EP1189928A1 - Methodes et compositions pour realiser une synthese regulee de polypeptides - Google Patents

Methodes et compositions pour realiser une synthese regulee de polypeptides

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Publication number
EP1189928A1
EP1189928A1 EP00930551A EP00930551A EP1189928A1 EP 1189928 A1 EP1189928 A1 EP 1189928A1 EP 00930551 A EP00930551 A EP 00930551A EP 00930551 A EP00930551 A EP 00930551A EP 1189928 A1 EP1189928 A1 EP 1189928A1
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European Patent Office
Prior art keywords
nca
amino acid
thf
group
block
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EP00930551A
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German (de)
English (en)
Inventor
Timothy J. Deming
Miaoer Yu
Scott A. Curtin
Jungyeon Hwang
Michael D. Wyrsta
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University of California
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University of California
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    • 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
    • C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
    • 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
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C07K1/084—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents containing nitrogen
    • 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
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/08—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents
    • C07K1/088—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using activating agents containing other elements, e.g. B, Si, As

Definitions

  • the present invention relates to the synthesis of amino-acid based polymers.
  • this invention relates to methods and compositions for the synthesis of amino-acid based polymers using catalysts under "living" conditions, that is conditions free of termination and chain transfer.
  • Synthetic polypeptides have a number of advantages over peptides produced in biological systems and have been used to make fundamental contributions to both the physical chemistry of macromolecules and the analysis of protein structures. See e.g. G.D. Fasman, Poly a- Amino Acids, Dekker, New York, (1967). Moreover, 2 synthetic peptides are both more cost efficient and can possess a greater range of material properties than peptides produced in biological systems.
  • Small synthetic peptide sequences are conventionally prepared using stepwise solid-phase synthesis.
  • Such solid phase synthesis makes use of an insoluble resin support for a growing oligomer.
  • a sequence of subunits, destined to comprise a desired polymer, are reacted together in sequence on the support.
  • a terminal amino acid is attached to the solid support in an initial reaction, either directly or through a keying agent.
  • the terminal residue is reacted, in sequence, with a series of further residues such as amino acids or blocked amino acid moieties to yield a growing oligomer attached to the solid support through the terminal residue.
  • unreacted reactant materials are washed out or otherwise removed from contact with the solid phase.
  • the cycle is continued with a pre-selected sequence of residues until the desired polymer has been completely synthesized, but remains attached to the solid support.
  • the polymer is then cleaved from the solid support and purified for use.
  • the foregoing general synthetic scheme was developed by R.B. Merrifield for use in the preparation of certain peptides. See e.g. See Merrifield's Nobel Prize Lecture “Solid Phase Synthesis", Science, Volume 232, pp. 341-347 (1986).
  • a major disadvantage of conventional solid phase synthetic methods for the preparation of oligomeric materials results from the fact that the reactions involved in the scheme are imperfect; no reaction proceeds to 100% completion. As each new subunit is added to the growing oligomeric chain a small, but measurable, proportion of the desired reaction fails to take place. The result of this is a series of peptides, nucleotides, or other oligomers having deletions in their sequence.
  • the result of the foregoing imperfection in the synthetic scheme is that as desired chain length increases, the effective yield of desired product decreases drastically, since increased chances for deletion occur. Similar considerations attend other types of unwanted reactions, such as those resulting from imperfect blocking, side reactions, and the like.
  • NCA ⁇ -aminoacid-N- carboxyanhydride
  • NCA polymerizations are much faster than amine initiated reactions. These polymerizations are poorly understood but are believed to propagate through either NCA anion or carbamate reactive species (see equations 3 and 4 below, respectively). See e.g. CH. Bamford, et al., Synthetic Polypeptides, Academic Press, New York, (1956).
  • NCA polymerizations employing conventional initiators are plagued by chain-breaking transfer and termination reactions which prevent formation of block copolymers.
  • chain-breaking transfer and termination reactions which prevent formation of block copolymers.
  • H.R. Kricheldorf a-Aminoacid-N-Carboxyanhydrides and Related Materials, Springer- Verlag, New York, (1987). Consequently, the mechanisms of NCA polymerization have been under intensive study so that problematic side reactions could be eliminated.
  • H.R. Kricheldorf in Models of Biopolymers by Ring-Opening Polymerization, Penczek, S. Ed., CRC Press, Boca Raton, (1990).
  • Block copolymers of amino acids have been less well studied, largely because our synthetic methods do not yet have fine enough control to produce well- defined structures. F. Cardinauz, et al., Biopolymers, 16:2005-2028 (1977). The same is true of the synthesis of block copolypeptides for use as biomaterials or as selective membranes - the potential advantages of the protein-like architectures have remained unrealized for want of adequate synthetic building blocks and tools.
  • biomedical applications such as drug delivery typically require water-soluble components to enhance their ability for circulation in vivo.
  • water-soluble polypeptides e.g., poly-L-lysine and poly-L- aspartate
  • Nonionic, water-soluble polypeptides are desired for biomedical applications since they avoid these problems, and can also display the stable secondary structures of proteins that influence biological properties.
  • all high molecular weight nonionic homopolypeptides >25 residues
  • naturally occurring amino acids are notoriously insoluble in water.
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • grafted onto polypeptides or other polymers to improve their properties in vivo.
  • PEG is nonionic, water-soluble, and most importantly not recognized by immune systems. It is believed that PEG imparts 6 biocompatibility through formation of a hydrated "steric barrier" at the surface of material that cannot be penetrated or recognized by biological molecules, such as proteolytic enzymes. As such, block or graft copolymer drug carriers containing PEG are able to circulate for long periods in the bloodstream without degradation.
  • Polypeptides are being considered for a variety of biomedical problems such as tissue engineering and drug delivery. Another consideration for these applications is the incorporation of endgroup functionality onto the chains, which is essential for targeting of the drug delivery complexes as well as substrate specific anchoring of these materials. These, and other features would be useful for controlling both the structure and the properties of polypeptide materials. Consequently, there is a need for novel methods and compositions which allow for the facile generation of peptides tailored to have specific desirable properties.
  • the present invention discloses novel methods and compositions which address the need for advanced tools to generate polypeptides having varied material properties.
  • the methods and initiator compositions for NCA polymerization disclosed herein allow the precise control of such polypeptide synthesis.
  • the methods of the invention allow successful peptide synthesis by utilizing the versatile chemistry of transition metals to mediate the addition of monomers to the active polymer chain-ends, and therefore eliminate chain-breaking side reactions in favor of the chain-growth process. In this way, the disclosed methods allow the formation of block copolymers.
  • One embodiment of the invention provides a method of making an amido- containing metallacycle comprising combining an amount of an a-aminoacid-N- carboxyanhydride monomer with an initiator molecule comprising a low valent transition metal-Lewis Base ligand complex so that an amido-containing metallacycle is formed.
  • An alternative embodiment of the invention provides a method of making an initiator molecule, which includes the step of combining an allyloxycarbonyl (alloc) protected amino acid amide and a low valent transition metal-Lewis base ligand complex so that an amido-amidate metallacycle is formed having the following general formula:
  • M is a low valent transition metal
  • L is a Lewis base ligand
  • one of R1 and R2 is an amino acid side group and the other is hydrogen
  • R3 is any functional end group capable of being attached to a primary amine group.
  • the R3 end group will typically be used to "tag" or functionalize the polypeptide chains, and is the main advantage associated with using this method.
  • this group will be a peptide, oligosaccha de, oligonucleotide, fluorescent molecule, polymer chain, small molecule therapeutic, chemical linker to attach the polypeptide to a substrate, chemical linker to act as a sensing moiety, or reactive linker to couple the polypeptide to larger molecules such as proteins, polysaccharides or polynucleotides
  • compositions consisting of five or six membered amido-containing metallacycles comprising molecules of the general formula
  • M is a low valent transition metal
  • L is a Lewis Base ligand
  • each of R1 , R2, R3, R5 and R6 (independently) is a moiety selected from the group consisting of the side chains of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, senne, threonine, tryptophan, tyrosine or valine
  • R4 is a hydrogen moiety or a polyaminoacid chain
  • R7 is a functional end group
  • the metal is a transition metal selected from the group consisting of nickel, palladium, platinum cobalt, rhodium, indium and iron and the Lewis Base ligand is selected from the group consisting of pyridyl ligands, diimine ligands, bisoxazo ne ligands, alkyl phosphine ligands, aryl phosphine ligands, tertiary amine ligands, isocyanide ligands and cyanide ligands
  • a related embodiment of the invention consists of a method of adding an aminoacid-N-carboxyanhydnde (NCA) to a polyaminoacid chain having an amido containing metallacycle end group by combining the NCA with the polyaminoacid chain so that the NCA is added to the polyaminoacid chain
  • NCA aminoacid-N-carboxyanhydnde
  • Another embodiment of the invention disclosed herein entails a method of polymerizing aminoacid-N-carboxyanhyd ⁇ de monomers by combining a NCA monomer with an initiator molecule complex comprised of a low valent transition metal-Lewis Base ligand
  • a specific embodiment of the invention disclosed herein entails a method of polymerizing aminoacid-N-carboxyanhydride monomers having a ring with a O-C 5 and a O-C 2 anhydride bond which consists of combining a first NCA monomer with an initiator molecule complex comprised of a low valent metal capable of undergoing an oxidative addition reaction wherein the oxidative addition reaction formally increases the oxidation state by two electrons; and an electron donor comprising a Lewis base.
  • the initiator molecule is then allowed to open the ring of the first NCA through oxidative addition across either the O-C 5 or O-C 2 anhydride bond and then combine with a second NCA monomer, to form an amido-containing metallacycle.
  • a third NCA monomer is then allowed to combine with the amido containing metallacyle so that the amido nitrogen of the amido containing metallacyle attacks the carbonyl carbon of the NCA .
  • the NCA is added to the polyaminoacid chain and the amido containing metallacyle is regenerated for further polymerization.
  • the efficiency of the initiator is controlled by allowing the reaction to proceed in a solvent selected for its ability to influence the reaction.
  • the solvent is selected from the group consisting of ethyl acetate, toluene, dioxane, acetonitrile, THF and DMF.
  • Another embodiment of the invention provides a method of making a block copolypeptide consisting of combining an amount of a first aminoacid-N- carboxyanhydride (NCA) monomer with an initiator molecule comprising a low valent transition metal-Lewis Base ligand complex so that a polyaminoacid chain is generated and then combining an amount of a second aminoacid-N- carboxyanhydride monomer with the polyaminoacid chain so that the second aminoacid-N-carboxyanhydride monomer is added to the polyaminoacid chain.
  • the initiator molecule combines with the first aminoacid-N-carboxyanhydride monomer to form an amido containing metallacycle intermediate of the general formula:
  • M is the low valent transition metal
  • L is the Lewis Base ligand; each of R1 , R2 and R3 independently is a moiety selected from the group consisting of the side chains of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; and
  • R4 is the polyaminoacid chain.
  • the invention provides block copolypeptide compositions having characteristics which have been previously unattainable through conventional techniques.
  • a specific embodiment of this invention consists of a polypeptide composition comprising a block polypeptide having a number of overall monomer units that are greater than about 100 amino acid residues and a distribution of chain-lengths at least about 1.01 ⁇ Mw/Mn ⁇ 1.25.
  • the polypeptide has a number of overall monomer units that are greater than about 250 amino acid residues.
  • the copolypeptide consists of a least 3 blocks of consecutive identical amino acid monomer units.
  • at least one of the block's components is g- benzyl-L-glutamate.
  • the present invention also discloses novel methods and compositions, which address the need for biocompatible materials having improved properties of biochemical stability, water solubility, and self assembly.
  • the methods of making amphiphilic block copolypeptides disclosed herein allow the synthesis and assembly of compositions containing well-defined vesicular structures, which are potentially valuable for biomedical applications, such as drug delivery.
  • One embodiment of the invention provides a method of making an amphiphilic block copolypeptide, which includes the steps of (1) generating a soluble block polypeptide by combining an amount of an oligo (ethyleneglycol) functionalized aminoacid-N-carboxyanhydride (EG-aa-NCA) monomer with an initiator molecule; and (2) attaching an insoluble block by combining the soluble block with a composition comprising at least one other amino acid NCA monomer.
  • the amino acid component of the EG-aa-NCA monomer is lysine, serine, cysteine, or tyrosine
  • the insoluble block can contain a mixture of amino acids, which includes one or more naturally occurring amino acids.
  • a related embodiment of the invention consists of a method of adding an aminoacid-N-carboxyanhydride (NCA) to a soluble block polypeptide having one or more oligo(ethyleneglycol)-terminated amino acid residues by combining the NCA with the polypeptide so that the NCA is added to the polypeptide.
  • NCA aminoacid-N-carboxyanhydride
  • the invention provides amphiphilic block copolypeptide compositions, which have improved characteristics of solubility, biochemical stability and biocompatibility.
  • the amphiphilic block copolypeptide includes a soluble block polypeptide having one or more oligo(ethyleneglycol)- terminated amino acid residues and an insoluble block comprised substantially of nonionic amino acid residues.
  • a specific embodiment of this invention is a polypeptide composition comprising: (1 ) a soluble block polypeptide having EG- lysine residues, and (2) an insoluble block polypeptide containing a mixture of two to three different kinds of amino acid components in a statistically random sequence.
  • the copolypeptide consists of a least 3 blocks, wherein one or more of the blocks is a soluble block polypeptide and another block is an insoluble block polypeptide.
  • the amphiphilic nature of the block copolypeptides provides yet another embodiment, which is a method of forming vesicles.
  • This method consists of suspending the amphiphilic block copolypeptides in an aqueous solution so that the copolypeptides spontaneously self assemble into vesicles.
  • smaller vesicles having a diameter of about 50 nm to about 500 nm can be formed by sonicating the suspension of larger vesicles.
  • the invention provides vesicle-containing compositions comprised of the amphiphilic block copolypeptides of the present invention and water.
  • the invention provides methods for making EG-functionalized amino acid monomers, which includes the step of combining an ethyleneglycol (EG) derivative with an amino acid having a reactive side group, e.g., lysine, serine, cysteine, and tyrosine.
  • EG ethyleneglycol
  • amphiphilic block copolypeptides are particularly attractive since the EG-amino acid domains will emulate certain desirable features of poly (ethyleneglycol), PEG.
  • PEG poly (ethyleneglycol), PEG.
  • PEG is well known for its bioinvisibility meaning that it is not recognized by immunological defense mechanisms in the body, and thus has found many useful applications in drug delivery, enzyme stabilization, tissue engineering, and implant surface modification.
  • NCAs amino acid-N-carboxyanhydrides
  • metals and ligands are described.
  • These initiators are substantially different in nature from all known conventional initiators used to polymerize NCAs and are also unique in being able to control these polymerizations so that block copolymers of amino acids can be prepared.
  • these initiators eliminate chain transfer and chain termination side reactions from these polymerizations resulting in narrow molecular weight distributions, molecular weight control, and the ability to prepare copolymers of defined block sequence and composition. All of these traits have previously been unobtainable using conventional initiator systems.
  • the initiators described herein are readily prepared in a single step from commercially available materials.
  • compositions disclosed herein allow the preparation of complex polypeptide biomatenals which have potential applications in biology, chemistry, physics, and materials engineering Potential applications include medicine (drug delivery, tissue engineering), "smart” hydrogels (environmentally responsive organic materials), and in organic/inorganic biomimetic composites (artificial bone, high performance coatings)
  • Figure 1 compares the abilities of different initiators to control molecular weight of PBLG as a function of initiator concentration in polymerizations of Glu- NCA A, phenethylamine initiator, B, b ⁇ pyN ⁇ (COD) initiator, C, sodium rerf-butoxide initiator, D, theoretical molecular weight calculated from [M]o/[l]o All polymerizations were run in anhydrous DMF at 25°C for 1 day in sealed tubes Molecular weight (Mn ) was determined by tandem GPC/light scattering in 0 1 M LiBr in DMF at 60 °C
  • Figure 2 is a chromatogram of a PBLGo 78-£>-PZLLo 22 diblock copolymer prepared by sequential addition of Lys-NCA and Glu-NCA to b ⁇ pyN ⁇ (COD) initiator in DMF
  • the polymer was injected directly into the GPC, eluted using 0 1 M LiBr in DMF at 60 °C through 10 5 A and 10 3 A Phe ⁇ omenex 5 ⁇ m columns, and detected with a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP
  • Figure 3 shows 2 chemical reaction schemes associated with ammo acid derived nickelacycles, intermediates in nickel initiator mediated polypeptide synthesis
  • Figure 4 shows 4 chemical reaction equations associated with ammo acid derived nickelacycles, intermediates in nickel initiator mediated polypeptide synthesis
  • Figure 5 shows chemical structures of some ligands used in NCA polymerization reactions
  • Figure 6 shows the formation of an amido-containing metallacycle by reaction of NCAs with a metal initiator
  • Figure 7 shows MALDI-MS of leucyl isoamylamide-C-terminated ol ⁇ go(phenylglyc ⁇ ne)s
  • a partial expansion of the spectrum is shown in the upper right Mass series were observed for (a) leuc e-OH terminated oligomers resulting from the hydrolysis of the terminal amide in TFA, (b) Leucme isoamylamide terminated oligomers resulting from intact end-functionalized chains, and (c) non- functionalized chains
  • 9a indicates the MH+ ion o f the nona(phenylglyc ⁇ ne) of the a series
  • the b and c series are labeled similarly
  • the ions of the various series also formed adducts with O atoms and C0 2 and are labeled as such
  • block copolypeptide refers to polypeptides containing at least two covalently linked domains ("blocks"), one block having ammo acid residues that differ in composition from the composition of ammo acid residues of another block
  • the number, length, order, and composition of these blocks can vary to include all possible ammo acids in any number of repeats
  • the total number of overall monomer units (residues) in the block copolypeptide is greater than 100 and the distribution of chain-lengths in the block copolymer is about
  • protection and “side-chain protecting group” as used herein refer to chemical substituents placed on reactive functional groups, typically nucleophiies or sources of protons, to render them unreactive as protic sources or nucleophiies
  • reactive functional groups typically nucleophiies or sources of protons
  • the ideal living polymerization is characterized by fast initiation and an absence of the termination and chain transfer steps that in most polymerization systems compete with propagation of the growing chain
  • all polymer chains begin growing at about the same time and continue to grow until the monomer has been exhausted
  • the average number of monomer residues per chain is then simply the molar ratio of monomer to initiator, and the distribution of chain lengths is described by Poisson statistics M Szwarc, Carbanions, Living Polymers, and Electron-Transfer Processes, Wiley, New York (1968)
  • these conditions have been met in the polymerization of NCAs by bipyNi (COD)
  • COD bipyNi
  • the distribution of chain lengths is narrow, consistent with Poission statistics, and the rate of polymerization is proportional to monomer concentration, indicating that the number of active chain ends remains constant throughout the reaction
  • the disclosed methods and compositions allow for the generation and manipulation of peptides in manners that have not previously been possible Living polymerizations allow the synthesis of polymers of predetermined molecular weights and narrow molecular-weight distribution, and, perhaps more importantly, the preparation of well-defined block copolymers in which long sequences of each of the individual monomer residues are linked together at a single site
  • Living polymerizations which once were reserved for a small subset of polymenzable monomers, can now be extended to NCAs and to the preparation of high-molecular- weight polypeptides and block copolypeptides with unusual and useful properties
  • compositions disclosed herein teach new ways to polymerize ammo acids and to add ammo acids to polyamino acid chains Further, the initiators and amido-containing metallacyle compositions disclosed herein allow the synthesis of block copolypeptides by eliminating of side reactions in favor of the chain-growth process (i e living polymerization), thus allowing multiple monomer additions to polyaminoacid chains While the specific methods and initiator and amido-containing metallacyle compositions disclosed represent preferred embodiments of this invention, as discussed below, other embodiments are also contemplated
  • NCAs a-amino acid-N-carboxyanhydrides
  • NCAs have been limited because of their complicated reactivity and tendency to uncontrollably polymerize.
  • the molecules described herein are a new class of initiators based on low valent metal-Lewis base complexes which are able to eliminate significant competing termination and transfer steps from NCA polymerizations and allow preparation of well-defined block copolypeptides.
  • a variety of illustrative initiator complexes useful in the generation of block copolypeptides are described herein such as those generated using bis-1 ,5- cyclooctadiene nickel (Ni(COD) 2 ) as the nickel source and 2,2'-bipyridyl (bipy) as the donor ligand component in tetrahydrofuran (THF) solvent.
  • Ni(COD) 2 bis-1 ,5- cyclooctadiene nickel
  • bipy 2,2'-bipyridyl
  • THF tetrahydrofuran
  • PR 3 Me, Et, Bu, cyclohexyl, phenyl]
  • R 2 PCH 2 CH 2 PR 2 [R Me, phe ⁇ yl]
  • a, a'-diimine ligands [
  • Example 4 in addition to bis-1 ,5-cyclooctadiene nickel (Ni(COD) 2 ), other sources of zerovalent nickel (e.g. Ni(CO) 4 ) as well as other low valent metals in the initiator complexes have been used successfully in these methods.
  • Illustrative metals useful in the generation of initiators are "low valent" transition metals, in particular the metals of Group VIII of the Periodic Table and illustrative examples of initiators using such metals is provided in Table 8. This group includes the metals, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd and Pt.
  • the "low valent" forms of the metals implies that the metals are in low oxidation states.
  • ligands which can be used to bind to the initiator metals complexes need to comprise a non nucleophilic electron donor comprising a Lewis base and can consists of a variety of groups which have this property including those that are pyridyl based (see e.g. entry 2-144, Table 7), diimine (see e.g. DIPRIM, 2-148), bisoxazoline (see e.g. DPOX, 3-2), alkyl phosphine (see e.g.
  • the ligands are bidentate (coordinate through 2 atoms) or are composed of 2 equivalents of monodentate ligands. Tridentate ligands can also be used (e.g. terpyridine).
  • the ligands generally are bound to the metal through N, P, or C atoms of the molecule. Other N or P donor ligands, similar to those mentioned above (i.e. neutral, non-nucleophilic, aprotic) can also support these initiators.
  • NCA monomers are well known in the art (see e.g. H.R. Kricheldorf, a-Aminoacid-N-Carboxyanhydhdes and Related Materials, Springer-Verlag, New York, (1987)).
  • the use of a variety of NCA monomers in methods of polypeptide synthesis is well known in the art.
  • the stepwise synthesis of polypeptides using NCAs (or derivatives thereof) is disclosed in U. S. Patent No. 3,846,399 (incorporated by reference herein).
  • U.S. Patent No. 4,267,344 discloses N-substituted N-carboanhydrides of a amino acids and their application in the preparation of peptides (incorporated by reference herein).
  • Another embodiment of the present invention involves the synthesis of unique oligo (ethyleneglycol) functionalized amino acids and their subsequent polymerization into oligo (ethyleneglycol) functionalized polypeptides.
  • the method of making these monomers includes the step of combining an ethyleneglycol (EG) derivative with an amino acid having a reactive side group, e.g., lysine, serine, cysteine, and tyrosine, to form an EG-functionalized amino acid.
  • EG ethyleneglycol
  • the EG derivative has the general formula (CH 3 OCH 2 CH 2 ) n X, where n amounts to about 1 to 3 EG repeats and X is a reactive group, such as chloroform ate, N-hydroxysuccidimydyl acetate, or a halide.
  • X is a reactive group, such as chloroform ate, N-hydroxysuccidimydyl acetate, or a halide.
  • the EG functionalized amino acids can then be converted to an NCA monomers for use in the synthesis of oligo (EG) functionalized polypeptides.
  • NCA monomers can be used to make polypeptide chains where the side chain of every residue is capped by a ethylene glycol oligomer; in effect, "PEG coated polypeptides".
  • Poly(ethyleneglycol), PEG is well known for its "bioinvisibility”, meaning that it is not recognized by immunological defense mechanisms in the body (non-antigenic), and thus has found many useful applications in drug delivery, enzyme stabilization, tissue engineering, and implant surface modification.
  • Such polymers are unusual in possessing excellent water solubility over broad pH ranges (2-13) and salt concentrations. Furthermore, the PEG "coating" strongly stabilizes the secondary structure of the polypeptides (beta-sheets and alpha- helices) such that the polymers possess stable secondary structures over broad pH and temperature ranges. These new polymers are attractive since they display most of the same properties of PEG (water solubility, biocompatibility), but possess completely different chain structures.
  • the serine and cysteine-derived polymers adopt beta-sheet structures and represent the first examples of water soluble polypeptides that form stable beta-sheet structures. As such their solution and mechanical properties are markedly different from PEG and thus provide an interesting alternative to PEG in biomedical materials.
  • the initiator complexes of the present invention can be synthesized by two different approaches, both of which entail the use of a low valent transition metal- Lewis Base ligand complex and result in the formation of an amido-containing metallacycle.
  • One embodiment of the invention provides a method of making an amido- containing metallacycle comprising combining an amount of an a-aminoacid-N- carboxyanhydride monomer with an initiator molecule comprising a low valent transition metal-Lewis Base ligand complex so that an amido-containing metallacycle is formed.
  • NCAs are unsymmetrical anhydrides, the oxidative-addition of NCAs can yield two distinct isome ⁇ c products
  • amido-amidate metallacycles generated from low valent transition metal precursors are active intermediates in the controlled polymerization of ⁇ -amino acid-N-carboxyanhydrides (NCAs).
  • NCAs ⁇ -amino acid-N-carboxyanhydrides
  • Another embodiment of the present invention entails new tandem addition reactions that allow the general synthesis of amido-amidate metallacycles useful for preparation of polypeptides containing a variety of defined endgroups.
  • This method of making an initiator molecule includes the step of combining an allyloxycarbonyl (alloc) protected amino acid amide and a low valent transition metal- Lewis base ligand complex so that an amido-amidate metallacycle is formed having the following general formula:
  • M is a low valent transition metal
  • L is a Lewis base ligand
  • one of R1 and R2 is an amino acid side group and the other is hydrogen
  • R3 is any functional end group capable of being attached to a primary amine group.
  • the alloc-amino acid amide has the general formula Alloc-NH- CH(R')C(O)NHR" where R' is an amino acid side group and R" is a functional end group.
  • the "alloc" group includes at least one allylic moieity, i.e., a carbon-carbon double bond bound to a saturated carbon.
  • the backbone "allylic" system is a key element which is required for the reaction to work. The allylic backbone is, in turn, coupled via an oxygen to the carbonyl bound to the nitrogen of the amino acid.
  • L-form their corresponding D-forms, or unnatural synthetic amino acid side-chains, providing that reactive functional groups (those that are either protic or nucleophilic - such as those of lysine, ornithine, cysteine, serine, histidine, arginine, glutamic or aspartic acid (i.e. amines, alcohols, sulfhydryls, imidazoles, guanidines, carboxylates)) are suitably protected (using standard peptide functional group protection) to eliminate their reactivity.
  • reactive functional groups such as those of lysine, ornithine, cysteine, serine, histidine, arginine, glutamic or aspartic acid (i.e. amines, alcohols, sulfhydryls, imidazoles, guanidines, carboxylates)
  • unnatural side-chains those that would react with the low-valent metal complexes (e.g. aryl-halides,
  • the R" group is crucial as this is the group that will typically be used to "tag” or functionalize the polypeptide chains, and is the reason and advantage for using this method.
  • This group can be virtually anything, bearing in mind the chemical limitations of functional groups as listed above for R'.
  • this group will be a peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer chain, small molecule therapeutic, chemical linker to attach the polypeptide to a substrate, chemical linker to act as a sensing moiety, or reactive linker to couple the polypeptide to larger molecules such as proteins, polysaccharides or polynucleotides.
  • NCAs ⁇ -amino acid-N-carboxyanhydrides
  • These initiators provide a facile method to synthesize complex copolypeptides where the polymer chain carboxy-terminus can be quantitatively functionalized with a wide range of substituents.
  • substituents can include, but are not limited to: polymers (polystyrene, poly(ethylene oxide)), peptides, oligosachharides, oligonucleotides, or other organic moieties.
  • the key feature utilized to connect these substituents to a polypeptide chain, and the only requirement of the substituents, is a primary amine group.
  • This feature allows the preparation of complex polypeptide biomaterials which have great potential applications in medicine (drug delivery, tissue engineering).
  • chain-end functional groups e.g. other polymers, fluorescent or radioactive tags, or biomolecules (peptide sequences, oligonucleotides, or oligo sachharides)
  • chain-end functional groups e.g. other polymers, fluorescent or radioactive tags, or biomolecules (peptide sequences, oligonucleotides, or oligo sachharides)
  • this methodology can be used for labelling polypeptide chains to analyze chain mobility/location in vivo/in vitro.
  • incorporation of endgroup functionalities, such as signaling or receptor groups, onto polypeptide chains is essential for targeting of drug delivery complexes as well as substrate specific anchoring of these materials.
  • the product nickelacycle was found to result from initial addition across the Alloc C-O bond, followed by a second addition across the N-H bond of the amide, not the allylic N-C bond (eq 7).
  • the product metallacycle, 1 retained the allyl substituent on nitrogen, as determined by FAB/MS, 1 H NMR of the hydrolysis product from reaction with HCI, and 13 C labeling studies.
  • the N-H addition was also verified by use of a N ⁇ -2-hexenyloxycarbonyl-amino acid allyl amide in the reaction which resulted in the formation of byproduct hexenes that were identified by 13 C ⁇ 1 H ⁇ NMR.
  • M is a low valent transition metal capable of undergoing an oxidative addition reaction
  • L is an electron donor such as a Lewis base
  • R# comprises any organic substituent not bearing free amine, hydroxyl, carboxylic acid, sulfhydryl, isocyanate, imidazole, or other highly protic or nucleophilic functionality. These functionalities may be present, however, if suitably chemically protected to render them unreactive as protic sources or nucleophiies.
  • Effective R substituents on the above structures exhibit a number of properties. For example, as disclosed in the examples below, the R substituents on the above structures are typically encompassed by the structures of the side chain substituents of amino acids or derivatives thereof.
  • R1 (and R4 in the center structure) is a proton.
  • each of R2 and R3 (and R5 and R6) are typically selected from the side chain substituents of amino acids.
  • one of the substituents (such as R1) is a proton (H), while the others can be different side chain group of a specific amino acid.
  • the placement of the proton (as either R2 or R3) is determined by the amino acid being of the L or D configuration.
  • the side chain will be one of those from the family of naturally occurring L- or D-amino acids, or synthetic amino acids or derivatives thereof.
  • Naturally occurring L- or D-amino acids e.g.
  • the side-chains of amino acids bearing polar functional groups e.g. NH 2 , COOH, SH, imidazole
  • polar functional groups e.g. NH 2 , COOH, SH, imidazole
  • the metal is a group VIII transition metal and the donor ligand(s) can be any of those given in Table 7.
  • the metal is nickel and the donor ligand is a 2,2'-bipyridyl (bipy) moiety.
  • the R2 or R3 group comprises an amino acid side chain selected from the group consisting of side-chain protected NCA formed from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, serine, threonine, tryptophan, and tyrosine or an amino acid side chain selected from the group consisting of side-chain NCA formed from alanine, glycine, isoleucine, leucine, phenylalanine, proline and valine.
  • a related embodiment of the invention consists of a method of adding an aminoacid-N-carboxyanhydride (NCA) to a polyaminoacid chain having an amido containing metallacycle end group comprising combining the NCA with the polyaminoacid chain so that the NCA is added to the polyaminoacid chain.
  • NCA aminoacid-N-carboxyanhydride
  • the amido containing metallacycle end group is of a formula as follows:
  • M is the low valent transition metal
  • L is the Lewis Base ligand
  • R1 comprises a constituent found in a side chain of an amino acid (e.g. a hydrogen for glycine or a methyl group for alanine etc.) selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;
  • an amino acid e.g. a hydrogen for glycine or a methyl group for alanine etc.
  • R2 comprises a constituent found in a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine;
  • R3 comprises a constituent found in a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; and
  • R4 is the polyaminoacid chain.
  • the metal group of the amido containing metallacycle is a transition metal selected from the group consisting of nickel, palladium, platinum, cobalt, rhodium, iridium and iron and the Lewis Base ligand is selected from the group consisting of pyridyl ligands, diimine ligands, bisoxazoline ligands, alkyl phosphine ligands, aryl phosphine ligands, tertiary amine ligands, isocyanide ligands, and cyanide ligands.
  • NCA aminoacid-N- carboxyanhydride
  • the NCA is an a-aminoacid-N-carboxyanhydhde selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.
  • Another embodiment of the invention disclosed herein entails a method of polymerizing aminoacid-N-carboxyanhydride monomers by combining a NCA monomer with an initiator molecule complex comprised of a low valent transition metal-Lewis Base ligand.
  • a specific embodiment of the invention disclosed herein entails a method of polymerizing aminoacid-N-carboxyanhydride monomers having a ring with a 0-C 5 and a 0-C 2 anhydride bond. The method consists of combining a first NCA monomer with an initiator molecule complex.
  • the complex is comprised of a low valent metal capable of undergoing an oxidative addition reaction, wherein the oxidative addition reaction formally increases the oxidation state by two electrons, and an electron donor comprising a Lewis base.
  • the initiator molecule opens the ring of the first NCA through oxidative addition across either the O-C 5 or O-C 2 anhydride bond and combines with a second NCA monomer to form an amido- containing metallacycle.
  • a third NCA monomer then combines with the amido containing metallacyle so that the amido nitrogen of the amido containing metallacyle attacks the carbonyl carbon of the NCA.
  • the [WHICH ONE? ⁇ NCA is then added to the polyaminoacid chain, and the amido containing metallacyle is regenerated for further polymerization.
  • the efficiency of the initiator is controlled by allowing the reaction to proceed in a solvent selected for its ability to influence the reaction.
  • the solvent is selected from the group consisting of ethyl acetate, toluene, dioxane, acetonitrile, THF and DMF.
  • Block copolymers have played an important part in materials science and technology because they allow the effective combination of disparate properties in a single material.
  • Block copolymers of styrene and dienes are rubbers at room temperature (a characteristic of the polydiene phase) but can be moulded at temperatures above the glass transition of the polystyrene phase. This distinguishes the block copolymers from most conventional rubbers, which must be chemically cross-linked (vulcanized) in order to withstand the stresses that they encounter in use. Because chemical cross-linking is irreversible, it must be done while making the final part; and cross-linked rubber is difficult to reprocess.
  • the 'cross-linking' step for styrene-diene block copolymers is instead a physical association of chains in the glassy polystyrene domains: the tendency of the two different chain sections to clump together, like with like. This association is robust enough to bear loads at room temperature, but is readily reversible upon heating.
  • One embodiment of the invention provides a method of making a block copolypeptide consisting of combining an amount of a first aminoacid-N- carboxyanhydride monomer with an initiator molecule comprising a low valent transition metal-Lewis Base ligand complex so that a polyaminoacid chain is generated and then combining an amount of a second aminoacid-N- carboxyanhydride monomer with the polyaminoacid chain so that the second aminoacid-N-carboxyanhydride monomer is added to the polyaminoacid chain.
  • the initiator molecule combines with the first aminoacid-N-carboxyanhydride monomer to form an amido containing metallacycle intermediate of the general formula:
  • M is the low valent transition metal
  • L is the Lewis Base ligand; and each of R1 and R2 and R3 independently consist of a side chain of an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; and
  • R4 is the polyaminoacid chain.
  • the low valent transition metal is selected from the group consisting of nickel, palladium, platinum, cobalt, rhodium, iridium and iron.
  • the Lewis Base ligand is selected from the group consisting of pyridyl ligands, diimine ligands, bisoxazoline ligands, alkyl phosphine ligands, aryl phosphine ligands, tertiary amine ligands, isocyanide ligands and cyanide ligands.
  • the first a-aminoacid-N- carboxyanhydride monomer is an NCA is an a-aminoacid-N-carboxyanhydhde selected from the group consisting of side-chain protected NCA formed from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, serine, threonine, tryptophan, and tyrosine or an amino acid side chain selected from the group consisting of side-chain NCA formed from alanine, glycine, isoleucine, leucine, phenylalanine, proline and valine.
  • NCA is an a-aminoacid-N-carboxyanhydhde selected from the group consisting of side-chain protected NCA formed from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, methi
  • the block copolypeptide has 10 consecutive identical amino acids per block, in a preferred embodiment, the block copolypeptide is composed of amino acid components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine.
  • the copolypeptide is a poly(e-benzyloxycarbonyl-L-Lysine- j/oc/c-g-benzyl-L-glutamate), PZLL-b-PBLG, diblock copolymer.
  • the copolypeptide is a poly(g-benzyl-L-glutamate-o/oc/ -e- benzyloxycarbonyl-L-Lysine-o/oc/ -g-benzyl-L-glutamate) triblock copolymer.
  • the number of consecutive monomer units (residues) in the block copolypeptide is greater than about 50 or 100 or 500 or 1000 (see e.g. the examples disclosed in Tables 1 and 4). In another related embodiment, the total number of overall monomer units (residues) in the block copolypeptide is greater than about 200, or greater than about 500, or greater than about 1000 (see e.g. the examples disclosed in Tables 1 and 4).
  • Illustrative embodiments of the invention that are disclosed in the examples below include diblock copolymers composed of amino acid components g-benzyl-L- glutamate and e-carbobenzyloxy-L-lysine.
  • the polymers were prepared by addition of Lys-NCA to bipyNi(COD) in DMF to afford living poly(e-carbobenzyloxy-L-lysine), PZLL, chains with organometallic end-groups capable of further chain growth.
  • Glu- NCA was added to these polymers to yield the PBLG-PZLL block copolypeptides.
  • Block copolymerizations were not restricted to the highly soluble polypeptides PBLG and PZLL.
  • Copolypeptides containing L-leucine and L-proline, both of which form homopolymers which are insoluble in most organic solvents (e.g. DMF) have also been prepared. Data for these copolymerizations are given in Table 1 in Example 2 below. Because of the solubilizing effect of the PBLG and PZLL blocks, all of the products were soluble in the reaction media indicating the absence of any homopolymer contaminants.
  • the block copolymers containing L-leucine were found to be strongly associating in 0.1 M LiBr in DMF, a good solvent for PBLG and PZLL. Once deprotected, the assembly properties of these materials are expected make them useful as tissue engineering scaffolds, drug carriers, and morphology-directing components in biomimetic composite formation.
  • embodiments of the present invention provide a number of novel methods and compositions for the generation of polypeptides having varied material properties.
  • the description and illustrative examples disclosed herein provide a number of exemplary embodiments of the invention.
  • Specific embodiments of the invention include methods of adding aminoacid-N- 41 carboxyanhydrides (NCAs) to polyaminoacid chains by exposing the NCA to solutions containing polyaminoacid chains having an amido amidate metallacyle end groups and reacting the NCA with the amido amidate metallacyle end group so that the NCA is added to the polyaminoacid chain.
  • NCAs aminoacid-N- 41 carboxyanhydrides
  • Addition embodiments include methods of controlling the polymerization of aminoacid-N-carboxyanhydrides by reacting NCAs with initiator molecules and allowing initiator complexes to regioselectively open the ring of the NCAs through oxidative addition across the O- C 5 or O-C 2 anhydride bond resulting a controlled polypeptide polymerization.
  • Other embodiments include methods for making amido-containing metallacycles are disclosed herein.
  • Additional embodiments of the invention include compositions for use in peptide synthesis and design including five and six membered amido- containing metallacycles and block copolypeptides.
  • polypeptides can be generated utilizing the methods disclosed herein wherein an initiator molecule combines with a first aminoacid-N-carboxyanhydride monomer to form an amido containing metallacycle intermediate of the five and six ring formulae disclosed herein.
  • polypeptides can be generated where the domains can either be repeats (2 or greater) of identical amino acids, or can be repeats (2 or greater) of mixtures of distinct amino acids, or a combination of the two.
  • the number, length, order, and composition of these domains can vary to include all possible amino acids in any number of repeats.
  • polypeptide could contain a sequence of, for example, 50 residues of leucine in one domain, followed by a statistical mixture of 20 valines and 20 glycines as the second domain, followed finally by a third domain of 40 phenylalanines.
  • Such polypeptides are substantially different than "statistically random" copolymers where the entire polypeptide is composed of statistical mixtures of amino acids in the chains, and there are no strict block domains.
  • these polypeptides have segregated domains where one statistical mixture will be separated from the others. For example in a statistical copolymer, the amino acids will be distributed statistically (basically at random) along the entire polypeptide chain.
  • a polypeptide chain can be constructed such that in one domain there will be a statistical mixture of leucine and glycine, followed by a second domain consisting of a statistical mixture of glycine and valine. Although both copolymers have statistical mixtures of residues along the chain, these polypeptide differ in that the valine and leucine residues are segregated into separate domains.
  • NCAs The living polymerization methods for NCAs that are disclosed herein will lead to various polypeptides and block copolypeptides having a variety of new and useful properties.
  • the disclosure provided herein demonstrates the successful synthesis of such materials, and creates a new family of polypeptides that link combination of acidic, basic and hydrophobic domains, all with excellent control of molecular architecture.
  • the prospects for application in biomedical engineering, drug delivery and selective separations are excellent.
  • these features allow the preparation of complex polypeptide biomaterials which have potential applications in biology, chemistry, physics, and materials engineering.
  • Yet another embodiment of the present invention entails the synthesis of amphiphilic block copolypeptides, which contain at least one water soluble block polypeptide ("soluble block") conjugated to a water-insoluble polypeptide domain (“insoluble block”).
  • the soluble block includes oligo (ethyleneglycol) terminated amino acid (EG-aa) residues, whereas the insoluble block is primarily composed of nonionic amino acid residues.
  • EG-aa oligo (ethyleneglycol) terminated amino acid
  • the insoluble block is primarily composed of nonionic amino acid residues.
  • the amphiphilic nature of the polymers gives them the ability to self-assemble into a variety of well-defined structures in aqueous solutions, not unlike the assembly of small molecule lipids and surfactants.
  • the amphiphilic block copolypeptides of the present invention contain one or more "soluble blocks.”
  • the amino acid composition of these soluble blocks includes one or more oligo(ethyleneglycol) functionalized amino acid residues.
  • Preferred oligo(ethyleneglycol) functionalized amino acid residues include EG-Lys, EG-Ser, EG-Cys, and EG-Tyr.
  • a most preferred soluble block consists of oligo(ethyleneglycol) terminated poly(lysine).
  • the amphiphilic block copolypeptides of the present invention also contain at least one "insoluble block,"which is covalently linked to the soluble block.
  • the insoluble block can contain a variety of amino acids residues or mixtures thereof, including the naturally occurring amino acids, omithine, or blocks consisting entirely of one or more D-isomers of the amino acids.
  • the insoluble block will typically be composed primarily of nonionic amino acid residues, which generally form insoluble high molecular weight homopolypeptides.
  • nonionic amino acids include, but are not limited to phenylalanine, leucine, valine, isoleucine, alanine, serine, threonine and glutamine.
  • any given insoluble block will usually contain 2 - 3 different kinds of amino acid components in a statistically random sequences.
  • the amphiphilic block copolypeptides oF the present invention are included in methods and compositions of matter, which form vesicular structures in aqueous solutions.
  • the method consists of suspending the amphiphilic block copolypeptides in an aqueous solution so that the copolypeptides spontaneously self assemble into vesicles.
  • the vesicle-containing compositions are comprised of the amphiphilic block copolypeptides of the present invention and water.
  • the vesicles range in controllable size from microns to less than a hundred nanometers in diameter, similar to liposomes yet more robust, which make them potentially valuable for biomedical/biotechnological applications (i.e. drug delivery).
  • smaller vesicles having a diameter of about 50 nm to about 500 nm can be formed by sonicating the suspension of larger vesicles.
  • the diameter of the vesicles can be controlled by adjusting the length and amino acid composition of the amphiphilic block copolypeptide (see, e.g., the examples below).
  • L-leucine isoamylamide hydrochloride, g-benzyl-L-glutamate NCA and L- leucine NCA were prepared according to literature procedures. M. Bodanszky, et al., The practice of Peptide Synthesis, 2 nd Ed., Springer, Berlin/Heidelberg, (1994); E.R. Blout, et al., J. Am. Chem Soc, 78:941-950 (1956); H. Kanazawa, et al., Bull. Chem Soc. Jpn., 51 :2205-2208 (1978). Hexanes, THF, and THF-d 8 were purified by distillation from sodium benzophenone ketyl. DMF and DMF-d 7 were purified by drying over 4A molecular sieves followed by vacuum distillation.
  • FTIR analysis of the crude reaction mixture confirmed the presence of (2,2'- bipyridyl)Ni( 13 CO) 2 [IR (THF): 1933, 1862 cm “1 (nCO, vs)] as well as 13 C-labeled polyleucine [IR (THF): 1613 cm “1 (nAmide I, vs); 1537 cm” 1 (nAmide II, vs)].
  • the reaction was also run in DMF-d 7 (0.5 mL) under otherwise identical conditions.
  • Glu NCA 50 mg, 0.2 mmol
  • DMF 0.5 mL
  • An aliquot of bipyNi(COD) 50 ml of a 40 mM solution in DMF
  • a stirbar was added and the flask was sealed, removed from the dry box, and stirred in a thermostated 25 °C bath for 16 hours.
  • Polymer was isolated by addition of the reaction mixture to methanol containing HCI (1 mM) causing precipitation of the polymer.
  • NCAs a-Amino acid-N-carboxyanhydrides
  • L 2 Ni(COD) a-Amino acid-N-carboxyanhydrides
  • These oxidative addition reactions were found to result in the addition across either the O-C 5 or the O-C 2 bond of the NCAs, ultimately giving, after addition of a second equivalent of NCA, chiral amido-amidate nickelacycles.
  • the donor ligands bound to the nickel (0) precursor were varied (e.g. alkyl phosphines, a,a'-diimines), the only products isolable from stoichiometric reactions with Glu-NCA in THF were some starting nickel(O) compound and poly(g- benzyl-L-glutamate), PBLG.
  • NCAs ⁇ -amino acid-N-carboxyanhydrides
  • Tandem gel permeation chromatography/light scattering was performed on an SSI Accuflow Series III liquid chromatograph pump equipped with a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP. Separations were effected by 10 5 A, 10 3 A, and 500 A Phenomenex 5 ⁇ columns using 0.1 M LiBr in DMF eluent at 60 °C. NMR spectra were measured on Bruker AVANCE 200MHz spectrometer. FAB Mass Spectrometry was performed at the facility in the Chemistry Department at the University of California, Santa Barbara.
  • MALDI mass spectra were collected using a Thermo BioAnalysis DYNAMO mass spectrometer running in positive ion mode with samples prepared by mixing solutions of analyte in TFA with solutions of 2,5- dihydroxybenzoic acid in TFA and allowing the mixtures to air dry. Fluorescence measurements were conducted on a SPEX FluoroMax.-2. Chemicals were obtained from commercial suppliers and used without purification unless otherwise stated. Alloc-L-amino amides, ⁇ -CBZ-L-lysine NCA, (S)-phenylglycine NCA, and ⁇ -benzyl-L- glutamate NCA were prepared according to literature procedures (Tirrell, J. G.; Fournier, M.
  • Hexanes, THF, and THF-d 8 were purified by first purging with dry nitrogen, followed by passage through columns of activated alumina 3 DMF and DMF-d were purified by drying over 4 A molecular sieves followed by vacuum distillation.
  • NCH R, R -CH?CH(CH 3 ) ? , 2
  • Glu NCA 50 mg, 0.2 mmol
  • DMF 1.0 mL
  • An aliquot of the initiator 100 ⁇ L of a 36 mM solution in DMF
  • a stir bar was added and the flask was sealed, removed from the dry box and stirred at 25 °C in a thermostated bath for 24 h.
  • Polymer was isolated by addition of the reaction mixture to methanol containing HCI (1 mM) causing precipitation of the polymer.
  • the initiators described above were generated using bis-1 ,5-cyclooctadiene nickel (Ni(COD) 2 ) as the nickel source in conjunction with a variety of donor ligand components.
  • Ni(COD) 2 bis-1 ,5-cyclooctadiene nickel
  • nickel-olefin complexes nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorus donor ligand complexes
  • nickel-olefin complexes nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorus donor ligand complexes
  • nickel nitrogen or phosphorus donor ligand complexes nickel-olefin complexes, nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorus donor ligand complexes
  • other donor ligands nitrogen or phosphorus based in particular
  • reaction solvents are logical extensions of this work.
  • transition metals specifically palladium, platinum, cobalt, rhodium and iridium, might also be used in the reaction with alloc-amino acid amides to form amido-amidate metallacycle initiators, and are thus additional potential modifications to this
  • EXAMPLE 3 Facile Synthesis of Block Copolypeptides of Defined Architecture- General Experimental Protocols and Reagents. Infrared spectra were recorded on a Perkin Elmer 1605 FTIR Spectrophotometer calibrated using polystyrene film. Tandem gel permeation chromatography/light scattering (GPC/LS) was performed on a Spectra Physics Isochrom liquid chromatograph pump equipped with a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP. Separations were effected by 10 5 A and lO ⁇ A Phenomenex 5 ⁇ columns using 0.1 M LiBr in DMF at 60 °C as eluent.
  • GPC/LS Tandem gel permeation chromatography/light scattering
  • Optical rotations were measured on a Perkin Elmer Model 141 Polahmeter using a 1 mL volume cell (1 dm length). NMR spectra were measured on a Bruker AMX 500MHz spectrometer. Chemicals were obtained from commercial suppliers and used without purification unless otherwise stated. (COD) 2 Ni was obtained from Strem Chemical Co., and 13 C-i-L-leucine and 13 C-phosgene were obtained from Cambridge Isotope Labs. g-Benzyl-L-glutamate NCA were prepared according to literature procedures. Hexanes, THF, and THF-d 8 were purified by distillation from sodium benzophenone ketyl. DMF and DMF-d 7 were purified by drying over 4A molecular sieves followed by vacuum distillation.
  • FTIR analysis of the crude reaction mixture confirmed the presence of (2,2'- bipyhdyl)Ni( 13 CO) 2 [IR (THF): 1933, 1862 cm “1 (nCO, vs)] as well as 13 C-labeled polyleucine [IR (THF): 1613 cm “1 (nAmide I, vs); 1537 cm “1 (nAmide II, vs)].
  • the reaction was also run in DMF-d 7 (0.5 mL) under otherwise identical conditions.
  • diblock copolymers composed of amino acid components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine were synthesized.
  • the polymers were prepared by addition of Lys-NCA to bipyNi(COD) in DMF to afford living poly(e-carbobenzyloxy-L-lysine), PZLL, chains with organometallic end-groups capable of further chain growth.
  • Glu-NCA was added to these polymers to yield the PBLG-PZLL block copolypeptides.
  • the evolution of molecular weight through each stage of monomer addition was analyzed 63 using gel permeation chromatography (GPC) and data are given in Table 1 below.
  • Block copolymerizations were not restricted to the highly soluble polypeptides PBLG and PZLL.
  • Copolypeptides containing L-leucine and L-proline, both of which form homopolymers which are insoluble in most organic solvents (e.g. DMF) were prepared. Data for these copolymerizations are given in Table 4 below. Because of the solubilizing effect of the PBLG and PZLL blocks, all of the products were soluble in the reaction media indicating the absence of any homopolymer contaminants.
  • the block copolymers containing L-leucine were found to be strongly associating in 0.1 M LiBr in DMF, a good solvent for PBLG and PZLL. Once deprotected, the assembly properties of these materials are expected to make them useful as tissue engineering scaffolds, drug carriers, and morphology-directing components in biomimetic composite formation.
  • the initiators described above were generated using bis-1 ,5-cyclooctadiene nickel (Ni(COD) 2 ) as the nickel source and 2,2'-bipyridyl (bipy) as the donor iigand component in tetrahydrofuran (THF) solvent.
  • Ni(COD) 2 bis-1 ,5-cyclooctadiene nickel
  • bipy 2,2'-bipyridyl
  • THF tetrahydrofuran
  • Other sources of zerovalent nickel e.g. Ni(CO) 4
  • other donor ligands e.g.
  • the use of other sources of zerovalent nickel e.g.
  • nickel-olefin complexes nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorous donor ligand complexes
  • nickel-olefin complexes nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorous donor ligand complexes
  • other donor ligands nitrogen or phosphorous based in particular
  • polymerization solvents are logical extensions of this work.
  • transition metals specifically palladium, platinum, cobalt, rhodium, iridium and iron are also able to polymerize NCA monomers.
  • metals in "Group VIII" i.e. Co, Rh, Ir, Ni, Pd, Pt, Fe, Ru, Os
  • Glu NCA 50 mg, 0.19 mmol
  • DMF dimethylformamide
  • An aliquot of (2,2'-bipyridyl)Ni(COD) 50 ml of a 40 mM solution in DMF, prepared by mixing equimolar amounts of 2,2'-bipyridyl and Ni(COD) 2 ) was then added via syringe to the flask. A stirbar was added, the flask was sealed and then stirred for 16 hours.
  • Glu NCA 50 mg, 0.19 mmol
  • DMF 0.5 mL
  • a 15 mL reaction tube which could be sealed with a TEFLONTM stopper.
  • An aliquot of (PMe 3 ) )Co 50TL of a 40 mM solution in DMF:THF (1 :1) was then added via syringe to the flask.
  • a stirbar was added, the flask was scaled and then stirred for 16 h.
  • Glu NCA 250 mg, 0.95 mmol
  • DMF dimethylformamide
  • An aliquot of (2,2'-bipyridyl)Ni(COD) 50 ml of a 40 mM solution in DMF, prepared by mixing equimolar amounts of 2,2'-bipyridyl and Ni(COD) 2 ) was then added via syringe to the flask. A stirbar was added, the flask was sealed and then stirred for 16 hours.
  • diblock and triblock copolymers were prepared by a procedure identical to that described above for either PZLL-b-PBLG and PBLG-b-PZLL-b-PBLG, except that either different monomers, or different amounts of monomers, were used for the individual polymerization reactions. Examples are given in Tables 4 (abovee) and Table 5(below). The nature of the amino acid monomer was found to be unimportant in limiting the effectiveness of these polymerizations. All amino acid NCAs tried were incorporated into block copolypeptides in any sequential order, as determined by the order of addition to the initiator.
  • Representative monomers include, but are not limited to: the naturally occurring L-amino acids, naturally occurring D-amino acids, a-disubstituted a-amino acids, racemic a-amino acids, and synthetic a-amino acids.
  • Block copolypeptides could be prepared using initiators other than (2,2'- bipyridyl)Ni(COD). The initiators given in Tables 7 and 8 below (except those that gave no yield of polymer) all were able to prepare block copolypeptides.
  • Infrared spectra were recorded on a Perkin Elmer 1605 FTIR Spectrophotometer calibrated using polystyrene film. Optical rotations were measured on a Perkin Elmer Model 141 Polarimeter using a 1 mL volume cell (1 dm length). NMR spectra and bulk magnetic susceptibility measurements (Evans method) were measured on a Bruker AMX 500MHz spectrometer. D.F. Evans, J. Chem. Soc, 2003-2009 (1959); J.K. Becconsal, J. Mol. Phys., 15:129-135 (1968). C, H, N elemental analyses were performed by the Microanalytical Laboratory of the University of California, Berkeley Chemistry Department.
  • Glu NCA 50 mg, 0.2 mmol
  • THF tetrahydrofura ⁇
  • An aliquot of (2,2'-bipyridyl)Ni(COD) 50 ml of a 40 mM solution in THF, prepared by mixing equimolar amounts of 2,2'-bipyridyl and Ni(COD) 2 ) was then added via syringe to the flask.
  • Glu NCA 50 mg, 0.2 mmol
  • DMF 0.5 mL
  • a reaction tube which could be sealed with a TEFLONTM stopper.
  • An aliquot of dmpeCoPhe 2 50TL of a 40 mM solution in DMF
  • a stirbar was added and the flask was sealed, removed from the dry box, and stirred in a thermostated 25°C bath for 16 h.
  • Polymer was isolated by addition of the reaction mixture to methanol containing HCI (1 mM) causing precipitation of the polymer. The polymer was then dissolved in THF and reprecipitated by addition to methanol.
  • Tetrahydrofuran (THF) hexane, N,N-d ⁇ methylformam ⁇ de, and diethyl ether were dried by passage through an alumina column under nitrogen prior to use All reactions were conducted under an anhydrous nitrogen atmosphere, unless otherwise noted
  • the chemicals were purchased from commercial suppliers and used without purification
  • Co(PMe 3 ) 4 was prepared according to the procedure of Klein, et al [Klein, et al , Methyltetrak ⁇ s(t ⁇ methylphosph ⁇ n)kobalt und seine De ⁇ vate, Chem Ber , 108 944-955 (1975), I ncorporated herein by reference]
  • the infrared spectra were recorded on a Perkin Elmer RX1 FTIR Spectrophotometer calibrated using polystyrene film 1 H NMR spectra were recorded on a Bruker AVANCE 200 MHZ spectrometer and were referenced to internal solvent resonances.
  • Tandem gel permeation chromatography/light scattering was performed on a SSI pump equipped with a Wyatt DAWN DSP light scattering detector and Wyatt Optilab DSP Separations were effected by 10 5 A, 10 4 A, and 10 3 A Phenomenex 5 ⁇ m columns using 0 1 M LiBr in DMF as eluent at 60°C Circular dichroism measurements were carried out on a Olis Rapid Scanning Mo ⁇ ochromator at room temperature The path length of the quartz cell was 1 0 mm and the concentration of peptide was 0 5 mg/mL MALDITOF mass spectra were collected using a Thermo BioAnalysis DYNAMO mass spectrometer running in positive ion mode with samples prepared by mixing solutions of analyte in THF with solutions of 2,5- dihydroxybenzoic acid in THF and allowing the mixture to air dry
  • N ⁇ -Cbz-L-tyrosine (5.00 g, 15.9 mmol) was dissolved in NaOH (0.634 g, 15.9 mmol) in water (100 mL).
  • (2-(2-methoxyethoxy)ethyl) chloroformate (4.34 g, 23.8 mmol) and 23.8 ml of 1 M NaOH were added simultaneously at 0 °C and the resulting mixture was stirred for one hour at this temperature. The mixture was then stirred for an additional three hours at ambient temperature.
  • the solution was acidified with 1 M HCI and extracted with ethyl acetate.
  • L-cysteine hydrochloride (2.00 g, 12.7 mmol) was dissolved in 1 M aqueous sodium bicarbonate (25.4 ml) and the solution was diluted with water (75 mL). The solution was cooled in an ice bath and then covered with ether (50 ml). (2-(2- methoxyethoxy)ethyl) chloroformate (2.31 g, 12.7 mmol) was added to the solution in one portion with vigorous stirring for one hour at 0 °C. The temperature was allowed to rise to 10 °C and stirred for an additionional two hours. The solvents were then removed under reduced pressure.
  • P poly (EG 2 -L lysine) domain
  • LV hydophobic domain composed of a random copolymer of L-leucine (L) and L-va ne (V) with an internal composition of 75% leucine and 25% valine
  • PLV10 is a diblock copolymer where the P block makes up 90 mole percent of the total polymer size and the LV block makes up 10% of the total copolymer

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Polyamides (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des méthodes et des compositions destinées à générer des polypeptides présentant des propriétés matérielles variées. Ces méthodes incluent un moyen de production de copolypeptides amphiphiles séquencés autoassemblés et des protocoles associés pour adjoindre des aminoacide-N-carboxyanhydrides (NCAs) à fonction oligo(éthylèneglyco) à des chaînes polyaminoacides. Des méthodes additionnelles incluent un moyen d'adjonction d'un groupe terminal à la terminaison carboxy de la chaîne polyaminoacide par réaction d'un amide d'acide aminé alloc-protégé avec un complexe aux ligands donneur de métal de transition, aux fins de former un métallacycle amido amidate pouvant être utilisé dans d'autres réactions de polymérisation. L'invention concerne en outre de nouvelles compositions qui s'utilisent dans la synthèse et la mise au point de peptides comprenant des métallacycles à cinq et six chaînons contenant amido et des copolypeptides séquencés.
EP00930551A 1999-05-10 2000-05-10 Methodes et compositions pour realiser une synthese regulee de polypeptides Withdrawn EP1189928A1 (fr)

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US187448P 2000-03-07
US19305400P 2000-03-29 2000-03-29
US193054P 2000-03-29
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US7041856B2 (en) * 2001-10-23 2006-05-09 Sumitomo Chemical Company, Limited Coupling catalyst and process using the same
JP4311069B2 (ja) 2003-01-17 2009-08-12 住友化学株式会社 カップリング化合物の製造方法
EP1449580B1 (fr) 2003-02-19 2005-12-28 Sumitomo Chemical Company, Limited Méthode de production d'un composé résultant de couplage transversal d'un halogènure alkylé et un composé organoboronique
CN109837255B (zh) * 2018-12-24 2023-03-14 深圳市安帝宝科技有限公司 一种化学修饰酮胺氧化酶的方法
FR3100248B1 (fr) * 2019-09-03 2022-01-07 Univ Bordeaux Procédé de préparation de polymères et copolymères contrôlés à base de peptides en solution aqueuse

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EP0612846A1 (fr) * 1993-01-28 1994-08-31 Amgen Inc. Analogues de G-CSF et méthodes pour les obtenir

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NZ250375A (en) * 1992-12-09 1995-07-26 Ortho Pharma Corp Peg hydrazone and peg oxime linkage forming reagents and protein derivatives
GB9317618D0 (en) * 1993-08-24 1993-10-06 Royal Free Hosp School Med Polymer modifications
FR2727117A1 (fr) * 1994-11-18 1996-05-24 Geffard Michel Utilisation de conjugues de la polylysine pour la preparation de medicaments utiles dans le traitement des maladies neurodegeneratives et des affections degeneratives a caractere autoimmun

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EP0612846A1 (fr) * 1993-01-28 1994-08-31 Amgen Inc. Analogues de G-CSF et méthodes pour les obtenir

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See also references of WO0068252A1 *

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