WO2003064452A2 - Method for the preparation of highly branched polypeptides - Google Patents

Method for the preparation of highly branched polypeptides Download PDF

Info

Publication number
WO2003064452A2
WO2003064452A2 PCT/EP2003/000899 EP0300899W WO03064452A2 WO 2003064452 A2 WO2003064452 A2 WO 2003064452A2 EP 0300899 W EP0300899 W EP 0300899W WO 03064452 A2 WO03064452 A2 WO 03064452A2
Authority
WO
WIPO (PCT)
Prior art keywords
nca
groups
amino
polypeptide
protected
Prior art date
Application number
PCT/EP2003/000899
Other languages
French (fr)
Other versions
WO2003064452A3 (en
Inventor
Harm-Anton Klok
Juan Rodriguez-Hernandez
Klaus MÜLLEN
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority to AU2003205707A priority Critical patent/AU2003205707A1/en
Publication of WO2003064452A2 publication Critical patent/WO2003064452A2/en
Publication of WO2003064452A3 publication Critical patent/WO2003064452A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • 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
    • C07K1/006—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length of peptides containing derivatised side chain amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for the preparation of branched polypeptides, to the polypeptides obtainable with said method and the use of the polypeptides as therapeutic or diagnostic agents.
  • Branched or dendritic oligomers and polymers of L-lysine have attracted attention for the construction of peptide antigens 111 and DNA delivery systems.
  • 121 Functionalization of the peripheral a- and e-amino groups of small dendritic oligo(L-lysine) scaffolds with one or different epitopes results in so-called multiple antigen peptides (MAPs), which are of interest for the development of vaccines and as diagnostic tools.
  • MAPs multiple antigen peptides
  • 111 Linear-dendritic di- and triblock copolymers of poly(ethylene glycol) and L-lysine dendrons have been reported, which can form water-soluble complexes with plasmid DNA. [21 These block copolymers show promise as carriers for gene delivery, since they combine the DNA complexing properties of cationic polymers with the water-solubility, non-immunogenicity and biocompatibility of poly(ethylene)glycol.
  • dendritic polypeptides have been generally prepared in a stepwise fashion, either in homogeneous solution or via solid-phase synthesis, from appropriately N", N e -diprotected building blocks.” "31 Provided the synthesis and purification are performed carefully, this strategy will afford well- defined and perfectly monodisperse dendrimers/dendrons. Synthesis and purification, however, are usually laborious, and a large number of steps is required to obtain high molecular weight material.
  • this object is achieved by a method for the preparation of branched polypeptides, in particular, highly branched or dendritic graft polypeptides, comprising the steps: (i) providing an cr-amino acid N-carboxyanhydride (NCA) having a protected primary amino group, which is different from the -NH 2 substituent, (ii) contacting the NCA with an initiator, thereby initiating a ring- opening polymerization of the NCA, (iii) removing the protective group,
  • NCA cr-amino acid N-carboxyanhydride
  • NCA ⁇ -amino acid N- carboxyanhydride
  • the method of the invention allows to repeat the deprotection/ring-opening polymerization cycle several times and to prepare highly branched or dendritic graft polypeptides. These polypeptides can be obtained in only a few reaction steps and are therefore an interesting alternative for the perfect polypeptide dendrimers that are prepared according to the state of the art in a time-consuming step-by-step fashion 1171 .
  • the principles of the inventive method are explained in detail in the following using the amino acid lysine as an example. However, it is to be noted that the inventive method can be performed using any amino acid having the characteristics as outlined in the claims.
  • the strategy which is also outlined in Scheme 1 , is based on the ring- opening (co)polymerization of an ⁇ -amino acid N-carboxyanhydride (NCA) having a protected primary amino group or of a mixture of NCAs in which at least one of the NCAs contains a protected primary amino group. If necessary, the side chains of the other NCA comonomers are protected with suitable orthogonal protective groups.
  • NCAs two orthogonally N-protected ⁇ -amino N-carboxyanhydrides (NCAs), e.g. two orthogonally N e -protected L-lysine N-carboxyanhydrides.
  • one of the monomers contains a primary amine group masked with a temporary protective group (TPG) which can be removed under relatively mild conditions.
  • TPG temporary protective group
  • the TPG group is fixed at the e-NH 2 group.
  • PPG permanent protective group
  • the e-NH 2 group of the other L-lysine monomer is masked with a permanent protective group (PPG) which is stable and not removable under the conditions applied for the removal of the TPG.
  • N e -protected L-lysine NCAs are elected.
  • N e -(tert- butoxycarbonyI)-L-lysine NCA(BOC-lys NCA) and N e -(benzyloxycarbonyl)-L- lysine NCA (Z-lys NCA) were used. Both, BOC-lys NCA [10] and Z-lys NCA 11 11 are easily prepared following published procedures.
  • BOC-lys NCA appears to be a suitable temporary protected monomer which, after deprotection, can act as a branching point and initiate the ring-opening copolymerization of a successive series of grafts.
  • a primary amine e.g. n-hexylamine
  • n-hexylamine is used to initiate the ring-opening copolymerization of the two NCA's to prepare the core of the targeted polypeptide.
  • Removal of the TPG generates a number of primary amine groups which can act as an initiator to graft a first generation of peptide arms onto the core.
  • Repetition of this ring-opening polymerization/deprotection cycle yields highly branched, or dendritic graft polypeptides.
  • the PPG can be removed in the very last step of the synthesis.
  • the PPG is removed to yield a polypeptide having free amino groups.
  • the method according to the invention is a new synthetic route which yields highly branched polypeptides with high molecular weights, e.g. > 10 kDa in a relatively small number of reaction steps.
  • the invention relates to a new synthetic strategy for the preparation of highly branched or dendritic graft polypeptides.
  • the method involves a repetitive sequence of NCA ring-opening polymerization and deprotection steps.
  • appropriately N e -protected L-lysine derivatives are used as branching points.
  • dendritic graft polypeptides e.g.- poly(L- lysine)s having a molecular weight of up to ⁇ 50 kDa were prepared in only 8 steps.
  • Such highly branched polypeptides are of interest as carriers for gene delivery and for the development of synthetic vaccines.
  • the method of the invention allows to prepare branched polypeptides, especially, highly branched or dendritic graft polypeptides.
  • the NCA used according to the invention is derived from an ⁇ -amino acid having an ⁇ -annino group forming the cyclic N-carboxyanhydride group and at least one further amino group which is/are different from the ⁇ -NH 2 substituent and which is/are protected by a protective group.
  • the NCA can be used alone or as a mixture, e.g.
  • NCAs a mixture of NCAs in which at least one of the NCAs contains a protected primary amino group.
  • the side chains of the other NCA comonomers are protected with suitable orthogonal protective groups.
  • a mixture of two or more ⁇ -amino acid N-carboxyanhydrides (NCAs) can be used.
  • functional groups at the side chains of the different monomers can be blocked with orthogonal protective groups. This allows to deprotect the functional group, in particular, a NH 2 -group, that acts as the branching point without affecting the other protective groups.
  • NCAs are used having orthogonally protected primary amino groups which are blocked by protective groups which are removable under distinct different conditions.
  • a mixture of NCAs is used, wherein comonomers are employed having side chains which do not interfere with or take part in a NCA ring-opening polymerization, such comonomers are e.g. Ala-NCA or Leu-NCA, having hydrocarbon side chains.
  • comonomers are e.g. Ala-NCA or Leu-NCA, having hydrocarbon side chains.
  • a first NCA with a first protective group and a second NCA with a second protective group being different from the first protective group are provided, wherein the first and the second NCAs are derived from the same amino acid.
  • the same amino group is blocked by different protective groups in each of the two NCAs.
  • Suitable amino acids, from which the NCAs can be derived and which can act as branching points include, but are not limited to lysine or ornithine.
  • the NCA or the mixture of NCAs is contacted with an initiator to start a ring-opening polymerization of the NCAs.
  • any initiator which starts the NCA ring-opening polymerization can be used.
  • Such initiators are well-known and available. Compounds having one or more amino groups are preferred. Suitable initiators include alkoxide anions, pyridines, C, to C 30 amines, e.g. tertiary, secondary or primary amines, in particular, C., to C 12 alkylamines, C, to C 12 alkyl diamines, amino acids, amino acids having at least two amino groups and dendrimers having free primary amino groups.
  • any polymer having one or more primary amine groups e.g. substituted poly(styrene), poly(ethy!ene oxide), saccharides etc. can also be used as initiator and result in hybrid materials.
  • the initiator used is an amino acid, from which an NCA according step (i) is derived, e.g. lysine or ornithine.
  • Suitable dendrimers having free primary amino groups comprise, for example, polypropyleneimine (PPI) and polyamidoamine (PAMAM) dendrimers.
  • Suitable protective groups are trifluoroacetyl, t-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl and 1 -methyI-1 -(3,5- dimethoxyphenyl) ethoxycarbonyl.
  • NCAs that act as branching point are copolymerized with NCAs containing functional groups that could interfere with the polymerization
  • protected NCAs are used, in particular, at least two NCAs which contain different protective groups each.
  • BOC t-butoxycarbonyl
  • Z benzyloxycarbonyl
  • PPG permanent protective group
  • the molar ratio of the first NCA having a temporary protected primary amino group to second NCAs having a permanent protected primary amino group or containing substituents that do not interfere with the NCA polymerization preferably ranges from 1 :20 to 5: 1 , more preferably from 1 : 10 to 1 : 1 and most preferably from 1 :5 to 1 :2.
  • the initiator in particular, free amino groups of the initiator cause a ring opening of the ⁇ -amino acid N-carboxyanhydrides (NCAs), thereby forming a peptide bond and generating again a free amino group, namely the ⁇ - amino group derived from the NCA.
  • NCAs ⁇ -amino acid N-carboxyanhydrides
  • a ring-opening polymerization of the NCAs proceeds.
  • protective groups are removed.
  • the protective group removed in this step is termed temporary protective group herein.
  • Protective groups orthogonal to this temporary protective group are not removed in step (iii), thus resulting in a polypeptide having free amino groups as well as side chain functionalities that do not interfere with the ring-opening polymerization process or which are protected with suitable permanent protective groups, e.g. permanent protected amino groups.
  • the free amino groups are derived from amino groups initially being protected by a temporary protective group.
  • step (iv) the same NCA or NCA mixture as employed in step (i) can be used. However, it is also possible to use a NCA or NCA mixture different from that of step (i), e.g. a mixture having a different ratio of constituents or different types of constituents.
  • a NCA or NCA mixture different from that of step (i), e.g. a mixture having a different ratio of constituents or different types of constituents.
  • new polypeptide chains are grafted onto to the polypeptide formed in the steps before by reacting the amino groups being protected in the first reaction steps by a temporary protective group with further ⁇ -amino acid N-carboxyanhydride (NCA) after removal of the temporary protective groups.
  • NCA ⁇ -amino acid N-carboxyanhydride
  • the chain length of linear segments can be easily adjusted by the molar ratio of ⁇ -amino acid N-carboxyanhydride (NCA) employed to free primary amine groups.
  • NCA ⁇ -amino acid N-carboxyanhydride
  • This molar ratio preferably ranges from 50: 1 to 5: 1 , more preferably from 30: 1 to 10: 1 in steps (ii) or/and (iv).
  • the molar ratio of NCA to free primary amine groups does not necessarily have to be the same during each grafting cycle but can be varied resulting in dendritic graft polypeptides with arm lengths that are different for each generation. Deprotection/grafting can be repeated as often as desired.
  • steps (iii) and (iv) are repeated one to ten times, more preferably three to eight times. However, it is also possible to repeat these steps twenty or more times depending on the desired size of the polypeptide to be formed.
  • steps (iii) and (iv) are repeated one to ten times, more preferably three to eight times. However, it is also possible to repeat these steps twenty or more times depending on the desired size of the polypeptide to be formed.
  • steps (iii) and (iv) are repeated one to ten times, more preferably three to eight times. However, it is also possible to repeat these steps twenty or more times depending on the desired size of the polypeptide to be formed.
  • the branching/grafting density of the dendritic graft polypeptides depends on the molar ratio between the temporary protected primary amine substituted NCA that acts as branching point and the other NCA comonomers.
  • the highest branching density one graft per amino acid residue, is obtained when the temporary protected primary amine substituted NCA is homopolymerized.
  • the branching density can be adjusted by the molar ratio of the monomers.
  • the temporary protected primary amine substituted NCA that acts as the branching point constitutes between 1 0- 60 mol% of the total monomer mixture.
  • the branching density does not need to be fixed throughout the synthesis, but can be varied from generation to generation.
  • polypeptides With the method of the invention fully protected, partially protected or totally deprotected polypeptides can be obtained. If the last step performed is a step (iv), fully protected polypeptides are obtained which contain at least one kind of protective group. If the polypeptide contains functional groups that could have interferred with the NCA ring-opening polymerization, preferably at least two types of orthogonal protective groups will be present. Partially protected polypeptides are obtained, if the last step performed is a step (iii), i.e. specifically temporary protective groups are removed and permanent protective groups that are eventually present remain in the molecule. It is especially preferred, however, to remove all protective groups, i.e.
  • the free amino groups of the dendritic graft polypeptide can be further functionalized to provide the molecules with a desired functionality.
  • the amino groups can be functionalized with one or more epitopes, resulting in multiple antigen peptides (MAPs) .
  • the free amino groups can also be used to attach synthetic polymers that possess a suitable end-group, e.g. a carboxylic acid end-functionalized poly(styrene) or poly(ethylene oxide), or can be modified with functional groups that can initiate radical or ring-opening polymerization reactions of vinyl-type and cyclic monomers, respectively. In this way, hybrid structures composed of a highly branched peptide core and a shell of a synthetic polymer become accessible. Such molecules are soluble in organic solvents and are of interest for storage, transport and release of small molecules and catalysis.
  • a further object of the present invention are branched polypeptides, in particular, dendritic graft polypeptides obtainable by the method described above.
  • Such polypeptides preferably contain units derived from ⁇ -amino acids having one or more primary amino groups in addition to the ⁇ -amino groups, wherein the additional amino groups are partially free and partially constitute branching points.
  • polypeptides can be prepared, wherein some of the amino groups serve as branching points (those provided with temporary protective groups) and other functional groups, in particular, amino groups can be obtained in free form after completion of the polymerizations (those, to which permanent protective groups were bound) .
  • the ratio of free amino groups to amino groups constituting branching points preferably ranges from 20: 1 to 1 :5, more preferably from 10: 1 to 1 : 1 .
  • polypeptides are of interest as vaccines and as diagnostic tools. Further, they can be used as carriers for gene delivery, since they have DNA-compIexing properties.
  • the invention therefore, also relates to a therapeutic or/and diagnostic agent containing a polypeptide as described above.
  • Scheme 1 shows the preparation of dendritic graft polypeptides via a repetitive sequence of NCA ring-opening polymerization and deprotection steps.
  • TPG temporary protective group
  • Fig.1 is part of the 700 MHz 1 H-NMR spectrum of the different dendritic graft poly(L-lysine)s recorded in D 2 O, and
  • Fig.2 shows GPC traces of the different dendritic graft poly(N e
  • the preparation of the dendritic graft poly(L-lysine)s was carried out in N,N-dimethylformamide (DMF), using a monomer mixture containing 20 mole% BOC-lys NCA and an initial molar ratio of NCA to primary amine groups of 20 throughout all polymerization steps.” 31
  • Treatment of this dendritic graft poly(Z-lys) with HBr/AcOH to remove the PPGs yielded the corresponding water-soluble polypeptide in quantitative yield.
  • Figure 1 shows an expansion of the methine region of the 1 H-NMR spectrum of the different dendritic graft poly(L-lysine)s.
  • the integral of the peak at ⁇ 4.30 ppm which can be assigned to the methine proton of the terminal ⁇ -amino acid of a branch, represents the number of grafting sites of that particular generation, or the number of arms of the next higher generation. Combination of these different integrals provides information about the number average degree of polymerization and the BOC-lys content of the arms that have been introduced in a particular generation.
  • Table 1 The results of the 1 H-NMR experiments are summarized in Table 1 .
  • I nteg rals were etermined relative to that of the triplet of the methyl group of the initiator moiety at 0.8 ppm, which was set equal to 3.
  • the 1 H-NMR spectra do not only give information about the number average degree of polymerization and the composition of the polypeptides, but also provide evidence for their branched topology. This becomes evident when comparing the integral of the methine protons of the terminal ⁇ -amino acid residues (labelled “C” in Fig.1 ) with that of the signals of the other methine protons (labelled “A” and “B” in Fig.1 ) . The ratio (A + B)/C would continuously increase with increasing degree of polymerization for a linear polypeptide. Table 1 , however, shows that for the polypeptides prepared according to the route outlined in Scheme 1 , this number is relatively small and slowly decreases from 1 8 to 1 3. This observation is consistent with a highly branched topology and a gradual decrease in the number average degree of polymerization per arm with increasing generation.
  • the first polypeptide dendrimers were prepared by stepwise coupling and deprotection of N ⁇ ,N ⁇ -di-(tert-butoxycarbonyl)-L-lysine: (a)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Polymers & Plastics (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to a method for the preparation of highly branched or dendritic graft polypeptides from alpha-amino acid N-carboxyanhydrides (NCA), to the polypeptides obtainable with said method and the use of the polypeptides as therapeutic or diagnostic agents.

Description

Method for the preparation of highly branched polypeptides
Description
The present invention relates to a method for the preparation of branched polypeptides, to the polypeptides obtainable with said method and the use of the polypeptides as therapeutic or diagnostic agents.
Branched or dendritic oligomers and polymers of L-lysine have attracted attention for the construction of peptide antigens111 and DNA delivery systems.121 Functionalization of the peripheral a- and e-amino groups of small dendritic oligo(L-lysine) scaffolds with one or different epitopes results in so-called multiple antigen peptides (MAPs), which are of interest for the development of vaccines and as diagnostic tools.111 Linear-dendritic di- and triblock copolymers of poly(ethylene glycol) and L-lysine dendrons have been reported, which can form water-soluble complexes with plasmid DNA.[21 These block copolymers show promise as carriers for gene delivery, since they combine the DNA complexing properties of cationic polymers with the water-solubility, non-immunogenicity and biocompatibility of poly(ethylene)glycol.
So far, dendritic polypeptides have been generally prepared in a stepwise fashion, either in homogeneous solution or via solid-phase synthesis, from appropriately N", Ne-diprotected building blocks.""31 Provided the synthesis and purification are performed carefully, this strategy will afford well- defined and perfectly monodisperse dendrimers/dendrons. Synthesis and purification, however, are usually laborious, and a large number of steps is required to obtain high molecular weight material.
Some earlier publications have reported the use of poly(L-Iysine) or copolymers of L-lysine and other σ-amino acids as macroinitiators to prepare graft polymers, which were termed multichain polypeptides.171 However, in all of these reports only a single grafting step was described. Further, dendritic graft poly(ethylene imine)151, poly(ethylene imine)-poly(2- ethyl-2-oxazoline) copolymers,151 poly(styrene),161 poly(butadiene)181 and poly(styrene)-poly(n-butyl methacrylate) copolymers[91have beendescribed.
Therefore, it was an object of the invention to provide a method for the simple preparation of branched polypeptides, by means of which, in particular, high-molecular compounds can be obtained in a few reaction steps.
According to the invention this object is achieved by a method for the preparation of branched polypeptides, in particular, highly branched or dendritic graft polypeptides, comprising the steps: (i) providing an cr-amino acid N-carboxyanhydride (NCA) having a protected primary amino group, which is different from the -NH2 substituent, (ii) contacting the NCA with an initiator, thereby initiating a ring- opening polymerization of the NCA, (iii) removing the protective group,
(iv) contacting the polypeptide obtained with an α-amino acid N- carboxyanhydride (NCA) having a protected primary amino group, thereby performing a ring-opening polymerization of the NCA, and (v) repeating steps (iii) and (iv) at least once.
The method of the invention allows to repeat the deprotection/ring-opening polymerization cycle several times and to prepare highly branched or dendritic graft polypeptides. These polypeptides can be obtained in only a few reaction steps and are therefore an interesting alternative for the perfect polypeptide dendrimers that are prepared according to the state of the art in a time-consuming step-by-step fashion1171. The principles of the inventive method are explained in detail in the following using the amino acid lysine as an example. However, it is to be noted that the inventive method can be performed using any amino acid having the characteristics as outlined in the claims.
The strategy, which is also outlined in Scheme 1 , is based on the ring- opening (co)polymerization of an σ-amino acid N-carboxyanhydride (NCA) having a protected primary amino group or of a mixture of NCAs in which at least one of the NCAs contains a protected primary amino group. If necessary, the side chains of the other NCA comonomers are protected with suitable orthogonal protective groups. Preferred is the use of two orthogonally N-protected σ-amino N-carboxyanhydrides (NCAs), e.g. two orthogonally Ne-protected L-lysine N-carboxyanhydrides. In this case one of the monomers contains a primary amine group masked with a temporary protective group (TPG) which can be removed under relatively mild conditions. Using e.g. lysine NCAs, the TPG group is fixed at the e-NH2 group. Functional groups present at the side chains of the other NCA comonomers, which could interfere with the polymerization, are protected with a permanent protective group (PPG) that is stable and is not removed under the conditions applied for the deprotection of the TPG. Using e.g. lysine NCAs, the e-NH2 group of the other L-lysine monomer is masked with a permanent protective group (PPG) which is stable and not removable under the conditions applied for the removal of the TPG.
Thus, for the realization of the synthesis depicted in Scheme 1 , preferably two orthogonally protected NCAs, in particular, Ne-protected L-lysine NCAs are elected. To explore the feasibility of the concept exemplarily Ne-(tert- butoxycarbonyI)-L-lysine NCA(BOC-lys NCA) and Ne-(benzyloxycarbonyl)-L- lysine NCA (Z-lys NCA) were used. Both, BOC-lys NCA[10] and Z-lys NCA11 11 are easily prepared following published procedures. Whereas the BOC groups are smoothly removed under the action of CF3COOH, Z-lys requires HBr/AcOH or hydrogenation for effective deprotection.1121 Thus, BOC-lys NCA appears to be a suitable temporary protected monomer which, after deprotection, can act as a branching point and initiate the ring-opening copolymerization of a successive series of grafts.
In the first synthetic step preferably a primary amine, e.g. n-hexylamine, is used to initiate the ring-opening copolymerization of the two NCA's to prepare the core of the targeted polypeptide. Removal of the TPG generates a number of primary amine groups which can act as an initiator to graft a first generation of peptide arms onto the core. Repetition of this ring-opening polymerization/deprotection cycle yields highly branched, or dendritic graft polypeptides. Following commonly accepted conventions, a dendritic graft polymer of generation n - 1 (G = n - 1 ) is obtained after n deprotection/grafting cycles. [5,6]
The PPG can be removed in the very last step of the synthesis. In a preferred embodiment the PPG is removed to yield a polypeptide having free amino groups. In sum, the method according to the invention is a new synthetic route which yields highly branched polypeptides with high molecular weights, e.g. > 10 kDa in a relatively small number of reaction steps.
Thus, the invention relates to a new synthetic strategy for the preparation of highly branched or dendritic graft polypeptides. The method involves a repetitive sequence of NCA ring-opening polymerization and deprotection steps. Preferably, appropriately Ne-protected L-lysine derivatives are used as branching points. In this way dendritic graft polypeptides, e.g.- poly(L- lysine)s having a molecular weight of up to ~ 50 kDa were prepared in only 8 steps. Such highly branched polypeptides are of interest as carriers for gene delivery and for the development of synthetic vaccines.
Generally, the method of the invention allows to prepare branched polypeptides, especially, highly branched or dendritic graft polypeptides. As starting material, an σ-amino acid N-carboxyanhydride (NCA = α-amino acid N-carboxyanhydride) having a protected primary amino group is used. The NCA used according to the invention is derived from an α-amino acid having an α-annino group forming the cyclic N-carboxyanhydride group and at least one further amino group which is/are different from the α-NH2 substituent and which is/are protected by a protective group. The NCA can be used alone or as a mixture, e.g. a mixture of NCAs in which at least one of the NCAs contains a protected primary amino group. If desired, the side chains of the other NCA comonomers are protected with suitable orthogonal protective groups. According to the present invention a mixture of two or more α-amino acid N-carboxyanhydrides (NCAs) can be used. In this case functional groups at the side chains of the different monomers can be blocked with orthogonal protective groups. This allows to deprotect the functional group, in particular, a NH2-group, that acts as the branching point without affecting the other protective groups. Preferably, NCAs are used having orthogonally protected primary amino groups which are blocked by protective groups which are removable under distinct different conditions. In another preferred embodiment a mixture of NCAs is used, wherein comonomers are employed having side chains which do not interfere with or take part in a NCA ring-opening polymerization, such comonomers are e.g. Ala-NCA or Leu-NCA, having hydrocarbon side chains. By selecting the type and amount of copolymer the chain length of each generation can be controlled.
It is also possible to use a mixture of two or more, preferably three or more NCAs, wherein at least two different NCAs derived from the same or different α-amino acid are employed having different TPGs. In this case sequential grafting to different, predefined sites of the polypeptide can be achieved.
In a preferred embodiment a first NCA with a first protective group and a second NCA with a second protective group being different from the first protective group are provided, wherein the first and the second NCAs are derived from the same amino acid. In this case the same amino group is blocked by different protective groups in each of the two NCAs. However, it is also possible to use different NCAs derived from different amino acids.
Suitable amino acids, from which the NCAs can be derived and which can act as branching points include, but are not limited to lysine or ornithine.
According to the inventive method the NCA or the mixture of NCAs is contacted with an initiator to start a ring-opening polymerization of the NCAs. To this end, any initiator which starts the NCA ring-opening polymerization can be used. Such initiators are well-known and available. Compounds having one or more amino groups are preferred. Suitable initiators include alkoxide anions, pyridines, C, to C30 amines, e.g. tertiary, secondary or primary amines, in particular, C., to C12 alkylamines, C, to C12 alkyl diamines, amino acids, amino acids having at least two amino groups and dendrimers having free primary amino groups. In addition, any polymer having one or more primary amine groups, e.g. substituted poly(styrene), poly(ethy!ene oxide), saccharides etc. can also be used as initiator and result in hybrid materials. In an especially preferred embodiment the initiator used is an amino acid, from which an NCA according step (i) is derived, e.g. lysine or ornithine. However, it is also possible to initiate the ring-opening polymerization by another amino acid. Suitable dendrimers having free primary amino groups comprise, for example, polypropyleneimine (PPI) and polyamidoamine (PAMAM) dendrimers.
Basically all groups which, under the ring-opening polymerization conditions of the NCAs, prevent the further amino group present in the amino acids employed from participating in said polymerization reaction can be used as protective group. Thus, the protective groups are stable under the polymerization conditions employed and are not split off. Suitable protective groups, for example, are trifluoroacetyl, t-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl and 1 -methyI-1 -(3,5- dimethoxyphenyl) ethoxycarbonyl. In case the primary amine substituted NCAs that act as branching point are copolymerized with NCAs containing functional groups that could interfere with the polymerization, it is essential according to the invention that protected NCAs are used, in particular, at least two NCAs which contain different protective groups each. Particularly preferred is the use of t-butoxycarbonyl (BOC) as a temporary protective group (TPG) and benzyloxycarbonyl (Z) as a permanent protective group (PPG) . When using said pair of orthogonally protective groups it is possible to split off the BOC protective group under conditions where the Z protective group is stable and not removed. In this way a particular type of protective group can be specifically split off in a partial deprotection step, while the protective group orthogonal thereto remains in the intermediate polypeptide formed. Preferably, a PPG is removed only after the final polymerization cycle.
The molar ratio of the first NCA having a temporary protected primary amino group to second NCAs having a permanent protected primary amino group or containing substituents that do not interfere with the NCA polymerization preferably ranges from 1 :20 to 5: 1 , more preferably from 1 : 10 to 1 : 1 and most preferably from 1 :5 to 1 :2.
The initiator, in particular, free amino groups of the initiator cause a ring opening of the α-amino acid N-carboxyanhydrides (NCAs), thereby forming a peptide bond and generating again a free amino group, namely the α- amino group derived from the NCA. Thus, a ring-opening polymerization of the NCAs proceeds. After this polymerization according to the invention protective groups are removed. The protective group removed in this step is termed temporary protective group herein. Protective groups orthogonal to this temporary protective group are not removed in step (iii), thus resulting in a polypeptide having free amino groups as well as side chain functionalities that do not interfere with the ring-opening polymerization process or which are protected with suitable permanent protective groups, e.g. permanent protected amino groups. The free amino groups are derived from amino groups initially being protected by a temporary protective group. By contacting the thus obtained polypeptide again with an NCA having a temporary protected primary amino group or mixtures thereof with suitable orthogonally protected NCA comonomers, if necessary, the ring- opening polymerization of the NCAs is started again. In this second reaction step the polypeptide obtained in step (iii) functions as an initiator for the ring-opening polymerization of the N-carboxyanhydrides (NCAs), since it contains free amino groups.
In step (iv) the same NCA or NCA mixture as employed in step (i) can be used. However, it is also possible to use a NCA or NCA mixture different from that of step (i), e.g. a mixture having a different ratio of constituents or different types of constituents. In step (iv) new polypeptide chains are grafted onto to the polypeptide formed in the steps before by reacting the amino groups being protected in the first reaction steps by a temporary protective group with further α-amino acid N-carboxyanhydride (NCA) after removal of the temporary protective groups.
The chain length of linear segments can be easily adjusted by the molar ratio of α-amino acid N-carboxyanhydride (NCA) employed to free primary amine groups. This molar ratio preferably ranges from 50: 1 to 5: 1 , more preferably from 30: 1 to 10: 1 in steps (ii) or/and (iv). During synthesis the molar ratio of NCA to free primary amine groups does not necessarily have to be the same during each grafting cycle but can be varied resulting in dendritic graft polypeptides with arm lengths that are different for each generation. Deprotection/grafting can be repeated as often as desired. Preferably, steps (iii) and (iv) are repeated one to ten times, more preferably three to eight times. However, it is also possible to repeat these steps twenty or more times depending on the desired size of the polypeptide to be formed. With the method of the invention it is easily possible to prepare high-molecular branched polypeptides having a molecular weight of greater than 10 kDa, in particular, greater than 30 kDa in only a few reaction steps. By using more repetitions of the deprotection and polymerization/grafting steps it is, however, also possible to prepare polypeptides having a molecular weight of greater than 100 kDa or even greater than 1000 kDa.
The branching/grafting density of the dendritic graft polypeptides depends on the molar ratio between the temporary protected primary amine substituted NCA that acts as branching point and the other NCA comonomers. The highest branching density, one graft per amino acid residue, is obtained when the temporary protected primary amine substituted NCA is homopolymerized. By copolymerization of this monomer with (mixtures of) other NCAs that are protected with permanent protective groups or carry substituents that do not interfere with the NCA polymerization, the branching density can be adjusted by the molar ratio of the monomers. Typically, the temporary protected primary amine substituted NCA that acts as the branching point constitutes between 1 0- 60 mol% of the total monomer mixture. The branching density does not need to be fixed throughout the synthesis, but can be varied from generation to generation.
With the method of the invention fully protected, partially protected or totally deprotected polypeptides can be obtained. If the last step performed is a step (iv), fully protected polypeptides are obtained which contain at least one kind of protective group. If the polypeptide contains functional groups that could have interferred with the NCA ring-opening polymerization, preferably at least two types of orthogonal protective groups will be present. Partially protected polypeptides are obtained, if the last step performed is a step (iii), i.e. specifically temporary protective groups are removed and permanent protective groups that are eventually present remain in the molecule. It is especially preferred, however, to remove all protective groups, i.e. both temporary protective groups and permanent protective groups that are eventually present, by treating the polypeptide under respective conditions after completion of the polymerization steps, i.e. when the polypeptide has the desired size. In this way a totally deprotected polypeptide is obtained which has free amino groups.
The free amino groups of the dendritic graft polypeptide can be further functionalized to provide the molecules with a desired functionality. For example, the amino groups can be functionalized with one or more epitopes, resulting in multiple antigen peptides (MAPs) . The free amino groups can also be used to attach synthetic polymers that possess a suitable end-group, e.g. a carboxylic acid end-functionalized poly(styrene) or poly(ethylene oxide), or can be modified with functional groups that can initiate radical or ring-opening polymerization reactions of vinyl-type and cyclic monomers, respectively. In this way, hybrid structures composed of a highly branched peptide core and a shell of a synthetic polymer become accessible. Such molecules are soluble in organic solvents and are of interest for storage, transport and release of small molecules and catalysis.
A further object of the present invention are branched polypeptides, in particular, dendritic graft polypeptides obtainable by the method described above. Such polypeptides preferably contain units derived from α-amino acids having one or more primary amino groups in addition to the α-amino groups, wherein the additional amino groups are partially free and partially constitute branching points. By the above-described manufacturing method polypeptides can be prepared, wherein some of the amino groups serve as branching points (those provided with temporary protective groups) and other functional groups, in particular, amino groups can be obtained in free form after completion of the polymerizations (those, to which permanent protective groups were bound) . The ratio of free amino groups to amino groups constituting branching points preferably ranges from 20: 1 to 1 :5, more preferably from 10: 1 to 1 : 1 .
These polypeptides are of interest as vaccines and as diagnostic tools. Further, they can be used as carriers for gene delivery, since they have DNA-compIexing properties. The invention, therefore, also relates to a therapeutic or/and diagnostic agent containing a polypeptide as described above.
The invention is further illustrated by the following Example and the Figures, wherein
Scheme 1 shows the preparation of dendritic graft polypeptides via a repetitive sequence of NCA ring-opening polymerization and deprotection steps. (TPG = temporary protective group, PPG
= permanent protective group) .
Fig.1 is part of the 700 MHz 1H-NMR spectrum of the different dendritic graft poly(L-lysine)s recorded in D2O, and
Fig.2 shows GPC traces of the different dendritic graft poly(Ne
(benzyloxycarbonyl)-L-lysine)s.
Example 1
The preparation of the dendritic graft poly(L-lysine)s was carried out in N,N-dimethylformamide (DMF), using a monomer mixture containing 20 mole% BOC-lys NCA and an initial molar ratio of NCA to primary amine groups of 20 throughout all polymerization steps."31 The deprotection/grafting cycle was repeated three times to ultimately afford a 2nd generation (G = 2) dendritic graft poly(Z-lys) . Treatment of this dendritic graft poly(Z-lys) with HBr/AcOH to remove the PPGs yielded the corresponding water-soluble polypeptide in quantitative yield. The polypeptides were analyzed with 1H-NMR spectroscopy and gel permeation chromatography (GPC). Figure 1 shows an expansion of the methine region of the 1H-NMR spectrum of the different dendritic graft poly(L-lysine)s.[14'15i Comparison of the integral of the signal at 0.9 ppm, which is due to the methyl group of the n-hexylamine initiator, with the sum of the integrals of the resonances representing the different methine protons yields the total number average degree of polymerization of the polypeptides. The integral of the peak at ~ 4.30 ppm, which can be assigned to the methine proton of the terminal α-amino acid of a branch, represents the number of grafting sites of that particular generation, or the number of arms of the next higher generation. Combination of these different integrals provides information about the number average degree of polymerization and the BOC-lys content of the arms that have been introduced in a particular generation. The results of the 1H-NMR experiments are summarized in Table 1 .
able 1 : Isolated yields and the results of the H-NMR characterization of the dendritic graft poly(L-lysine)s.
olymer Yield'1' 1 H-NMR integrals12' DPn (3) M DPn/arm(b) Total number of BOC-lys
(%) (-) (-) (g/mol) (-) branching content'6' points (-) (mol %) (1 ) Isolated
A B D (A + B)/C yi e ld , afte r ore 74 18 0 1 1 18 20 2670 20 6 25 precipitation
10 60 84 6 6 n.d. 15 96 12400 13 14 10 a nd freeze- i1 62 204 17 13.5 n.d. 16 235 30200 10 25 8 drying,
!2 54 300 22.5 24.5 n.d. 13 347 44600 5 n.d. n.d. (2) I nteg rals were etermined relative to that of the triplet of the methyl group of the initiator moiety at 0.8 ppm, which was set equal to 3.
3) Total number average degree of polymerization of the polypeptide.
4) Total number average molecular weight of the polypeptide.
5) Number average degree of polymerization of the arms added in a particular generation.
6) BOC lys content of the arms added in a particular generation.
The 1H-NMR spectra do not only give information about the number average degree of polymerization and the composition of the polypeptides, but also provide evidence for their branched topology. This becomes evident when comparing the integral of the methine protons of the terminal α-amino acid residues (labelled "C" in Fig.1 ) with that of the signals of the other methine protons (labelled "A" and "B" in Fig.1 ) . The ratio (A + B)/C would continuously increase with increasing degree of polymerization for a linear polypeptide. Table 1 , however, shows that for the polypeptides prepared according to the route outlined in Scheme 1 , this number is relatively small and slowly decreases from 1 8 to 1 3. This observation is consistent with a highly branched topology and a gradual decrease in the number average degree of polymerization per arm with increasing generation.
The data presented in Table 1 and Fig.1 clearly prove the feasibility of the synthetic strategy outlined in Scheme 1 and show that dendritic graft poly(L-lysine)s with number average molecular weights up to ~ 50 kDa can be prepared in only 8 steps. The 1 H-NMR data are supported by the results of the GPC analysis,1161 which are shown in Fig.2. All GPC traces possess a monomodal character and the average molecular weights (see fable 2), which can be calculated from the chromatograms rapidly increase with increasing generation. However, since the elution times were converted to molecular weights using a calibration curve constructed with linear poly(ethylene oxide) standards, the GPC experiments can only provide qualitative support. Table 2: The results of the GPC analysis of the different poly(Z-lys) dendritic graft polymers.
Polymer Mn (g/mol) W Mw (g/mol)( ) Mw/Mn (-)
.Core 2550 4600 1.8 GO 18800 31900 1.7 G1 60700 102200 1.7 G2 212900 409900 1.9
(1) Number-average molecular weight.
(2) \Λ/eight~average molecular weight.
Table 1 , however, also indicates that the synthesis is not completely free of side reactions. This is illustrated by the continuous decrease both in the number average degree of polymerization of the branches and in the BOC- lys content in these arms with increasing generation number. Although a 20-fold excess of NCA with respect to the number of grafting sites was used throughout the synthesis, the number average degree of polymerization per branch was found to decrease from ~ 20 for the core to ~ 5 for the G2 dendritic graft poly (L-lysine). This decrease in the DPn is accompanied by a decrease in the isolated yields. These findings might be interpreted in terms of a reduced accessibility of the grafting sites for the NCA monomers with increasing generation number. References and Notes
[1] See e.g.: (a) D.N.Posnett, H.McGrath, J.P.Tam, J.Biol.Chem.1988,
263, 1719-1725. (b) J.P.Tam, Proc.Natl.Acad.Sci. USA 1988, 85, 5409-5413. (c) J.P.Tam, J. Immunol. Methods 1996, 196, 17-32.
[2] (a) T.M. Chapman, G.L.Hillyer, E.J.Mahan, K.A.Schaffer,
J.Am.Chem.Soc. 1994, 116, 11195-11196. (b) J.S.Choi, E.J.Lee,
Y.H.Choi, Y.J.Yeong, J.S.Park, Bioconjugate Chem. 1999, 10, 62-
65. (c) J.S.Choi, D.K.Joo, C.H.Kim, K.Kim, J.S.Park, J.Am.Chem.Soc.2000, 122, 474-480.
[3] The first polypeptide dendrimers were prepared by stepwise coupling and deprotection of Nσ,Nε-di-(tert-butoxycarbonyl)-L-lysine: (a)
R.G.Denkewalter, J.F.Kolc, W.J.Lukasavage (Allied Corp.), U.S. US
4,410,688, 1983 [Chem.Abstr. 1984, 100 103907p]. (b) R.G.Denkewalter, J.F.Kolc, W.J.Lukasavage (Allied Corp.), U.S. US
4,289,872, 1981 [Chem.Abstr. 1985, 102 79324q]. (c)
S.M.Aharoni, N.S.Murthy, Polym.Commun. 1983, 24, 132-136.
[4] For reviews on the synthesis and polymerization of α-amino acid N- carboxyanhydrides, see e.g.: (a) H.R.Kricheldorf, α-Amino acid N- carboxyanhydrides and related heterocycles, Springer-Verlag: Berlin
1987. (b) T.J.Deming, Adv. Mater. 1997, 9, 299-311. (c)
T.J.Deming, J.Polym.Sci. Part A: Polym.Chem. 2000, 38, 3011-
3018.
[5] D.A.Tomalia, D.M.Hedstrand, M.S.Ferritto, Macromolecules 1991, 24, 1435-1438.
[6] M.Gauthier, M.Moller, Macromolecules 1991, 24, 4548-4553.
[7] See eg.: (a) M.Sela, S.Fuchs, R.Arnon, Biochem. J. 1962, 85, 223-
235. (b) A.Yaron, A.Berger, Biochim.Biophys.Acta 1965, 107, 307-
332. (c.) M.Sakamoto, J.Po/ym.Sci.:Po/ym. Chem. Ed. 1978, 16, 1107-1122. (d) M.Sakamoto, Y.Kuroyanagi, R.Sakamoto,
J.Polym.Sci.: Polym.Chem. Ed. 1978, 16, 2001-2017. [8] M.A.Hempenius, W.Michelberger, M.Mδller, Macromolecules 1997, 30, 5602-5605.
[9] R.B.Grubbs, C.J. Hawker, J.Dao, J . M . J . Frechet, Angew.Chem.lnt.Ed.Engl. 1997, 36, 270-272. [10] R.Wilder, S.Mobashery, .Orβf.CΛetr?. 1992, 57, 2755-2756.
[11] D.S.Poche, M.J.Moore, J.L.Bowles, Synth. Commun. 1999, 29, 843-854.
[12] T.W.Greene, P.G.M.Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc., New York 1999. [13] Polymerizations were conducted in dry DMF by adding the initiator (either neat, in case of n-hexylamine, or dissolved in DMF, in case of the macroinitiators) to a DMF solution containing the appropriate amount of monomer (~ 0.2 g/mL). After 5 days, the polymers were isolated by precipitation in Et2O. BOC groups were removed by treating the protected precursor polypeptide with CF3COOH for 15-
30 min at room temperature, followed by precipitation in NaHCO3 (aq) and freeze-drying.
[14] 1H-NMR experiments were performed at room temperature on a Bruker WS700 spectrometer. D2O was used as the solvent, and the residual proton signal was taken as internal standard.
[15] 1H-NMR resonances were assigned according to earlier published data on L-lysine oligomers: W.N.E. van Dijk-Wolthuis, L.van de
Water, P. van de Wetering, M.J.van Steenbergen, J.J.Kettenes-van den Bosch, W.J.W.Schuyl, W.E.Hennink, Macromol.Chem.Phys. 1997, 198, 3893-3906.
[16] GPC experiments were performed at 60°C with a setup consisting of a Waters 510 pump and a series of three PSS SDV columns with pore sizes of 500, 104 and 106 A. A 0.1 M solution of LiBr in DMF was used as the mobile phase and sample elution was monitored with simultaneous UV-Vis and refractive index detection. Elution times were converted to molecular weights using a calibration curve constructed from narrow polydispersity poly(ethylene oxide) standards. [17] An alternative approach for the preparation of highly branched polypeptides involves end-capping the NCA polymerization with the active ester of an Nσ,Nf-diprotected lysine derivative. Removal of the protective groups generates two primary amine groups at the peptide's N-terminus that can be used to initiate the polymerization of a next generation of arms, see: A.C.Birchall, M. North, Chem. Commun. 1 998, 1 335. This synthetic approach, however, is fundamentally different from the present method of invention and is also much less flexible, e.g. in varying the grafting density of the polymer.

Claims

Claims
1 . A method for the preparation of branched polypeptides comprising the steps:
(i) providing an α-amino acid N-carboxyanhydride (NCA) having a protected primary amino group, (ii) contacting the α-amino acid N-carboxyanhydride with an initiator, thereby initiating a ring-opening polymerization of the α-amino acid N-carboxyanhydride,
(iii) removing the protective group, (iv) contacting the polypeptide obtained with an α-amino acid N- carboxyanhydride having a protected primary amino group, thereby performing a ring-opening polymerization of the α- amino acid N-carboxyanhydride, and
(v) repeating steps (iii) and (iv) at least once.
2. The method according to claim 1 , wherein in step (i) or step (iv) a mixture of an α-amino acid NCA having a protected primary amino group with NCAs which contain no functional groups that could interfere with NCA polymerization or which are orthogonally protected, is used.
3. The method according to claim 1 or 2, further comprising a step (vi) deprotecting the polypeptide.
4. The method according to any of claims 1 to 3, wherein at least one NCA derived from lysine is used.
5. The method according to any of the preceding claims, wherein the protective groups are selected from trifluoracetyl, t-butoxycarbonyl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl and 1 -methyl-1 - (3,5-dimethoxyphenyl)ethoxycarbonyl.
6. The method according to any of the preceding claims, wherein a first NCA having a temporary protected primary amino group and a second NCA having a permanent protected primary amino group are employed, wherein the molar ratio of the first NCA having a temporary protected primary amino group to the second NCA having a permanent protected primary amino group ranges from 1 :20 to 5: 1 .
7. The method according to any of the preceding claims, wherein an initiator having one or more amino groups is used.
8. The method according to any of the preceding claims, wherein an initiator is used, selected from alkoxides, organometallic initiators, C,-C12 alkylamine, C.,-C12 alkyldiamine, C C12 substituted secondary and tertiary amines, amino acids, amino acids having at least two amino groups, dendrimers having free primary amino groups and polymers having one or more free primary amine group.
9. The method according to any of the preceding claims, wherein the same or a different NCA mixture is employed in steps (i) and (iv) .
1 0. The method according to any of the preceding claims, wherein the initial molar ratio of NCA to free primary amine groups ranges from 50: 1 to 5: 1 in steps (ii) or/and (iv) .
1 1 . The method according to any of the preceding claims, wherein in step (iii) specifically one of the protective groups is removed.
1 2. The method according to any of the preceding claims, wherein steps (iii) and (iv) each are repeated one to ten times.
1 3. The method according to any of the preceding claims, wherein a branched polypeptide having a molecular weight of greater than 10 kDa is prepared.
14. The method according to any of the preceding claims, wherein in steps (i) or/and (iv) additional NCAs which are derived from amino acids having no further amino group besides the α-amino group and/or NCAs which have further free amino groups and/or NCAs which have further orthogonally protected amino groups are provided.
1 5. The method according to any of the preceding claims, further comprising a step (vii) functionalizing free amino groups.
1 6. Branched polypeptide obtainable by the method according to any of claims 1 -1 5.
7. Branched polypeptide according to claim 1 6, which contains units derived from σ-amino acids having one or more primary amino groups in addition to the α-amino groups, wherein the amino groups are partially free and partially constitute branching points.
8. Branched polypeptide according to claim 1 6 or 1 7, wherein the ratio of free amino groups to amino groups constituting branching points ranges from 20: 1 to 1 :5.
9. Therapeutic agent containing a polypeptide according to any of claims 1 6 to 18.
20. Diagnostic agent containing a polypeptide according to any of claims 16 to 18.
21. Use of a polypeptide according to any of claims 16 to 18 for the manufacture of a pharmaceutical agent being a vaccine or/and a gene delivery tool.
PCT/EP2003/000899 2002-01-29 2003-01-29 Method for the preparation of highly branched polypeptides WO2003064452A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003205707A AU2003205707A1 (en) 2002-01-29 2003-01-29 Method for the preparation of highly branched polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP02002204.2 2002-01-29
EP02002204 2002-01-29

Publications (2)

Publication Number Publication Date
WO2003064452A2 true WO2003064452A2 (en) 2003-08-07
WO2003064452A3 WO2003064452A3 (en) 2004-01-22

Family

ID=27635784

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2003/000899 WO2003064452A2 (en) 2002-01-29 2003-01-29 Method for the preparation of highly branched polypeptides

Country Status (2)

Country Link
AU (1) AU2003205707A1 (en)
WO (1) WO2003064452A2 (en)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006114528A1 (en) * 2005-04-28 2006-11-02 Centre National De La Recherche Scientifique Method of preparing grafted polylysine dendrimers
WO2007060119A1 (en) * 2005-11-25 2007-05-31 Basf Se Production and use of highly functional, highly branched or hyperbranched polylysines
CN102153654A (en) * 2009-12-31 2011-08-17 中国疾病预防控制中心病毒病预防控制所 Branched polypeptide using immune active peptide as vector and derivatives and application of branched polypeptide
CN102153742A (en) * 2011-01-21 2011-08-17 中国科学院长春应用化学研究所 Poly-amino acid grafted copolymer and method for preparing same
CN104524584A (en) * 2015-02-04 2015-04-22 中国科学院长春应用化学研究所 Step-by-step responsive nano-carrier as well as preparation method and application thereof
WO2020128089A1 (en) 2018-12-21 2020-06-25 The Royal College Of Surgeons Ireland Star polypeptides
WO2022096518A1 (en) 2020-11-05 2022-05-12 Covestro (Netherlands) B.V. Compositions suitable for enhancing the flexural properties of objects containing vegetable fibers
WO2024008939A1 (en) 2022-07-08 2024-01-11 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024008938A1 (en) 2022-07-08 2024-01-11 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024008940A1 (en) 2022-07-08 2024-01-11 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024038153A1 (en) 2022-08-19 2024-02-22 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024038152A1 (en) 2022-08-19 2024-02-22 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109970881B (en) * 2019-03-01 2021-06-11 暨南大学 3D printing controlled-release nitric oxide nano stent material, and preparation method and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4289872A (en) * 1979-04-06 1981-09-15 Allied Corporation Macromolecular highly branched homogeneous compound based on lysine units

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4289872A (en) * 1979-04-06 1981-09-15 Allied Corporation Macromolecular highly branched homogeneous compound based on lysine units

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
HERNANDEZ JUAN RODRIGUEZ ET AL: "Synthesis and ring-opening (Co)polymerization of L-lysine N-carboxyanhydrides containing labile side-chain protective groups" J POLYM SCI PART A;JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY MAY 1 2003, vol. 41, no. 9, 1 May 2003 (2003-05-01), pages 1167-1187, XP002250743 *
JOON SIG CHOI ET AL: "Synthesis of a barbell-like triblock copolymer, poly(L-lysine) dendrimer-block-poly(ethylene glycol)-block-poly(L-lysine) dendrimer, and its self-assembly with plasmid DNA" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 122, no. 3, 26 January 2000 (2000-01-26), pages 474-480, XP002250768 UNITED STATES ISSN: 0002-7863 *
KLOK H-A ET AL: "STAR-SHAPED FLUORESCENT POLYPEPTIDES" JOURNAL OF POLYMER SCIENCE, INTERSCIENCE PUBLISHERS, XX, vol. 39, no. 10, 2001, pages 1572-1582, XP001061864 *
KLOK HARM-ANTON ET AL: "Dendritic-graft polypeptides" MACROMOLECULES;MACROMOLECULES NOV 5 2002, vol. 35, no. 23, 5 November 2002 (2002-11-05), pages 8718-8723, XP002250744 *
RODRIGUEZ-HERNANDEZ JUAN ET AL: "Highly branched poly(L-lysine)." BIOMACROMOLECULES, vol. 4, no. 2, March 2003 (2003-03), pages 249-258, XP002250742 ISSN: 1525-7797 *
ROY R ET AL: "SOLID-PHASE SYNTHESIS OF DENDRITIC SIALOSIDE INHIBITORS OF INFLUENZA A VIRUS HAEMAGGLUTININ" JOURNAL OF THE CHEMICAL SOCIETY, CHEMICAL COMMUNICATIONS, CHEMICAL SOCIETY. LETCHWORTH, GB, no. 24, 21 December 1993 (1993-12-21), pages 1869-1872, XP000616525 ISSN: 0022-4936 *
SAKTHIVEL T ET AL: "Synthesis and physicochemical properties of lipophilic polyamide dendrimers." PHARMACEUTICAL RESEARCH (NEW YORK), vol. 15, no. 5, May 1998 (1998-05), pages 776-782, XP009014408 ISSN: 0724-8741 *
SELA M ET AL.: "Multichain Polyamino acids" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 78, 20 February 1956 (1956-02-20), pages 746-751, XP002250775 United States *
SELA M ET AL.: "Studies on the Chemical Basis of Antigenicity of Proteins" BIOCHEMICAL JOURNAL, vol. 85, 1962, pages 223-235, XP009015349 *
STIRIBA S-E ET AL: "DENDRITIC POLYMERS IN BIOMEDICAL APPLICATIONS: FROM POTENTIAL TO CLINICAL USE IN DIAGNOSTICS AND THERAPY" ANGEWANDTE CHEMIE. INTERNATIONAL EDITION, VERLAG CHEMIE. WEINHEIM, DE, vol. 41, no. 8, 15 April 2002 (2002-04-15), pages 1329-1334, XP001111698 ISSN: 0570-0833 *
TAM J P: "Synthetic peptide vaccine design: Synthesis and properties of a high-density multiple antigenic peptide system" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 85, August 1988 (1988-08), pages 5409-5413, XP002131244 ISSN: 0027-8424 cited in the application *
TOTH G K ET AL: "BRANCHED POLYPEPTIDES AS ANTIGENS FOR INFLUENZA VIRUS HEMAGGLUTININ AND T-CELL RECEPTOR SUBUNITS" PEPTIDE RESEARCH, NATICK, MA, US, vol. 6, no. 5, 1 September 1993 (1993-09-01), pages 272-280, XP000607497 ISSN: 1040-5704 *
VOYER NORMAND ET AL: "The use of peptidic frameworks for the construction of molecular receptors and devices." TETRAHEDRON, vol. 51, no. 34, 21 August 1995 (1995-08-21), pages 9241-9284, XP002250745 ISSN: 0040-4020 *
WILDER R ET AL: "The use of triphosgene in the preparation of N-carboxy-alpha-amino acid anhydrides" JOURNAL OF ORGANIC CHEMISTRY, AMERICAN CHEMICAL SOCIETY. EASTON, US, vol. 57, no. 9, 24 April 1992 (1992-04-24), pages 2755-2756, XP002154840 ISSN: 0022-3263 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080206183A1 (en) * 2005-04-28 2008-08-28 Central National De La Recherche Scientifique Method of Preparing Grafted Polylysine Dendrimers
FR2885130A1 (en) * 2005-04-28 2006-11-03 Centre Nat Rech Scient PROCESS FOR PREPARING POLYLYSIN DENDRIMERES GRAFTS
WO2006114528A1 (en) * 2005-04-28 2006-11-02 Centre National De La Recherche Scientifique Method of preparing grafted polylysine dendrimers
CN102276827A (en) * 2005-11-25 2011-12-14 巴斯夫欧洲公司 Modified polylysine
US8378049B2 (en) 2005-11-25 2013-02-19 Basf Aktiengesellschaft Production and use of highly functional, highly branched or hyperbranched polylysines
EP2194063A2 (en) 2005-11-25 2010-06-09 Basf Se Production and use of modified polylysines
US7786240B2 (en) 2005-11-25 2010-08-31 Basf Aktiengesellschaft Production and use of highly functional, highly branched or hyperbranched polylysines
EP2194063A3 (en) * 2005-11-25 2010-09-08 Basf Se Production and use of modified polylysines
EP2332969A2 (en) 2005-11-25 2011-06-15 Basf Se Production of highly functional, highly branched or hyperbranched polylysines
US8846842B2 (en) 2005-11-25 2014-09-30 Basf Aktiengesellschaft Production and use of highly functional, highly branched or hyperbranched polylysines
EP2332969A3 (en) * 2005-11-25 2011-11-02 Basf Se Production of highly functional, highly branched or hyperbranched polylysines
WO2007060119A1 (en) * 2005-11-25 2007-05-31 Basf Se Production and use of highly functional, highly branched or hyperbranched polylysines
JP2009517489A (en) * 2005-11-25 2009-04-30 ビーエーエスエフ ソシエタス・ヨーロピア Production and use of highly functionalized hyperbranched or hyperbranched polylysine
CN102153654A (en) * 2009-12-31 2011-08-17 中国疾病预防控制中心病毒病预防控制所 Branched polypeptide using immune active peptide as vector and derivatives and application of branched polypeptide
CN102153654B (en) * 2009-12-31 2012-10-10 中国疾病预防控制中心病毒病预防控制所 Branched polypeptide using immune active peptide as vector and derivatives and application of branched polypeptide
CN102153742B (en) * 2011-01-21 2012-11-21 中国科学院长春应用化学研究所 Poly-amino acid grafted copolymer and method for preparing same
CN102153742A (en) * 2011-01-21 2011-08-17 中国科学院长春应用化学研究所 Poly-amino acid grafted copolymer and method for preparing same
CN104524584A (en) * 2015-02-04 2015-04-22 中国科学院长春应用化学研究所 Step-by-step responsive nano-carrier as well as preparation method and application thereof
CN104524584B (en) * 2015-02-04 2017-06-23 中国科学院长春应用化学研究所 A kind of nano-carrier for responding step by step, its preparation method and its application
WO2020128089A1 (en) 2018-12-21 2020-06-25 The Royal College Of Surgeons Ireland Star polypeptides
WO2022096518A1 (en) 2020-11-05 2022-05-12 Covestro (Netherlands) B.V. Compositions suitable for enhancing the flexural properties of objects containing vegetable fibers
WO2024008939A1 (en) 2022-07-08 2024-01-11 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024008938A1 (en) 2022-07-08 2024-01-11 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024008940A1 (en) 2022-07-08 2024-01-11 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024038153A1 (en) 2022-08-19 2024-02-22 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature
WO2024038152A1 (en) 2022-08-19 2024-02-22 Covestro (Netherlands) B.V. Compositions for fibreboards with enhanced properties upon fast-curing at low temperature

Also Published As

Publication number Publication date
AU2003205707A1 (en) 2003-09-02
WO2003064452A3 (en) 2004-01-22

Similar Documents

Publication Publication Date Title
AU741166B2 (en) Heterofunctionalized star-shaped poly(ethylene glycols) for protein modification
WO2003064452A2 (en) Method for the preparation of highly branched polypeptides
Habraken et al. How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 C? Effect of temperature on homo-, block-and graft (co) polymerization
Bulmus RAFT polymerization mediated bioconjugation strategies
Hernández et al. Synthesis and ring‐opening (co) polymerization of L‐lysine N‐carboxyanhydrides containing labile side‐chain protective groups
Klok et al. Dendritic− graft polypeptides
CN107556497B (en) A kind of preparation method and application of hybridized hydrogel material
Inoue et al. Preparation and Conformation of Hexaarmed Star Poly (. beta.-benzyl-L-aspartates) Synthesized Utilizing Hexakis (4-aminophenoxy) cyclotriphosphazene
CN116355201B (en) One-pot method for preparing copolymerized amino acid based on in-situ purification
Zhao et al. Well-defined (co) polypeptides bearing pendant alkyne groups
Karadag et al. Synthesis of poly (2-ethyl-2-oxazoline)-block-polypeptide copolymers by combination of ring-opening polymerization of oxazoline and polycondensation of activated urethane derivatives of α-amino acids
Whitton et al. Arborescent micelles: Dendritic poly (γ‐benzyl l‐glutamate) cores grafted with hydrophilic chain segments
US20090131589A1 (en) Controlled molecular weight amino acid polymers having functionalizable backbones and end groups and processes for preparing the same
Kricheldorf et al. Secondary structure of peptides, 11. How to synthesize helical poly (L‐phenylalanine)?
Sartore et al. Low molecular weight end-functionalized poly (N-vinylpyrrolidinone) for the modification of polypeptide aminogroups
Bulmuş et al. Conjugates of poly (N‐isopropyl acrylamide‐co‐acrylic acid) with alanine monopeptide, dipeptide, and tripeptide
De Marre et al. Synthesis of macromolecular Mitomycin C derivatives
Kim et al. Synthesis of diblock copolymer, methoxypoly (ethylene glycol)-block-polyamidoamine dendrimer and its generation-dependent self-assembly with plasmid DNA
Kricheldorf et al. Secondary structure of peptides. 20. Primary-secondary structure relationships of Ala/Gly/Val copolypeptides
Alsehli et al. Arborescent Polypeptides for Sustained Drug Delivery
Tinajero-Díaz et al. Polypeptide-based materials prepared by ring-opening polymerisation of anionic-based α-amino acid N-carboxyanhydrides: A platform for delivery of bioactive-compounds
Lu et al. Semitelechelic poly [N-(2-hydroxypropyl)-methacrylamide] for biomedical applications
WO2018016513A1 (en) Method for producing polymer having amino group at end
Daly et al. λ-Benzyl-L-Glutamate Graft Copolymers of Cellulose and Poly [Arylene Ether Sulfone]
Gevrek et al. Design and synthesis of maleimide group containing polymeric materials via the Diels-Alder/retro Diels-Alder strategy

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP