WO2011007133A2 - Macromolécules modifiées par un polymère - Google Patents

Macromolécules modifiées par un polymère Download PDF

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WO2011007133A2
WO2011007133A2 PCT/GB2010/001338 GB2010001338W WO2011007133A2 WO 2011007133 A2 WO2011007133 A2 WO 2011007133A2 GB 2010001338 W GB2010001338 W GB 2010001338W WO 2011007133 A2 WO2011007133 A2 WO 2011007133A2
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group
polymer
compound
groups
substituted
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PCT/GB2010/001338
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WO2011007133A3 (fr
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David Haddleton
Matthew Wynn Jones
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Warwick Effect Polymers Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/585—Calcitonins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins

Definitions

  • the invention relates to a process for covalently modifying biological macromolecules, to the macromolecules themselves and to uses thereof
  • the invention relates, in particular, to a process for chemically modifying biological macromolecules comprising disulfide b ⁇ dges or sulphydryl groups Background
  • Cysteine residues not involved in disulfide bridges are known as targets for the site- specific covalent attachment of polymers to synthesise protein-polymer conjugates ⁇ - Functional polymers bearing maleimide, orthopy ⁇ dyl disulfide, vmyl sulfone and iodoacetamide end-groups have all been used to target cysteine residues for conjugation to peptides
  • Free thiol groups are relatively rare in native proteins and other biological macromolecules and they can be introduced by genetic engineering or by the reduction of disulfide bridges 2
  • "Click chemistry" is a concept introduced in 2001 by Sharpless and co-workers, 3 which describes few classes of near-perfect chemical transformations that share a number of important properties such as very high efficiency in terms of both conversion and selectivity with very mild reaction conditions, the use of non-toxic reagents and solvents, and a simple work up Amongst these reactions, thiol-ene reactions have recently emerged as very powerful click" processes.
  • a first aspect of the invention provides a method of attaching a moiety to a biological macromolecule, the biological molecule comprising a sulphydryl group or disulphide bond, comprising reacting:
  • A is selected from an ester group -CO-O-, keto or aldehyde group -CO-, -CN-, -CO-NR.2 IV (where each R [V may be independently selected and may be H or -F-Q wherein at least one R 1V is -F-Q), substituted or non-substituted aryl, phenyl or allyl;
  • B is H or a carbon atom, and where B is H, Y, X, E and Z are not present;
  • E and F are independently selected and may or may not be present and may be a bond or a linking group
  • X and Y are independently selected and may be H or methyl;
  • Z is a group which is not a leaving group;
  • Q is H, a side group or a functional group; wherein at least one or both of Q and/or Z are functional moieties selected from a polymer, a branched moiety comprising one or more hydroxyl groups (wherein said hydroxyl groups are subsequently reacted with a polymer comprising a group capable of reacting with the hydroxyl group(s) to attach the polymer to the compound), alkyne groups (wherein the alkyne group is subsequentially reacted with an azide-containing further functional group to attach the further functional group to the compound), a moiety comprising a radically transferable group for use as an atom transfer radical polymerisation initiator; and most preferably B is H or together B, Y, X, E and Z form a methyl group and/or A and F make -CO-O- or -CO-N-.
  • a method of attaching a polymer to a biological macromolecule, the biological macromolecule comprising a sulphydryl group or a disulphide bond comprising reacting
  • A is selected from an ester group -CO-O-, keto or aldehyde group -CO-, -CN-, -CO-NR.2 IV
  • each R IV may be independently selected and may be H or -F-Q wherein at least one
  • R IV is -F-Q), substituted or non-substituted aryl, phenyl or allyl;
  • B is H or a carbon atom and when B is H, Y, X, E and Z are not present;
  • E and F are independently selected and may be a bond or a linking group
  • X and Y are independently selected atoms or groups and may be, for example, H or methyl;
  • Z is a group which is not a leaving group and may be a polymer
  • R is H, or a side group
  • Z and R are polymer(s) or comprise a polymer.
  • R may comprise a polymer.
  • a further aspect of the invention provides a method of adding a functional group to a biological macromolecule, the biological molecule comprising a sulphydryl group or a disulphide bond comprising reacting; (a) the biological macromolecule; with
  • A is selected from an ester group -CO-O-, keto or aldehyde group -CO-, -CN-, -CO-NR 2 IV
  • each R IV may be independently selected and may be H or -F-Q wherein at least one R IV is -F-Q), substituted or non-substituted aryl, phenyl or allyl;
  • B is H or a carbon atom and when B is H, Y, X, E and Z are not present;
  • E and F are independently selected and may be a bond or a linking group
  • X and Y are independently selected atoms or groups and may be, for example, H or methyl;
  • Z is a group which is not a leaving group
  • R' is a moiety comprising an alkyne group
  • a still further aspect of the invention allows polymers to be indirectly attached to the macromolecule via the addition of a compound containing hydroxyl groups which may then be used to react with, for example, carboxylate groups on the polymer, to attach the polymer, via the compound, to the biological macromolecule.
  • a further aspect of the invention provides a method of attaching a polymer to a biological macromolecule, the biological macromolecule comprising a sulphydryl group or a disulphide bond, comprising reacting;
  • A is a carbon-carbon double bond
  • A is selected from an ester group -CO-O-, keto or aldehyde group -CO-, -CN-, -C0-NR 2 IV (where each R ⁇ v may be independently selected and may be H or -F-Q wherein at least one R ⁇ v is -F-Q), substituted or non-substituted aryl, phenyl or allyl;
  • B is H or a carbon atom and when B is H, Y, X, E and Z are not present;
  • E and F are independently selected and may be a bond or a linking group
  • X and Y are independently selected atoms or groups and may be, for example, H or methyl;
  • Z is a group which is not a leaving group
  • R" is a branched moiety comprising two or more hydroxyl groups; and (d) reacting the hydroxyl groups of the compound attached to the biological molecule with a polymer comprising a group capable of reacting the hydroxyl groups to attach the polymer to the compound.
  • the method of the invention may be used to produce a macro initiator for Atom Transfer Radical Polymerisation or Transition Metal Mediated Radical Polymerisation. These techniques are generally well known in the art (see for example WO 97/47661, WO 96/30421 and WO 97/18247).
  • Z is a group which is not a leaving group and Q is a branched or non-branched linear alkyl or ester (typically with 1 to IO carbons) substituted with a radically transferable atom or group, such as a halogen, including F, Cl or Br.
  • the macroinitiator may be used as an initiator to initiate the polymerisation of an olefinically unsaturated compound.
  • This may be used, for example, to produce block, ABA, or statistical (co)polymers attached to the biological molecule.
  • a and F do not together make vinyl pyridine.
  • B is H or together B, Y, X, E and Z form a methyl group.
  • a and F preferably make -CO-O- or -CO-N-R 2 .
  • a and/or F may form a selectively cleavable link.
  • Such links are generally well known in the art. They may be broken enzymatically, hydro lytically, by exchange reactions, heat, salt, pH or UV or other ionising radiations.
  • the biological macro molecule is preferably selected from a polypeptide, a polynucleotide or a carbohydrate.
  • the polypeptide preferably comprises at least five, at least ten, at least fifteen, at least twenty amino acids
  • the polynucleotide preferably comprises nine, at least ten, at least fifteen, at least twenty, at least twenty-five, or at least thirty nucleotide residues
  • the carbohydrate preferably comprises five or more sugar residues, preferably more than ten, more than fifteen or more than twenty sugar residues
  • Biomolecule may be an aptamer, DNA, RNA or PNA (peptide nucleic acid)
  • the biological molecule may comprise a naturally occurring sulphydryl residue or disulphide bond
  • sulphydryl residues may also be introduced into the biological molecule, for example by incorporation of a cysteine residue in proteins, oligonucleotides, aptamers or siRNA
  • the method is not used to cross-link, for example, two or more proteins or other biological molecules together to form a scaffold
  • the compound comprises a carbon-carbon double bond in the form of a vinyl residue This reacts with the sulphydryl group or disulphide bond
  • the sulphydryl group or disulphide bond may be reduced and/or catalysed using a suitable reducing agent and/or catalyst
  • reducing agents and catalysts are generally known in the art. They are generally basic compounds, including amines such as primary, secondary or tertiary amines and organo phosphines
  • the preferred organo phosphines include t ⁇ s(Z-carboxyethyl)phosphine (TECP) and dimethylphenyl phosphine (DMPP)
  • Primary amines include- RNH 2 where R is preferably alkyl or aryl
  • the primary amine may also be provided by the biological molecule. That is, for example, if the biological molecule comprises lysine residues, then the lysine residues will catalyse the attachment of the vinyl group to the sulphydryl groups
  • Preferred amines include n-butyl amine and benzyl amines
  • One advantage of the current system is that the same compound may not only catalyse the reduction of, for example, the disulphide bond, but also catalyse the attachment of the vinyl group to the reduced sulphur atom in substantially a single step process.
  • A the electron withdrawing moiety, assists the reaction of the vinyl residue with the sulphydryl group or disulphide bond.
  • A is selected from: an ester group - CO-O-, keto or aldehyde -CO-, -CN-, -CO-NR 2 - (where R 2 may be H or a further polymer), and a substituted or non-substituted aryl group, phenyl or allyl.
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • R 2 may be H or a further polymer
  • a substituted or non-substituted aryl group phenyl or
  • Group Z is a group which is not a leaving group. That is, where present, it remains attached to group B via the bond or linking group (E) during the reaction.
  • Z may be, for example, H. It may also be a straight - or branched - chain, substituted or non- substituted - Ci to C ⁇ o alkyl group or a substituted or non-substituted - aryl group. Z may also be a polymer.
  • Z may be polyethylene glycol (PEG) or:
  • R 4 may be H or a pendant side chain.
  • This may be polymer such as a polyalkylene group, a polyalkylene (e.g. polyethylene) glycol group, or a sugar or other functional group optimally attached via a linker.
  • the linker may comprise, for example, a triazole, such as a 1, 4 triazole obtained from clicking the side chain onto the polymer backbone via Huisgen 1, 3 - dipolarcycloaddition.
  • Q may be a side group, such as H, or alkyl, such as methyl or ethyl, a and b are integers of 1 to 50, especially 2 to 30, 3 to 25, 4 to 20. R may be the same as R 4 .
  • RAFT reverse addition-fragmentation chain transfer polymerisation
  • TMM-LRP transition metal mediated living radical polymerisation
  • ATRP atom transfer radical polymerisation
  • vinyl containing groups such as (meth) acrylate groups
  • a hydroxyl functional polymer might be esterified with an acryloyl halide or a methacryloyl halide or an alternative (meth)acrylating group such as an acid or anhydride.
  • catalytic chain transfer polymerisation (CCTP) inherently leaves a vinyl group towards one end of the polymer and additionally a residue of the monomers used
  • a methacrylate terminated diethylene glycol undergoes CCTP using a colbalt-containing catalyst.
  • the polymer (“Z") is made of a backbone derived from methacrylate, with diethylene glycol residues attached. It will be noted that adjacent to the electron withdrawing group is a further diethylene glycol residue derived from the monome ⁇ c units. Hence, R may be a residue derived from the monomeric units making up a polymer at Z where present.
  • the or each polymer is water soluble or water dispersible. That is the presence of the polymer when attached to the biological macromolecule allows the dispersal of the biological macromolecule within an aqueous environment.
  • the polymer may be linear, branched, dendritic, comb-shaped or star-shaped.
  • the polymer comprises one or more polyalkylene groups, such as polyethylene glycol or polypropylene glycol.
  • Comb-shaped polymers consisting of polyethylene glycol methacrylate are commercially available under the trade mark PoIyPEG from Warwick Effect Polymers Limited. Methods of making such comb polymers are generally shown in WO 2004/1 13394.
  • the polymer may comprise a backbone, the polymer comprising a plurality of pendent side groups. Such pendent side groups may be attached to the backbone by a triazole group. Such polymers may be made via click chemistry, as described in WO 2007/104948.
  • the pendent groups are polyalkylene glycol groups, such as polyethylene glycol, or comprise sugar moieties.
  • E and F are independently selected and may be a bond or a linking group.
  • E and F are independently selected and may be a bond or a straight or a branched linking group.
  • E and F may be selected from a bond, a Ci to C 20 straight - or branched - chain, substituted or non-substituted alkylene groups optionally interrupted by one or more sulphur (such as -SO 2 -), oxygen or nitrogen atoms (such as -NH- or -N(alkyl) -), aldehyde or ester groups; and substituted- or non-substituted aryl or heteroaryl.
  • E or F may be - O - CH 2 -.
  • R" is a branch moiety comprising two or more hydroxyl groups. The Applicant has identified that this allows the attachment of compounds, such as polymers, onto the biological macromolecule via the compound.
  • the invention also provides a kit for use in a method according to the invention comprising a compound selected from:
  • a compound capable of reducing a disulphide bond or sulphydyl group of a catalyst capable of catalysing a reaction of the carbon-carbon double bond compound such as that of (i), with a disulphide or sulphydyl group on the biological macromolecule preferably the compound is selected from:
  • A is selected from an ester group -CO-O-, ⁇ eto or aldehyde group -CO-, -CN-, -CO-NR 2
  • each R IV may be independently selected and may be H or -F-Q wherein at least one
  • R IV is -F-Q), and substituted or non-substituted aryl, phenyl or allyl;
  • B is H or a carbon atom and when B is H, Y, X, E and Z are not present;
  • E and F are independently selected and may be a bond or a linking group
  • X and Y are independently selected atoms or groups and may be, for example, H or methyl;
  • Z is a group which is not a leaving group
  • R is H, or a side group
  • Z and R are polymer(s);
  • R' is a moiety comprising an alkyne group
  • R" is a branched moiety comprising two or more hydroxyl groups
  • the compound is selected from:
  • n is an integer of 1 or more
  • P is a biological macromolecule
  • A is selected from an ester group -CO-O-, keto or aldehyde group -CO-, -CN-, -CO-NR 2
  • each R IV may be independently selected and may be H or -F-Q wherein at least one R IV is -F-Q), and substituted or non-substituted aryl, phenyl or allyl;
  • B is H or a carbon atom and when B is H, Y, X, E and Z are not present;
  • E and F are independently selected and may be a bond or a linking group
  • X and Y are independently selected atoms or groups and may, for example, be H or methyl;
  • Z is a group which is not a leaving group and may be a polymer
  • R is H, or a side group
  • Z and R are polymer(s).
  • R' is a moiety comprising an alkyne group
  • R" is a branched moiety comprising two or more hydroxyl groups
  • G is a functional group
  • n as defined above may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 30.
  • the individual components are preferably as defined as above.
  • the compounds may be used with biological macromolecules comprising sulphydryl groups or disulphide groups.
  • the Inventors have unexpectedly found that it is possible to attach polymers, such as PEG, to disulphide groups of at least some polypeptides, such as salmon calcitonin, without substantially affecting the activity of the protein. That is, it is possible to attach two separate compounds to the now separated sulphur atoms from the disulphide bond.
  • Medicaments comprising the compounds of the invention, optimally in combination with pharmaceutically acceptable carriers are also provided. Methods of making medicaments utilising the compounds of the invention are also provided.
  • the compounds of the invention are typically soluble in biologically compatable media such as serum, blood, intra-and-extra cellular fluid, saline and phosphate-buffered saline.
  • biologically compatable media such as serum, blood, intra-and-extra cellular fluid, saline and phosphate-buffered saline.
  • FIG. 1 Ci 8 RP-HPLC monitoring of the one-pot 2-steps thiol-ene click process.
  • Figure 2. MALDI ToF mass spectrum of sCT-(mPEG 475 ) 2 after trytpic digestion (matrix: DHB-sodium trifluoroacetate). Both major and minor mass distributions correspond to the (Cysi-Lys ⁇ )-(mPEG475)2 fragment, associated with Na + and H + ions, respectively. Spectra were recorded in reflectron mode in order to analyse the isotopic pattern of the conjugates.
  • Figure 3. ELISA measurement of the increase of intracellular cyclic AMP induced in T47D human breast cancer cells bearing sCT receptors by sCT-(mPEG475) 2 and native sCT.
  • Salmon calcitonin was used as a model disulfide bridge- containing peptide.
  • Salmon calcitonin is a 32 aminoacid calcitropic hormone currently administered for the treatment of a number of hypercalcemia-related diseases, including postmenopausal osteoporosis and Paget's Disease.
  • sCT presents a disulfide bridge— Cys'-Cys - that can be reduced to give two sulphydryl units available for thiol functionalisation.
  • TCEP tris(2-carboxyethyl)phosphine
  • J.A. Burns, J.C. Butler, J. Moran and G. M. Whitesides, Journal of Organic Chemistry, 1991, 56, 2648-2650 a commercially available water-soluble phosphine was tested both as the reducing agent for the opening of the Cys'- Cys 7 bridge of sCT and as a catalyst for the subsequent thiol-ene PEGylation of the two resulting sulphidryl units (Scheme 1).
  • Scheme 1 tris(2-carboxyethyl)phosphine
  • the mixture was then diluted with water and the excess of mPEG acrylate and TCEP catalyst was removed by extensive dialysis (MWCO 3 kDa) against water.
  • MWCO 3 kDa extensive dialysis
  • the one-pot two-steps process was found to proceed efficiently even in the presence of air, despite free cysteine residues are known to reform disulfide bridges in the presence of oxygen.
  • MALDI-ToF mass spectroscopy of the conjugate showed one mass distribution corresponding to a sCT-(mPEG4 75 ) 2 species.
  • sCT sequence includes primary amine groups, namely Lys", Lys 18 and the Cys 1 N-terminus, which could in principle undergo aza-Michael addition with the mPEG acrylate conjugating Polymers employed in this study (although at pH neutral and below thiols are known for reacting one to three orders of magnitude faster than amines in Michael-type processes) (M.A. Gauthier, M.I. Gibson and H.-A. Klok, Agnew. Chem. Int. Ed, 2009, 48, 48-58).
  • Ci8 RP-HPLC analysis indicated that, in spite of the presence of a relatively large excess of conjugating polymer (7 eq.), a biohybrid material containing exatly two mPEG chains was formed in less than 2 h.
  • the phosphine-mediated one-pot thiol-ene click method developed here appears to be very general and could find application for the site-selective pegylation of a number of peptides and proteins in which, as for sCT, the disulfide bridge targeted for polymer conjugation is not essential for retaining their bioactivity.
  • it allowed for quantitative substrate modification in a relatively short reaction time, a factor that can be very important for peptides with limited stability in solution.
  • Methanol, DEGMEMA and H 2 O were degassed by bubbling with nitrogen for approximately 2 hrs prior to use.
  • Methanol (25 ml) and H 2 O (46 ml) were added to CVA (0.28 g).
  • DEGMEMA (29.41 ml) was added to CoBF (3.06 mg).
  • 3.2 ml of the CoBF/monomer solution was sy ⁇ nged into the water/initiator solution and the feed started at a rate of 1 ml every 3 mins.
  • the reaction flask was subsequently cooled in the fridge to terminate the polymerisation.
  • the sample flask was heated to 3O 0 C to stop the solution freezing.
  • TCEP HCl (7.1 mg) was dissolved in water (1.0 mL). A 100 ⁇ L aliquot (2.5 ⁇ mol of TCEP HCl, 1.7 eq) of this solution was taken and added to a vial containing a solution of salmon calcitonin (5.0 mg) in water (0.4 mL) and the resulting solution was stirred at ambient temperature. RP-HPLC analysis revealed that quantitative reduction of the Cys'-Cys 7 disulfide bridge occurred after 30 minutes. Poly DEGMEMA (average M n ⁇ 3200, 36.4 mg) was dissolved in phosphate buffer (0.500 mLpH 7.0) and was added to the reduced peptide solution and left to stir at ambient temperature.
  • the solution was then diluted with water and dialysed (2 kDa MWCO) membrane against pure water for 5 days.
  • Scheme 4 shows the addition of alkyne groups to disulphide groups.
  • PR3 is phosphine catalyst.
  • Cu AAC copper catalysed azide-alkyne cycloaddition.
  • PolyPEG - methacrylate may also be added:

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Abstract

L'invention porte sur des procédés et des composés permettant l'addition d'un polymère à une macromolécule biologique, la macromolécule biologique comprenant un groupe sulfhydryle ou une liaison disulfure qui est amené à réagir avec une fraction vinylique attachée au polymère.
PCT/GB2010/001338 2009-07-13 2010-07-13 Macromolécules modifiées par un polymère WO2011007133A2 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2011102685A2 (fr) 2010-02-19 2011-08-25 Samsung Electronics Co., Ltd. Procédé et appareil destinés à transmettre un contenu vidéo compressé par codec
CN111318185A (zh) * 2018-12-17 2020-06-23 中国石油化工股份有限公司 一种增强亲水性的石墨烯过滤膜材料的涂装工艺

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Publication number Priority date Publication date Assignee Title
WO2011102685A2 (fr) 2010-02-19 2011-08-25 Samsung Electronics Co., Ltd. Procédé et appareil destinés à transmettre un contenu vidéo compressé par codec
CN111318185A (zh) * 2018-12-17 2020-06-23 中国石油化工股份有限公司 一种增强亲水性的石墨烯过滤膜材料的涂装工艺
CN111318185B (zh) * 2018-12-17 2022-04-05 中国石油化工股份有限公司 一种增强亲水性的石墨烯过滤膜材料的涂装工艺

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