimide terminated polymer as a pale orange oil. Comparison of the integration of the maleimide vinyl signals and the terminal OCH3 of the PEG side-chains confirmed that the maleimide function did not decompose during the deprotection step. References

1. D. M. Haddleton, M. C. Grossman, B. H. Dane, D. J. Duncalf, A. M. Henning, D. Kukulj and A. J. Shooter, Macromolecules, 1999, 32, 2110. 2. R. N. Keller and W. D. Wycoff, Inorg. Synth., 1947, 2, 1. 3. James R. Dudley, Jack T. Thurston, Frederic C. Schaefer, Dagfrid HoIm- Hansen, Clarence J. Hull, and Pierrepont Adams, J. Am. Chem. Soc, 1951, 73, 2986.