EP1784430A1 - Initiateur de polymerisation de radicaux vivants comprenant un groupe fonctionnel capable de reagir avec des polypeptides ou analogues, polymere en peigne obtenu avec cet initiateur, conjugues polypeptidiques et medicaments obtenus a base de cet initiateur - Google Patents

Initiateur de polymerisation de radicaux vivants comprenant un groupe fonctionnel capable de reagir avec des polypeptides ou analogues, polymere en peigne obtenu avec cet initiateur, conjugues polypeptidiques et medicaments obtenus a base de cet initiateur

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Publication number
EP1784430A1
EP1784430A1 EP04743240A EP04743240A EP1784430A1 EP 1784430 A1 EP1784430 A1 EP 1784430A1 EP 04743240 A EP04743240 A EP 04743240A EP 04743240 A EP04743240 A EP 04743240A EP 1784430 A1 EP1784430 A1 EP 1784430A1
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EP
European Patent Office
Prior art keywords
initiator
mmol
solution
mpeg
mol
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EP04743240A
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German (de)
English (en)
Inventor
David Haddleton
Francois Lecolley
Lei Tao
Guiseppe Mantovani
Adrian Carmichael
Adam Peter Jarvis
Andrew Gregory Steward
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Warwick Effect Polymers Ltd
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Warwick Effect Polymers Ltd
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Publication of EP1784430A1 publication Critical patent/EP1784430A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F290/00—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
    • C08F290/02—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups on to polymers modified by introduction of unsaturated end groups
    • C08F290/06—Polymers provided for in subclass C08G
    • C08F290/062—Polyethers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F299/00—Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers
    • C08F299/02—Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers from unsaturated polycondensates
    • C08F299/022—Macromolecular compounds obtained by interreacting polymers involving only carbon-to-carbon unsaturated bond reactions, in the absence of non-macromolecular monomers from unsaturated polycondensates from polycondensates with side or terminal unsaturations
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/01—Atom Transfer Radical Polymerization [ATRP] or reverse ATRP

Definitions

  • 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).
  • 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.
  • PEG poly (ethylene glycol), which is known by the abbreviation PEG
  • 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.
  • PEGylation 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.
  • 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.
  • PEG-INTRONTM is an ⁇ -interferon product produced by Schering-Plough and Enzon, Inc. which is used to treat hepatitis C and cancer.
  • ProthecanTM 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.
  • 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.
  • 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.
  • 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.
  • 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 trichlorophenyl carbonate
  • carbonylimidazole Compounds such as tresylate are known to bind to proteins via nucleophilic attack.
  • cysteine residues on proteins or polypeptides include maleimides, vinylsulphones, pyridyl sulphides and iodoacetamides.
  • 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.
  • 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).
  • 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.
  • biological substances for example, proteins and polypeptides, nucleic acids (DNA and RNA), carbohydrates and fats
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the first aspect of the invention provides a method of producing a comb polymer comprising the steps of:
  • an initiator compound comprising a homolytically cleavable bond
  • 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.
  • 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.
  • 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).
  • poly (ethylene glycol) may be used.
  • 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).
  • mPEG monomethoxy PEG
  • This is synthesised by an ionic ring opening polymerisation initiated with methoxide ions and is commercially available.
  • 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.
  • 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.
  • 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.).
  • 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.
  • olefinically unsaturated moieties examples 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, ⁇ -methyl styrene; vinyl halides such as vinyl chloride and vinyl fluoride;
  • 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.
  • any suitable linkage groups for example via a methyl ether linkage.
  • 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.
  • 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.
  • the initiator compound (ii) is selected from:
  • 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 moiety which is capable of reacting with the protein or polypeptide has the formula:
  • n integer of 0 to 10
  • Y is an aliphatic or aromatic moiety
  • X ' where R 1 is H, methyl, ethyl, propyl or butyl, X is a halide, especially Cl or Br.
  • the initiator (ii) has a formula:
  • n is an integer of 0 to 10
  • 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).
  • the catalyst comprises a ligand which is any N-, O-, P- or S- containing compound which can coordinate in a ⁇ -bond to a transition metal or any carbon-containing compound which can coordinate in a ⁇ -bond to the transition metal, such that direct bonds between the transition metal and growing polymer radicals are not formed.
  • a ligand which is any N-, O-, P- or S- containing compound which can coordinate in a ⁇ -bond to a transition metal or any carbon-containing compound which can coordinate in a ⁇ -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
  • 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:
  • 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
  • n an integer of 1 to 2.
  • the metal ion may be attached to a coordinating ligand, such as (CH 3 CNV Y may be chosen from Cl, Br, F, I, NO 3 , PF 6 , BF 4 , SO 4 , CN, SPh, SCN, SePh or triflate (CF 3 SO 3 ). Copper (I) triflate may be used. This is available in the form of a commercially available benzene complex (CF 3 SO 3 CUhCoHe.
  • a coordinating ligand such as (CH 3 CNV Y may be chosen from Cl, Br, F, I, NO 3 , PF 6 , BF 4 , SO 4 , CN, SPh, SCN, SePh or triflate (CF 3 SO 3 ). Copper (I) triflate may be used. This is available in the form of a commercially available benzene complex (CF 3 SO 3 CUhCoHe.
  • the especially preferred compound used is CuBr.
  • A may be F, Cl, Br, I, N, O 3 , SO 4 or CuX 2 (where X is a halogen).
  • the transition metal may be selected from Cu + , Cu 2+ , Fe 2+ , Fe 3+ , Ru 2+ , Ru 3+ , Cr 2+ , Cr 3+ , Mo 2+ , Mo 3+ , W 2+ , W 3+ , Mn 3+ Mn 4+ , Rh 3+ , Rh 4+ , Re 2+ , Re 3+ , Co + , Co 2+ , V 2+ , V 3+ , Zn + , Zn 2+ , Au + , Au 2+ , Ag + and Ag 2+ .
  • the organodiimine has a formula selected from:
  • Ri , R 2 , Rio, Rii, Rn and Ri 3 may be a Ci to C 20 alkyl, hydroxyalkyl or carboxyalkyl, in particular C] to C 4 alkyl, especially methyl or ethyl, n-propylisopropyl, n-butyl, sec-butyl, tert butyl, cyclohexyl, 2-ethylhexyl, octyl decyl or lauryl.
  • Preferred ligands include:
  • R14 Hydrogen. C. to C 10 branched chain alkyi. carboxy- or hydroxy- C 1 to C 10 alkyl.
  • the catalyst is
  • the organodiimine is N-(n-propyl)-2-pyridylmethanimine (NMPI), N-ethyl-2- pyridyl methanimine or N-(n-ethyl)-2-pyridylmethanimine.
  • NMPI N-(n-propyl)-2-pyridylmethanimine
  • NMPI N-ethyl-2- pyridyl methanimine
  • N-(n-ethyl)-2-pyridylmethanimine N-(n-ethyl)-2-pyridylmethanimine.
  • 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.
  • a block co-polymer may be produced by additionally polymerising one or more different olefinically unsaturated monomers.
  • 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, ⁇ -methyl
  • 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.
  • 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, tol
  • the reaction temperature may be carried out from -20 to greater than 200 0 C, especially +5 to 13O 0 C.
  • WO 97/47661 shows examples of living radical polymerisation and the typical conditions that may be used.
  • 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.
  • the components are the catalyst of formula: [ML 1n ] n+ A" ' (defined above) are at a ratio of catalyst : initiator of 3 : 1 to 1 : 100.
  • 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.
  • 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 apply 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.
  • an inert atmosphere such as nitrogen or argon
  • 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.
  • 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 comb polymer may incorporate a fluorescently-labelled monomer.
  • 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.
  • 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:
  • 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 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.
  • the moiety capable of reacting with a protein or polypeptide has a formula:
  • Y is an aliphatic or aromatic moiety
  • R' is H, methyl, ethyl, propyl or butyl
  • X is a halide, especially Cl or Br.
  • the initiator has a formula of:
  • n is an integer of 0 to 10
  • X is a halide, especially Cl or Br.
  • the initiator especially has the formula:
  • 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.
  • 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 NH 2 group, if the reaction conditions are not controlled.
  • 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,
  • 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.
  • the comb polymer has an average total molecular weight of 2,000-80,000, especially 20,000-40,000.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • LRP living radical polymerisation
  • NHS N-hydroxysuccinimide
  • FIG. 1 shows SEC curves for NHS functionalised poly (MMA), solid curve, and the produce (N-benzylamide functionalised poly (MMA), dashed curve).
  • FIG 22 Kinetic plot for the hydrolysis of N-succinimidyl terminated poly(MPEG(395)MA initiated by 8 in different buffers.
  • FIG. 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 32 SDS-PAGE for the conjugation of ⁇ oly(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.
  • 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).
  • ° kp[Pol*] rate constant of propagation x [active propagating polymer chains] from first order kinetic plot.
  • b determined by the 1 H NMR peak intensity ratio on a BrUker DPX 300 MHz c ⁇ Kn-octyl)-2-pyridylmethanimine used as the ligand ⁇ 10 mole % HEMA/90 mole % MMA
  • 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.
  • dNbpy 4,4'-di(5-nonyl)-2.2'-bipyridyl
  • 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 0 C for 4.5 hours. The catalyst was removed from the samples by passing through a column of activated basic alumina prior to SEC.
  • N-hydroxysuccinimide-functionalised polymers as these polymers could not be passed over basic alumina.
  • N-benzylamide functional groups may be added and can be used to reach with free amide groups of the sort found in proteins.
  • N-hydroxysuccinimide initiator (7) (NHS-Br)
  • 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.
  • N-hydroxysuccinimide functionalised poly(PEGMA) were purified by two consecutive purifications from a Toluene solution in diethyl ether.
  • 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 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 30 0 C in 80% toluene solution.
  • Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate) was freeze dried prior to use to remove all water.
  • 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 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 0 C in 80% toluene solution.
  • 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 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 0 C in 80% toluene solution.
  • Bisomer S20W (50% aqueous solution of methoxypolyethyleneglycol methacrylate) was freeze dried prior to use to remove all water.
  • 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 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 0 C in 66% toluene solution.
  • Bisomer MPEG550MA was used as provided.
  • 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 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 0 C in 66% toluene solution.
  • 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.
  • Table 6 Data for the polymerization of methoxypolyethyleneglycol methacrylate (1080) with an initiator derived from N-hydroxy succinimide at 90 0 C in 66% toluene solution.
  • Bisomer SlOW 50% aqueous solution of methoxypolyethyleneglycol methacrylate was freeze dried prior to use to remove all water.
  • 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.
  • Table 7 Data for the polymerization of methoxypolyethyleneglycol methacrylate (628) with an initiator derived from N-hydroxy succinimide at 30 0 C in 66% toluene solution.
  • Bisomer MPEG550MA was used as provided.
  • 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.
  • Table 8 Data for the polymerization of methoxypolyethyleneglycol methacrylate (628) with an initiator derived from N-hydroxy suceinimide at 50 0 C in 66% toluene solution.
  • Bisomer MPEG550MA was used as provided.
  • 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
  • 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 0 0 C with an ice bath before the dropwise addition of a solution of N 5 N 1 - Dicyclohexylcarbodiimide (6.70 g, 32.68 mmol) in 50 mL of anhydrous DCM. After addition, the mixture was stirred at room temperature overnight.
  • 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 0 0 C 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).
  • 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 0 C 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).
  • Tritylthiolether propanol 50 g, 0.150 mol
  • triethylamine 31.3 mL, 0.225 mol
  • anhydrous tetrahydrofuran 125 mL
  • 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.
  • Potassium hydroxide (30 g, 0.51 mol) was suspended in ethylene glycol (100 ml) and the mixture was heated to 115 0 C 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 0 C 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 MgSO 4 .
  • the anhydride, 4,10-dioxa-tricyclo[5.2.1.0 2>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.
  • 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.
  • MPEG(395)MA 10 mL, 22.74 x 10 '3 mol
  • N-( «-ethyl)-2- ⁇ yridylmethanimine (0.64 mL, 4.54 x 10 "3 mol)
  • toluene 10 mL
  • 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-pyridylmethar ⁇ nine (0.114 g, 0.797 mmol) was added.
  • Table 4 Data for the polymerization of MPEG(550)MA with initiator 8 at 50/70 0 C in 73 w/v% toluene solution.
  • 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.
  • Table 5 Data for the polymerization of MPEG(550)MA with initiator 8 at 90 0 C in 73 w/v% toluene solution.
  • 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 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 50 0 C in 67 v/v% toluene solution.
  • 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.
  • Table 7 Data for the polymerization of MPEG(550)MA with initiator 8 at 50 0 C in 67 v/v% toluene solution.
  • 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.
  • Table 8 Data for the polymerization of MPEG(550)MA with initiator 8 at 50 0 C in 67 v/v% toluene solution. Sample Time Conversion Mn PDi
  • 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.
  • Table 9 Data for the polymerization of MPEG(550)MA with initiator 8 at 50 0 C in 67 v/v% toluene solution.
  • 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.
  • Table 10 Data for the polymerization of MPEG(550)MA with initiator 8 at 50 0 C in 67 v/v% toluene solution.
  • fPEGJ/flJ/fCuJ/fLJ 6.4/1/1/2 in 67 v/v% toluene solution at 70 0 C
  • 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.
  • Table 11 Data for the polymerization of MPEG(550)MA with initiator 8 at 70 0 C in 67 v/v% toluene solution.
  • 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 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 50 0 C in 67 v/v% toluene solution.
  • 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.
  • 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 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 0 C in 80 w/v% toluene solution.
  • 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 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 50 0 C in 80 w/v% toluene solution.
  • 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.
  • MPEG(395)MA 10 mL, 22.74 mmol
  • N-(n- ⁇ ropyl)-2- ⁇ yridyhnethanimine (0.71 mL, 4.54 mmol)
  • 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).
  • 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.
  • MPEG(395)MA 10 mL, 22.74 mmol
  • N-(n-ethyl)-2-pyridylmethanimine (0.64 mL, 4.54 mmol
  • 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 0 C. Samples were removed periodically using degassed syringes and quenched in liquid nitrogen for conversion and molecular weight analysis.
  • 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.
  • MPEG(395)MA 10 mL, 22.74 mmol
  • N-( «-propyl)-2-pyridylmetharrimine (0.71 mL, 4.54 mmol)
  • anisole 10 mL
  • 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.
  • Table 19 Data for the polymerization of MPEG(550)MA with initiator 7 at 30 0 C in 66 w/v% toluene solution.
  • 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.
  • Table 20 Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0 C in 66 w/v% toluene solution.
  • 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 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 0 C in 66 w/v% toluene solution.
  • 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.
  • Table 22 Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0 C in 66 w/v% toluene solution.
  • 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.
  • 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 50 0 C in 62 w/v% toluene solution.
  • Initiator 7 (0.05 g, 0.189 mmol), Cu(I)Br (0.027 g, 0.189 mmol, 1 eq), 2,2'-bi ⁇ yridyl (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.
  • Table 24 Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0 C in 67 w/v% toluene solution using 2,2'-bipyridyl ligand.
  • 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.
  • Table 25 Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0 C in 67 w/v% toluene solution using 4,4'-dinonyl-2,2'-dipyridyl ligand.
  • 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.
  • Table 26 Data for the polymerization of MPEG(550)MA with initiator 7 at 50 0 C in 67 w/v% toluene solution using 1,1,4,7,10,10-hexamethyltriethylenetetramine ligand. Sample Time Conversion Mn PDi
  • 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.
  • Table 27 Data for the polymerization of MPEG(55O)MA with initiator 7 at 50 0 C in 67 w/v% toluene solution using N,N,N',N",N"-pentamethyldiethylenetriamine ligand. Sample Time Conversion Mn PDi
  • 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 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 0 C in 66 w/v% toluene solution.
  • 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.
  • Table 29 Data for the polymerization of MPEG(IOOO)MA with initiator 7 at 50 0 C in 75 w/v% toluene solution.
  • 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.
  • 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.
  • Table 31 Data for the polymerization of MPEG(IOOO)MA with initiator 7 at 50 0 C in 75 w/v% toluene solution.
  • 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 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 0 C in 80 w/v% toluene solution.
  • 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 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.
  • 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 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 0 C in 80 w/v% toluene solution.
  • 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.
  • Table 35 Data for the polymerization ofMPEG(1000)MA with initiator 7 at 90 0 C in 66 w/v% toluene solution.
  • [PEG]/[I]/[Cu]/[L] 19.2/1/1/2 in 80 w/v% toluene solution at 50/70 0
  • Initiator 7 (0.67 g, 2.53 mmol)
  • Cu(I)Br (0.36 g, 2.53 mmol, 1 eq)
  • MPEG(2000)MA 101.24 g, 0.049 mol
  • a magnetic follower was 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.
  • Table 36 Data for the polymerization of MPEG(2000)MA with initiator 7 at 50/70 0 C in 80 w/v% toluene solution.
  • 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.
  • Table 37 Data for the polymerization of MPEG(2000)MA with initiator 7 at 50/70 0 C in 75 w/v% toluene solution.
  • [PEG]/[I]/[Cu]/[L] 28.8/1/1/2 in 75 w/v% toluene at 50/70 0 C.
  • Initiator 7 (0.44 g, 1.67 xW 3 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.
  • [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.
  • Table 39 Data for the polymerization of MPEG(550)MA with initiator 5 at 50/90 0 C in 75 w/v% toluene solution.
  • 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.
  • Table 40 Data for the polymerization ofMPEG(1000)MA with initiator 5 at 50 0 C in 75 w/v% toluene solution.
  • [PEG]/[I]/[Cu]/[L] 28.8:1/1/2 in 67 w/v% acetone at 50 0 C 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.
  • Table 41 Data for the polymerization of MPEG(2000)MA with initiator 9 at 50 0 C in 67 w/v% acetone solution.
  • Initiator 6 (0.119 g, 3.333 Xl(T 4 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.
  • Table 42 Data for the polymerization of MPEG(2000)MA with initiator 6 at 30 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 43 Data for the polymerization of MPEG(2000)MA with initiator 6 at 30 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 44 Data for the polymerization of MPEG(2000)MA with initiator 6 at 30 0 C in 67 w/v% toluene solution.
  • [PEGl/ffl/fCuJ/IL] 25/1/1/2 in 67 w/v% toluene at 50 0
  • Initiator 10 (0.81 g, 1.68 ⁇ l ⁇ "3 mol)
  • Cu(I)Br (0.24 g, 1.68 xlO "3 mol)
  • 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.
  • Table 45 Data for the polymerization of MPEG(395) with initiator 10 at 50 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 46 Data for the polymerization of MPEG(550)MA with initiator 11 at 50 0 C in 61 v/v% toluene solution.
  • 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 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 0 C in 67 v/v% toluene solution.
  • [PEG]/[I]/[Cu]/[L] 12/1/1/2 in 80 w/v% toluene solution at 50/70 0
  • Initiator 11 0.1 g, 0.369 mmol
  • Cu(I)Br 0.053 g, 0.369 mmol, 1 eq
  • MPEG(2000)MA 9.24 g, 4.44 mmol
  • a magnetic follower was 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 N-ethyl-2-pyridylmethanimine (0.10 g, 0.758 mmol) was added.
  • 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 0 C in 80 w/v% toluene solution.
  • 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.
  • 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 (4O 0 C) overnight.
  • Table 50 Data for the polymerization of MPEG(IOOO)MA with initiator 12 at 70 0 C in 66 v/v% toluene solution.
  • 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)
  • 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.
  • 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.
  • 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 (40 0 C) overnight.
  • Table 51 Data for the polymerization of MPEG(IOOO)MA with initiator 12 at 50 0 C in 66 v/v% toluene solution.
  • 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.
  • Table 52 Data for the polymerization of MPEG(550)MA with initiator 13 at 30 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 53 Data for the polymerization of MPEG(550)MA with initiator 13 at 50 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 54 Data for the polymerization of MPEG(550)MA with initiator 13 at 50 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 55 Data for the polymerization of MPEG(550)MA with initiator 13 at 50 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 56 Data for the polymerization of MPEG(550)MA with initiator 13 at 70 0 C in 67 w/v% toluene solution.
  • 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.
  • Table 57 Data for the polymerization of MPEG(550)MA with initiator 13 at 50/90 0 C in 67 w/v% toluene solution.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne un procédé de production d'un polymère en peigne qui consiste : (à) utiliser : (i) plusieurs monomères linéaires, ramifiés ou en étoile, substitués ou non substitués, et présentant un fragment oléfiniquement insaturé, ledit fragment pouvant subir une polymérisation par addition ; (ii) un composé initiateur, qui comprend une liaison obtenue par clivage homolytique ; (iii) un catalyseur pouvant catalyser la polymérisation du monomère ; et (b) à amener le catalyseur à catalyser, en combinaison avec l'initiateur, la polymérisation de plusieurs monomères afin d'obtenir le polymère en peigne. L'invention concerne également des catalyseurs et des polymères pouvant être obtenus par ce procédé. De préférence, le polymère en peigne peut se lier à des protéines et peut être produit à partir de monomères qui sont des alcoxy polyéthers, par exemple le polyalkylène glycol ou le polytétrahydrofurane.
EP04743240A 2004-07-06 2004-07-06 Initiateur de polymerisation de radicaux vivants comprenant un groupe fonctionnel capable de reagir avec des polypeptides ou analogues, polymere en peigne obtenu avec cet initiateur, conjugues polypeptidiques et medicaments obtenus a base de cet initiateur Withdrawn EP1784430A1 (fr)

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