JP2003503511A - Methods and compositions for controlled polypeptide synthesis - Google Patents

Abstract

  (57) [Summary] Disclosed herein are methods and compositions for producing polypeptides having various material properties. The method includes a means for self-assembly of the amphiphilic block copolypeptide and a related protocol for adding oligo (ethylene glycol) functionalized amino acid-N-carboxylic anhydrides (NCAs) to the polyamino acid chain. A further method is to terminate the carboxy group of a polyamino acid chain by reacting an alloc-protected amino acid amide with a transition metal-donor ligand complex, thereby forming an amide-amidate metallacycle for use in further polymerization reactions. Including means for adding to the ends. Also disclosed are novel compositions for use in the synthesis and design of peptides, including 5- and 6-membered amide-containing metallacycles and block copolypeptides.

Description

Detailed Description of the Invention

(Cross Reference Regarding Related Application) This application is a part-pending application of nonprovisional application number 09 / 272,109 filed on March 19, 1999, and the application is filed on March 19, 1998. Provisional application number 60 /
078,649 is claimed to be priority under Article 119 (e). No. 60 / 133,304, filed May 10, 1999, as well as provisional application No. 60 / 133,305, filed May 10, 1999, filed March 7, 2000. Claiming priority under provisional application no. 60 / 187,488 and provisional application no. 60 / 193,054 filed March 29, 2000 under section 119 (e). The contents of the above provisional and non-provisional applications are incorporated herein by reference.

References to Federally Sponsored Research The present invention is based on the National Science Fo
grant) Grant number DMR9632716, CHE97019
69 and 9701969, The Office of Nav.
al Research) Grant No. N00014-96-0729 and the Department of Defense Grant DA
It was conducted with government support from AH04-96-1-004. The government has certain rights in this invention. (Background of the Invention)

TECHNICAL FIELD This invention relates to the synthesis of polymers based on amino acids. In particular, the present invention
Using the catalyst under "living" conditions, i.e., in the absence of termination and chain transfer,
Methods and compositions for synthesizing amino acid-based polymers.

DESCRIPTION OF RELATED ART Synthetic polypeptides have many advantages over peptides produced in biological systems and make a fundamental contribution to both the physicochemical and protein structural analysis of macromolecules. Is used as. For example, GD Fasman, Poly a
-Amino Acids, Dekker, New York (1967). In addition, synthetic peptides are more cost effective and may have a wider range of material properties than peptides produced in biological systems.

Small synthetic peptide sequences are typically less than 100 residues in length and are conventionally prepared using stepwise solid phase synthesis. Such solid phase synthesis utilizes an insoluble resin support for the growing oligomers. An array of subunits defined to make up the desired polymer is reacted sequentially on the support. The terminal amino acid is attached to the solid support directly in the first reaction or via a coupling agent. The terminal residue, in turn, is reacted with a series of additional residues such as amino acids or blocked amino acid moieties and attached to the solid support via the terminal residue to give a growing oligomer. At each step of the synthetic scheme, unreacted reactant material is washed away or otherwise removed from contact with the solid phase. The cycle is continued with residues of preselected sequence until the desired polymer has been fully synthesized but is still attached to the solid support. At that point, the polymer is cleaved from the solid support and purified for use. The general synthetic scheme described above is followed by RB Merri for use in the preparation of certain peptides.
Developed by field. For example, Merrifield's Nobel Prize Lecture "Solid Phase Synthesis", Science, 232, 341-347 (1986).
)checking.

A major drawback of conventional solid phase synthesis methods for preparing oligomeric materials is the incompleteness of the reactions used in the above schemes; the fact that none of the reactions proceed to 100% completion. . The addition of each new subunit to the growing oligomer chain does not result in the desired reaction in a low but measurable ratio. The result is a series of peptides, nucleotides, or other oligomers with deletions in their sequence. As a result of the imperfections in the synthetic scheme, the effective yield of the desired product drops sharply as the chance of deletion increases with the length of the desired chain. Similar considerations are followed for other types of undesired reactions, such as those that are the result of incomplete blocking, side reactions and the like. The fact that purification is very difficult when the number of undesired polymer species resulting from the respective reactions not occurring is very important, if not equivalent. For example, if the polypeptide is amino acid residue 100
If it is desired to have one, it may result in as many as 99 separate peptide species with one amino acid residue deletion, and more than one deletion residue, a side reaction product. An even higher number of undesired polymers with etc. may occur.

Due to the above problems associated with the solid phase method, those skilled in the art will use other protocols for peptide synthesis. For example, synthetic copolymers having a narrow molecular weight distribution, controlled molecular weight, and block and star structures can be prepared using so-called living polymerization techniques. For example, O.Webster, Science, 251: 88.
7-893 (1991). In these polymerizations, the chains grow linearly by successive additions of monomers and there is no chain scission migration and termination reaction. The active end groups of the growing polymer chain are not deactivated (ie, they are
Remains "living"), the chain continues to grow as long as the monomer is present. Chain length during living polymerization is controlled by adjusting the monomer to the stoichiometry of the initiator. Under conditions where all chains grow at the same rate, living polymers have a narrow distribution of chain lengths. Complex sequences, such as block copolymers, are then constructed by stepwise adding different monomers to this growing chain. AN
oshay et al., Block Copolymers, Academic Pre
SS, New York, (1977).

[0008] Chemical synthesis of high molecular weight polypeptides is carried out using α-amino acid-N-carboxylic acid anhydride (
Most directly achieved by ring-opening polymerization of NCA) monomers (see Equation 1 below). For example, HR Kricheldorf, Models of Biop.
Polymers by Ring-Opening Polymerizati
on, Penczek, S. Ed., CRC Press, Boca Raton
, (1990). Generally, NCA polymerization is based on two types of initiators, either nucleophiles (typically primary amines) or strong bases (typically sodium alkoxides). Can be classified into the following categories (see Equation 1 below). Polymerization initiated by nucleophiles is believed to grow via primary amine end groups (see Equation 2 below). These polymerizations show the complex kinetics of an initial slow primary process followed by accelerated monomer consumption, suggesting multiple growing species with different reactivities. For example, M. Idelson et al., J.
See Am. Chem Soc., 80: 2387-2393 (1958). Due to the large number of side reactions, these initiators are typically limited to the formation of low molecular weight polymers (10 kDa <Mn <50 kDa) containing a substantial fraction of molecules with a degree of polymerization less than 10. Therefore, the polymer has an extremely wide molecular weight distribution (Mw / Mn = 4-10).
) Has. For example, RD Lundberg et al., J. Am. Chem Soc.
, 79: 3961-3972 (1957).

[0009]

[Chemical 3]

Strong base initiated NCA polymerization is much faster than amine initiated reactions. Little is known about these polymerizations, but it is believed that they grow via either the NCA anion or the carbamate reactive species (see equations 3 and 4 below, respectively). For example, CH Bamford et al., Synthetic
Polypeptides, Academic Press, New Y
See ork, (1956).

[0011]

[Chemical 4]

A major limitation of NCA polymerizations using conventional initiators is due to the fact that they are hindered by chain scission migration and termination reactions that prevent the formation of block copolymers. For example, HR Kricheldorf, a-Aminoacid
-N-Carboxyanhydrides and Related Mat
initials, Springer-Verlag, New York, (198
See 7). As a result, the NCA polymerization mechanism has been rigorously studied so as to eliminate the problematic side reaction. For example, HR Kricheldo
rf, Models of Biopolymers by Ring-Ope
Ning Polymerization, Penczek, S., CRC
See Press, Boca Raton, (1990). These studies
It is exposed to serious hindrance due to the complexity of polymerization, which can proceed by multiple routes. further,
The high sensitivity of NCA polymerization to reaction conditions and impurities also led to conflicting data in the literature, resulting in controversy regarding different hypothetical mechanisms. H. Sekiguchi, Pure and Appl. Chem., 53
: 1689-1714 (1981); H. Sekiguchi et al., J. Poly.
Sci. Symp., 52: 157-171 (1975).

The problems associated with existing peptide synthesis methods pose various problems to those skilled in the art.
For example, the strand break transfer reaction that occurs in NCA polymerization eliminates systematic control of peptide molecular weight. Moreover, block copolymers cannot be prepared using such methods. Block copolymers of amino acids have not been well studied, and many of the causes are that our synthetic methods cannot be controlled with sufficient precision to generate well-defined structures. F. Cardinauz et al., Bi
opolimers, 16: 2005-2028 (1977). The same is true for block copolypeptide synthesis for use as a biological material or as a selective membrane, but the potential benefit of this proteinaceous structure is the lack of suitable synthetic building blocks and tools. Therefore, it has not been realized yet. For example, biomedical applications, such as drug delivery, usually require water-soluble ingredients to enhance their in vivo circulating capacity. The problem with common water-soluble polypeptides (eg, poly-L-lysine and poly-L-asparagine) is that they exhibit pH-dependent solubility and circulatory life due to aggregation by biopolymers of opposite charge. Is a limited polyelectrolyte. Nonionic water-soluble polypeptides are desirable for biomedical applications because they can avoid these problems, and may also exhibit stable secondary structure of proteins that affect their biological properties. However, all high molecular weight nonionic homopolypeptides (> 25 residues) derived from naturally occurring amino acids are highly insoluble in water.

One approach to produce nonionic, water-soluble polypeptides uses polyethylene glycol (PEG), which is usually grafted onto polypeptides or other polymers to improve their properties in vivo. To do. PEG is nonionic, water soluble and, more importantly, not recognized by the immune system. PEG is believed to confer biocompatibility by forming a hydrating "steric barrier" on the surface of the material that is impermeable to or unrecognized by biological molecules such as proteolytic enzymes. . Thus, block or graft copolymer drug carriers containing PEG can circulate in the bloodstream for extended periods of time without degradation.

The drawback of grafting PEG onto a polypeptide is, despite its attractive properties, the expensive amino- or carboxylato.
Requiring a functionalized molecule for conjugation, it usually must be short (<5000 Da) to ensure high functionality. Therefore, we develop an alternative method for producing nonionic water-soluble polypeptide building blocks that also incorporate into the polypeptide the attractive properties of biochemical stability, self-assembly and water solubility. In particular, there is still a lot of interest.

Polypeptides have been investigated for a variety of biomedical problems such as tissue engineering and drug delivery. Another challenge in these applications is the incorporation of end group functionality on the chain, which is essential for targeting drug delivery complexes as well as substrate-specific anchoring of these materials. These and other features will also be useful for controlling both the structure and properties of the polypeptide material. As a result, there is a need for new methods and compositions that allow the ready production of peptides that have been treated to have specific desired properties.

DISCLOSURE OF THE INVENTION The present invention discloses novel methods and compositions that meet the need for advanced tools for producing polypeptides with modified material properties. The methods and initiator compositions disclosed herein for NCA polymerization allow for precise control of such polypeptide synthesis. In particular, the method of the present invention succeeds in peptide synthesis by utilizing the versatile chemistry of transition metals that mediate the addition of monomers to the active chain ends of polymers, thus eliminating chain scission side reactions. And favors the chain growth process. Thus, the disclosed method allows the formation of block copolymers. Furthermore, by attaching the active terminus of the growing polymer to the metal center, its reactivity towards the monomer can be precisely controlled through the diversity of metal and ancillary ligands attached to this metal. A wide range of selective chemical transformations and polymerizations catalyzed by transition metal complexes demonstrate the versatility of this approach.

One aspect of the invention is a method of making an amide-containing metallacycle, wherein an amount of a-amino acid-N-carboxylic acid anhydride monomer (a-aminoacines) is used.
dN-carboxyanhydride monomer) is combined with an initiator molecule comprising a low valent transition metal-Lewis base ligand complex to form an amide-containing metallacycle.

Another aspect of the invention is a method of making an initiator molecule, which comprises combining an allyloxycarbonyl (alloc) protected amino acid amide with a low valent transition metal-Lewis base ligand complex, The following general formula:

[0020]

[Chemical 5]

[Wherein M is a low-valent transition metal and L is a Lewis base ligand;
One of R1 and R2 is an amino acid side group and the other is hydrogen; and R3 is any functional end group that can be attached to a primary amine group]. A method is provided that includes forming. The R3 end group is typically used to "tag" or functionalize the polypeptide chain, which is a major advantage associated with using this method. Typically, this group is a peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer chain, therapeutic chemical linker of a small molecule that attaches a polypeptide to a substrate, a chemical linker that acts as a sensing molecule, or A reactive linker that attaches a polypeptide to a large molecule, such as a protein, polysaccharide or polynucleotide. Another aspect of the invention is the general formula:

[0022]

[Chemical 6]

[Wherein M is a low-valent transition metal; L is a Lewis base ligand; each of R 1, R 2, R 3, R 5 and R 6 is (independently) alanine, arginine, asparagine. , Aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,
A moiety selected from the group consisting of the side chains of phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine; R4 is a hydrogen moiety or a polyamino acid chain and R7 is a functional end group] A composition comprising a 5- or 6-membered amide-containing metallacycle containing a molecule of In a preferred embodiment of these compositions, the metal is a transition metal selected from the group consisting of nickel, palladium, platinum, cobalt, rhodium, iridium and iron and the Lewis base ligand is a pyridyl ligand, diimine ligand, bisoxazoline. Ligand, alkylphosphine ligand, arylphosphine ligand,
It is selected from the group consisting of tertiary amine ligands, isocyanide ligands and cyanide ligands.

A related aspect of the invention comprises a method of adding an amino acid-N-carboxylic acid anhydride (NCA) to a polyamino acid chain having an amide-containing metallacycle end group,
In order to add the NCA to the polyamino acid chain, the NCA is combined with the polyamino acid chain.

Another aspect of the invention disclosed herein is amino acid-N- by combining an NCA monomer with an initiator molecular complex composed of a low valent transition metal-Lewis base ligand. A method of polymerizing a carboxylic acid anhydride monomer is included.
Specific embodiments of the invention disclosed herein is a O-C ₅ and O-C ₂ process for polymerizing amino -N- carboxylic acid anhydride monomer having a ring holding anhydride bonds, It consists of combining an initiator molecular complex composed of a low-valent metal capable of undergoing an oxidative addition reaction with a first NCA monomer, wherein the oxidative addition reaction is an electron containing two electrons and a Lewis base. Included is a method of explicitly increasing the oxidation state by a donor. The initiator molecule then opens the ring of the first NCA by oxidative addition via either an O—C ₅ or an O—C ₂ anhydride bond, and then combines with a second NCA monomer to form an amide-containing metalla. Form a cycle. Subsequently, when the third NCA monomer is combined with the amide-containing metallacycle, the amide nitrogen of the amide-containing metallacycle attacks the carbonyl carbon of the NCA. Therefore, NCA is added to the polyamino acid chain and the amide-containing metallacycle is regenerated for further polymerization. In a preferred embodiment of the invention, the efficiency of the initiator is controlled to drive the reaction in a solvent selected for its ability to influence the reaction.
In a particular embodiment of the present invention the solvent is selected from the group consisting of ethyl acetate, toluene, dioxane, acetonitrile, THF and DMF.

Another aspect of the invention is that an amount of a first amino acid-N-carboxylic acid anhydride (
NCA) monomer is combined with an initiator molecule containing a low valence transition metal-Lewis base ligand complex to form a polyamino acid chain, followed by an amount of a second amino acid-
Provided is a method for producing a block copolypeptide, which comprises combining an N-carboxylic acid anhydride monomer with the polyamino acid chain and adding a second amino acid-N-carboxylic acid anhydride monomer to the polyamino acid chain. . In a preferred embodiment of this method, the initiator molecule is combined with a first amino acid-N-carboxylic anhydride monomer and has the general formula:

[0027]

[Chemical 7]

[Wherein M is a low-valent transition metal; L is a Lewis base ligand; R 1, R 2 and R 3 are each independently alanine, arginine, asparagine, aspartic acid, cysteine , Glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine side chain; and R4 is a polyamino acid. A chain] of the amide-containing metallacycle intermediate.

In yet another aspect, the invention provides a block copolypeptide composition having features not achieved by the prior art. A particular embodiment of the present invention is about 100
Many full-length monomer units of one or more amino acid residues and at least about 1.
A polypeptide composition comprising a block polypeptide having a chain length distribution of 01 <Mw / Mn <1.25. In a related aspect, the polypeptide is about 250
It has a large number of full-length monomer units that are more than one amino acid residue. In a specific embodiment, the copolypeptide consists of at least 3 blocks of consecutive identical amino acid monomer units. In a particular embodiment of the present invention, at least one of the block components
The first is g-benzyl-L-glutamate.

The present invention also discloses novel methods and compositions that meet the need for biocompatible materials with improved properties such as biochemical stability, water solubility and self-assembly. The methods of making the amphipathic block copolypeptides disclosed herein allow for the synthesis and assembly of compositions with well-defined vesicular structures, such as drug delivery. Important for biomedical applications.

One aspect of the present invention is a method of producing an amphipathic block copolypeptide, comprising: (1) an amount of oligo (ethylene glycol) functionalized amino acid-N-carboxylic acid anhydride (EG- aa-NCA) monomer is combined with an initiator molecule to form a soluble block polypeptide; and (2) the soluble block is combined with a composition comprising at least one other amino acid NCA monomer. Thereby binding the insoluble block. In a preferred aspect of the method, the amino acid component of the EG-aa-NCA monomer is
Lysine, serine, cysteine, or tyrosine, while the insoluble block can contain a mixture of amino acids, including one or more naturally occurring amino acids.

A related aspect of the invention is to combine soluble blocks with one or more oligo (ethylene glycol) -terminating amino acid residues by combining the NCA with the polypeptide such that the NCA is added to the polypeptide. Amino acid-N in polypeptide
-A method of adding a carboxylic acid anhydride (NCA).

In yet another aspect, the invention provides amphipathic block copolypeptide compositions having improved properties of solubility, biochemical stability and biocompatibility. Amphiphilic block copolypeptides include a soluble block polypeptide having one or more oligo (ethylene glycol) -terminating amino acid residues and an insoluble block composed of substantially nonionic amino acid residues. Specific embodiments of the invention include (1) a soluble block polypeptide having an EG-lysine residue; and (2) an insoluble block containing a mixture of 2-3 different amino acid components that are statistically random sequences. -It is a polypeptide composition containing a polypeptide. In another specific embodiment, the copolypeptide consists of at least three blocks, one or more of the blocks being a soluble block polypeptide and the other block being an insoluble block polypeptide.

The amphipathic nature of block copolypeptides provides yet another aspect, which is a method of forming vesicles. This method consists of suspending the amphipathic block copolypeptide in an aqueous solution and allowing the copolypeptide to spontaneously self-assemble as vesicles. In a specific embodiment, smaller vesicles having a diameter of about 50 nm to about 500 nm can be formed by sonicating a suspension of larger vesicles.

In a related aspect, the invention provides a vesicle-containing composition composed of an amphipathic block copolypeptide of the invention and water.

In another related aspect, the present invention is a method of making an EG-functionalized amino acid monomer, wherein the ethylene glycol (EG) derivative is, for example, lysine, serine,
Methods are provided that include the step of combining cysteine and an amino acid having a reactive side group such as tyrosine.

Methods and compositions for making amphipathic block copolypeptides are particularly attractive because the EG-amino acid domain is comparable to certain desirable properties of poly (ethylene glycol), or PEG. For example, PEG is well known for its biological invisibility, which is not recognized by the body's immune defense mechanisms, and thus has found many useful applications in drug delivery, enzyme stabilization, tissue engineering, and implant surface modification.

As an example of the preferred embodiments of the present invention, based on various metals and ligands,
A series of initiators that polymerize amino acid-N-carboxylic acid anhydrides (NCAs) into block copolypeptides have been described. These initiators are substantially different in nature from all known conventional initiators used in NCAs polymerization, and also in the ability to control their polymerization so that block copolymers of amino acids can be prepared. Be unique. Specifically, these initiators have the ability to eliminate chain transfer and chain termination side reactions from these polymerizations, resulting in narrow molecular weight distribution, molecular weight control, and the ability to prepare copolymers of well-defined block sequence and composition. Occurs. All of these characteristics have hitherto not been obtainable using conventional initiator systems.
Moreover, the initiators disclosed herein are easily prepared in a single step from commercially available materials.

The discovery of this new class of initiators, and methods of using them, eliminates side reactions from NCA polymerization and further allows the preparation of well-defined block copolypeptides. The formation of a representative example of our initiator is based on the NCA monomer being a zero-valent nickel complex, bipyNi (COD) [bipy = 2,2 ′].
-Bipyridyl, COD = 1,5-cyclooctadiene]. This reaction is similar to the known oxidative addition of cyclic anhydrides to zero-valent nickel, resulting in an acylcarboxylate divalent nickel complex (see equation 5 below).

[0040]

[Chemical 8]

This reaction is similar to these known oxidative addition reactions, but there is no precedent for the reactions that occur during the formation of the molecules disclosed herein.

The methods and compositions of initiators disclosed herein allow the preparation of complex polypeptide biological materials with potential applications in biology, chemistry, physics and materials engineering. To do. Potential applications include medical (drug delivery, tissue engineering),
Included are "smart" hydrogels (organic materials that are environmentally reactive), and organic / inorganic biomimetic composites (artificial bone, high performance coatings).

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions The term “block copolypeptide” as used herein refers to a polypeptide that comprises at least two covalent binding domains (“blocks”), one block. Have amino acid residues of different composition than the composition of the amino acid residues of another block.
The number, length, order and composition of these blocks can be any number of repeats.
) Can be changed to include all possible amino acids. Suitably, the total number of full length monomeric units (residues) in the block copolypeptide is greater than 100 and the chain length distribution in the block copolymer is about 1.01 <Mw / Mn <about 1.25. [Where Mw / Mn = weight average molecular weight divided by number average molecular weight]. The terms "protecting" and "side chain protecting group" are used herein to refer to chemical substituents placed on a reactive functional group that is typically a nucleophile or a source of protons and refer to them as a proton. Make it non-reactive as a protic source or nucleophile. The selection and placement of these substituents is described in literature procedures, M. Bodanszky et al., The p.
ractice of Peptide Synthesis, 2nd edition, Spr
Inger, Berlin / Heidelberg (1994).

II. Review Ideal living polymerizations are characterized by rapid initiation, and the absence of termination and chain transfer steps that compete with the growth of growing chains in most polymerization systems. When these conditions are realized, all polymer chains begin to grow almost simultaneously and continue to grow until the monomer is depleted. The average number of monomer residues per chain is then simply the molar ratio of monomer to initiator and the chain length distribution is explained by Poisson statistics. M. Szwarc, Carbanions, Living Poly
mers, and Electron-Transfer Process
S. Wiley, New York (1968). As disclosed herein, these conditions were met in the polymerization of NCAs with bipyNi (COD). The chain length distribution is narrow, consistent with Poisson statistics, showing that the polymerization rate is proportional to monomer concentration, indicating that the number of active chain ends remains constant throughout the reaction. What makes living polymerization so powerful for block copolymer synthesis is
There is no end and no movement. Since the growing chain remains active after the monomer is depleted, adding a second monomer at this stage results in the growth of a second block that is different in composition from the first. Appropriate selection of monomers makes it possible to fabricate various combinations of the above properties, such as rubber-like glassy; hydrophilic and hydrophobic; and conductive and insulating. By providing examples of NCAs that are polymerized with zero-valent nickel catalysts under "living" polymerization conditions, i.e., without termination and chain transfer, the disclosed methods and compositions have not previously been possible. In a different manner, it allows the production and manipulation of peptides. Living polymerization enables the synthesis of polymers of predetermined and narrow molecular weight distribution, and perhaps more importantly, a well-defined block copolymer in which a long sequence of each individual monomer residue is attached at a single site. Can be prepared. The advantages of living polymerization, previously reserved for a small subset of polymerizable monomers, can be extended to NCAs to prepare high molecular weight polypeptides and block copolypeptides with unusual and useful properties. Can also be expanded.

The methods and compositions disclosed herein teach novel methods for polymerizing amino acids and adding amino acids to polyamino acid chains. In addition, the initiator and amide-containing metallacycle compositions disclosed herein allow the synthesis of block copolypeptides by side reaction elimination, which favors the chain growth process (ie, living polymerization), thus Allows the addition of multiple monomers to the amino acid chain. The specific methods and initiators and amide-containing metallacycle compositions disclosed represent preferred embodiments of the invention as described below, and other embodiments are also encompassed.

The following examples discuss general features for forming active metal initiators, as well as a means of measuring initiator efficiency (see, eg, Example 3). Also disclosed herein are parameters for generating effective initiators, as well as assays that assess the activity of various initiator complexes in the disclosed methods, and their ability to function. Further disclosed herein are a number of different initiator compositions that have been evaluated for their ability to act in the system.
Further, the examples illustrate the effect of different solvents on different polypeptide addition reactions. One of skill in the art can also use these protocols to construct new potential initiator molecules and evaluate their ability to function in the disclosed methods. The protocols disclosed herein can also be used to assess the activity of different amide-containing metallacycles, and their ability to function in the disclosed methods.

In providing a novel means for polymerizing amino acids and adding amino acids to polyamino acid chains, the disclosed methods and compositions overcome some of the problems associated with complex polypeptide synthesis. Successful block copolypeptide synthesis requires elimination of side reactions that favor the chain growth process (ie, living polymerization), which results in
Allows the addition of multiple monomers to each chain. LJ Fetters, “Mo
nodisperse Polymers ", Encyclopedia of
Polymer Science and Engineering, Second Edition, Wiley-Interscience, New York, 10: 19-2.
5 (1987); O. Webster, "Living Polymeriza.
motion methods ", Science, 251: 887-893 (19).
91). This problem has been addressed by utilizing the diverse chemistry of transition metals to mediate the addition of monomers to the active polymer chain ends. TJ Deming, "P
oleptide Materials: New Synthetic
Methods and Applications ”Adv.
als, 9: 299-311 (1997). A wide range of selective chemical conversions and polymerizations catalyzed by transition metal complexes demonstrate the potential of this approach. JP
Collman et al., Principles and Applications.
of Organotransition Metal Chemistry
, 2nd Edition, University Science, Mill Valley,
(1987). The oxidative addition of cyclic carboxylic acid anhydrides to nickel (0) was first described by Uhl.
ig and co-workers. E. Uhlig et al., Z. Anorg.
Allg. Chem., 465: 141-146 (1980). When succinic anhydride is added to L ₂ Ni (COD), a 6-membered acylcarboxylate nickelacycle is first formed, which decarbonylates above ambient temperature and is a stable 5-membered alkylcarboxylate complex. To form. L ₂ = donor ligand (s); COD = 1,5-cyclooctadiene; bipy = 2,2′-bipyridyl. With asymmetric anhydrides, the region specificity of oxidative addition was found to vary with donor ligand (L ₂ ) and solvent. AM Castano
Et al., Organometallics, 13: 2262-2268 (1994).
. When NCA is oxidatively added to nickel (0) across an asymmetric anhydride bond, the region specificity of the addition is important in determining the nature and reactivity of the product. Due to the high stability of the resulting 5-membered metallacycle, decarbonylation is expected to be preferable to decarboxylation for both initial products (Scheme 1 of Figure 3).
See). E. Uhlig et al., Z. Anorg. Allg. Chem., 465: 1.
41-146 (1980). The addition of NCAs to nickel (0) is of interest as the resulting metal amide or metal carbamate complex may prove useful as a reactive, chiral synthetic intermediate.

The reaction chemistry of a-amino acid-N-carboxylic acid anhydrides (NCAs) is based on the fact that these molecules are potential precursors for aligning specific peptides, polypeptides, and other amino acid-containing compounds. , Has been studied. HR Kricheldo
rf, a-amino acid-N-carboxylic acid anhydride and related materials, Spring
er-Verlag, New York, (1987); HR Krichel.
dorf, Models of Biopolymers by Ring-O
penning Polymerization, Penczek, S., CR
C Press, Boca Raton, (1990). NCAs are attractive peptide building blocks because they are easily prepared from amino acids and they do not show racemization at the chiral a-carbon either during preparation or in subsequent reactions. WE Hanby et al., Nature, 161: 132 (1948).
); A. Berger et al., J. Am. Chem Soc., 73: 4084-408.
8 (1951). However, the use of NCAs has been limited due to their complex reactivity and uncontrollable tendency to polymerize.

Attempts to control polymerization using traditional amine-initiated metal coordination complexes have been described in the art. TJ Deming, “Transition Metal”
-Amine Initiators for Preparation of
Well-defined Poly (g-benzyl-L-glutam
ate) ”J. Am. Chem. Soc., 1997, 119: 2759-2760.
(1997). The use of metal amine complexes to polymerize g-benzyl-L-glutamate N-carboxylic anhydride, Glu-NCA, as described herein, results in a narrow molecular weight distribution (Mw / Mn = 1. It allows the preparation of poly (g-benzyl-L-glutamate), ie PBLG, having a molecular weight of 0.5-1.10), allowing some control of the molecular weight. However, typical problems inherent in primary amine-initiated polymerizations (ie slow growth and chain transfer reactions) have prevented the use of these initiators for block copolypeptide preparation. It was

Living polymerization of NCAs and synthesis of block copolypeptides using nickel initiators has been reported. TJ Deming, Nature, 390:
386-389 (1997). This reference discloses a stoichiometric reaction in which NCAs are oxidatively regionally added to a zero-valent nickel source to form a complex, which in turn rearranges into an unprecedented amide-containing metallacycle. is doing. This Nickela cycle, when complexed with a donor ligand, becomes an effective NCA polymerization initiator.

III. Methods and Compositions of the Invention The molecules described herein, unlike initiators known in the art, represent a new class of initiators based on low valent metal-Lewis base complexes. , They can eliminate the important competitive steps of termination and migration from NCA polymerization, allowing the preparation of well-defined block copolypeptides.

Donor ligand / transition metal complex bis-1,5-cyclooctadiene nickel (Ni (COD) ₂ ) as a nickel source and 2,2'-bipyridyl (bipy) as tetrahydrofuran (T
Disclosed herein are various exemplary initiator complexes useful in the production of block copolypeptides, such as those produced using HF) as a donor ligand component in a solvent. As discussed below and shown in Tables 7 and 8, other sources of zero-valent nickel (eg, nickel-olefin complexes, nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and PR ₃ [R = Me , Et
, Bu, cyclohexyl, phenyl], R ₂ PCH ₂ CH ₂ PR ₂ [R = Me, phenyl], a, a′-diimine ligand [1,10-phenanthroline, neocuproine], diamine ligand [tetra The use of other specific ligands such as methylene diamine], and isocyanide ligands [tert-butyl isocyanide and related nickel nitrogen or phosphorus donor ligand complexes] may work in the complexes of the invention to initiate their polymerization. Can be done. In addition to bis-1,5-cyclooctadiene nickel (Ni (COD) ₂ ),
As shown in Example 4 below, other sources of zero-valent nickel (eg, Ni (C
O) ₄ ) as well as other low valent metals in the initiator complex have also been used successfully in these methods. Exemplary metals useful for initiator formation are "low valence" transition metals, especially those of Group VIII of the Periodic Table, and exemplary examples of initiators using such metals are shown in Table 8. Show. This group includes metals Fe, Ru, Os, Co, Rh, Ir, Ni.
, Pd, and Pt. The "low valence" form of a metal means that the metal is in a low oxidation state. For Ni, Pd, Pt, Co and Fe, this means a zero valence (0) oxidation state. For Ir and Rh, this means a monovalent (+1) oxidation state. For Ru and Os this is
It means a divalent (+2) oxidation state. See the complexes in Table 8 for related examples. The term "low valent metal" extends to other metals in their oxidation states where they may undergo two electronic oxidations.

In addition to using 2,2′-bipyridyl (bipy) as the donor ligand component, a variety of other donor ligands can also be used in the initiator complex, as shown in Example 4 below. . As shown in Table 7, ligands that can be used to attach to the initiator metal complex must include a non-nucleophilic electron donor, including a Lewis base, and are pyridyl-based (eg, No. 2-144 of Table 7). ), Diimines (see, for example, DIPRIM 2-148), bisoxazolines (eg, DPOX,
3-2), alkylphosphine (see, for example, dmpe, 2-148)
, Arylphosphines (see, for example, PPh3, 2-151), tertiary amines (see, for example, tmeda, 3-10), isocyanides or cyanides (see, for example, 3-34), or combinations of these ligands. Including what is
It can be composed of various groups with this property. Generally, the ligand is either a bidentate (coordinated by two atoms) or is composed of two equivalents of a monodentate ligand. Tridentate ligands can also be used (eg, terpyridine). Generally, the ligand is the N of the molecule.
Binds to the metal via the P, P, or C atom. Other N or P donor ligands can also support these initiators, similar to those described above (ie, neutral, non-nucleophilic, aprotic).

NCA Monomers NCA monomers are well known in the art, as exemplified in the references cited above (eg, HR Kricheldorf, a-Aminoacid-N-
Carboxyanhydrides and Related Material
als, Springer-Verlag, New York, (1987)). Moreover, the use of various NCA monomers in polypeptide synthesis methods is well known in the art. For example, stepwise synthesis of polypeptides using NCAs (or derivatives thereof) is disclosed in US Pat. No. 3,846,399 (incorporated herein by reference). Further, U.S. Pat. No. 4,267,344 is
Disclosed are N-substituted N-carbohydrates of amino acids and their application in peptide preparation (incorporated herein by reference).

Another aspect of the invention involves the synthesis of unique oligo (ethylene glycol) -functionalized amino acids and their subsequent polymerization into oligo (ethylene glycol) -functionalized polypeptides. Methods of making these monomers include, for example, combining an ethylene glycol (EG) derivative with an amino acid having a reactive side group such as lysine, serine, cysteine and tyrosine to form an EG-functionalized amino acid. . EG derivatives have the general formula _{(CH 3 OCH 2 CH 2)} n X, [ wherein, N represents, approximately 1-3 EG repeated body, X is chloroformate, N-
Hydroxysuccimidyl acetic acid,
Or a reactive group such as halide]. The EG-functionalized amino acid can then be converted to the NCA monomer used in the synthesis of oligo (EG) -functionalized polypeptides.

A generalized diagram for the synthesis of oligo (EG) -functionalized serines, tyrosine or cysteine is shown in Scheme I (below).

[0057]

[Chemical 9]

[0058]   A more detailed synthetic route is shown in Scheme II and Example 5 (below).

[0059]

[Chemical 10]

Further, a preferred method for synthesizing oligo (EG) -functionalized lysine is shown in Scheme III (below).

[0061]

[Chemical 11]

These amino acid derivatives and polymers are novel compositions of matter, which polymers have unique properties that make them potentially valuable for biomedical / biotechnology applications. . Oligo-EG functionalized NCA monomers can be used to produce polypeptide chains in which the side chains of all residues are capped with ethylene glycol oligomers, or "PEG-coated polypeptides." Poly (ethylene glycol), PEG is well known for its "biological invisibility", which is not recognized by the body's immunological defense mechanisms (
non-antigenic) and thus finds many useful applications in drug delivery, enzyme stabilization, tissue engineering, and implant surface modification.

Such polymers are unusual in that they have excellent water solubility over a wide pH range (2-13) and salt concentration. In addition, PEG "coating"
Secondary structure of the polypeptide so that the polymer has stable secondary structure over a wide pH and temperature range (beta sheet and alpha helix)
Strongly stabilize. These new polymers, because they exhibit most of the same properties of PEG (water solubility, biocompatibility) but have completely different chain structures,
It is attractive. Serine and cysteine derived polymers represent the first example of a water soluble polypeptide that adopts a beta sheet structure and forms a stable beta sheet structure. As such, their solution and mechanical properties are significantly different from PEG, providing an interesting PEG substitute in biomedical materials.

Preparation of Amide-Containing Metallacycles The initiator complexes of the present invention can be synthesized by two different approaches, both of which involve the use of low valent transition metal-Lewis base ligand complexes, including amide-containing metallacycles. Bring formation.

Transition Metal / Donor Ligand + NCA Monomer One aspect of the present invention is to provide an amount of a-amino acid-N-carboxylic anhydride monomer with an initiator molecule containing a low valent transition metal-Lewis base ligand complex. Provided is a method of making an amide-containing metallacycle comprising combining to form an amide-containing metallacycle. The formation of these initiators consists of NCA monomer,
It results from an unprecedented reaction with a low valent metal-Lewis base complex such as the zero-valent nickel complex bipyNi (COD) [bipy = 2,2′-bipyridyl; COD = 1,5-cyclooctadiene]. This reaction is similar to the oxidative addition of cyclic anhydrides to zero-valent nickel, resulting in a divalent nickel metallacycle (see equation 6 below). E. Uhlig et al., □ Reactionion cyc
lischer Carbonaeurean hydride mit (a
, A (-Dipyridyl)-(cyclooctadiene-1,5) -n
ickel □ Arg.Allg.Chem., 465: 141-146 (19)
80); K. Sano et al., "Preparation of Ni-or P".
t-containing Cyclic Esters by Oxidat
Ive Addition of Cyclic Carboxylic Anhydrides and Their Properties ”, Bu
ll. Chem. Soc. JPn., 57: 2741-2747 (1984);
M. Castano et al., “Reactivity of Nickelacyc.
le Derived from Aspartic Acid: Alkyla
editions, Insertions, and Oxidations "Orga
nonmetallics, 13: 2262-2268 (1994).

[0066]

[Chemical 12]

However, there is no precedent for activation and polymerization of NCAs by oxidative ring opening of anhydrides. BipyNi of L-proline NCA lacking a proton bound to nitrogen
The success of the polymerization with (COD) supports the hypothesis of oxidative addition across the anhydride bond, rather than the reaction at the N—H bond. In this regard, the initial oxidative addition to the NCA involves the addition of anhydride bonds (eg O—C ₅ for nickel, cobalt and iron, and O—C ₅ and O— for rhodium and iridium.
It is observed that this can occur on either side of (both C ₂ ).

Since NCAs are asymmetric anhydrides, the oxidative addition of NCAs can give rise to two different isomeric products. In practicing one aspect of the present invention, the addition of NCAs to nickel was found to be completely regioselective for ring opening across the O—C ₅ bond. The reaction of bipyNi (COD) with ¹³ C ₂ -L-leucine NCA and ¹³ C ₅ -L-leucine NCA results in oxidative addition products and bipyN
This yielded i (CO) ₂ , which were examined by ¹³ C NMR and FTIR spectroscopy. BipyNi ( ¹² derived from reaction with ¹³ C ₂ -L-leucine NCA
CO) ₂ detection (FTIR (THF): n (CO) = 1978, 1904 cm ⁻¹
), And bipyNi ( ¹³ C derived from the reaction with ¹³ C ₅ -L-leucine NCA.
O) ₂ Detection (FTIR (THF): n ( CO) = 1934,1862cm -1; 1 3 C NMR (DMF-d 7): d 198 (Ni- C O)) , the region having chemical product Identified. Dimethylformamide (DMF), a good solvent for polypeptides
In), this addition product was found to be fully active for polymerizing additional NCA monomers.

Donor Ligand / Transition Metal + alloc-Amino Acid Amide As described above, the amide-amidate metallacycle produced from a low valent transition metal precursor is prepared from α-amino acid-N-carboxylic acid anhydrides (NCAs). It is an active intermediate in controlled polymerization. The limitation of this method is that active growing species
in situ, and therefore does not allow controlled functionalization of the polypeptide chain ends. Due to this cause, we sought an alternative method for directly synthesizing these types of initiators.

Accordingly, another aspect of the invention involves a novel tandem addition reaction that allows the general synthesis of amide-amidate metallacycles useful in the preparation of polypeptides containing a variety of well-defined end groups. . This method of making an initiator molecule involves combining an allyloxycarbonyl-protected amino acid amide with a low-valent transition metal-Lewis base ligand complex and has the following general formula:

[0071]

[Chemical 13]

[Wherein M is a low-valent transition metal, L is a Lewis base ligand,
One of R1 and R2 is an amino acid side group, the other is hydrogen, and R3 is any functional end group that can be attached to a primary amine group]. Form.

Alloc-amino acid amides have 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. An "alloc" group contains at least one allylic moiety, ie, a carbon-carbon double bond attached to a saturated carbon. The backbone "allylic" system is a key element required for the reaction to work. The allylic skeleton is then attached via oxygen to the carbonyl attached to the nitrogen of the amino acid. The R ′ group can be a side chain functionality found in any of the twenty natural amino acids (L-forms), their corresponding D-forms or non-natural synthetic amino acid side chains, and reactive functional groups (lysine, ornithine, Of cysteine, serine, histidine, arginine, glutamic acid or aspartic acid (ie amines, alcohols, sulfhydryls,
Those that are either protic or nucleophilic (such as imidazoles, guanidines, carboxylates) are properly protected (using standard peptide functional group protection) and eliminate their reactivity Provide what to do. Among the unnatural side chains,
Those that react with low valent metal complexes (eg aryl-halides, allylic esters, isothiocyanates or isocyanates) cannot be used either. The R "group is important because it is the group commonly used to" tag "or functionalize polypeptide chains, and is the cause and advantage of using this method. Considering the chemical limitation of the functional group as described above with respect to R ′, this group is
It can be virtually anything. Typically, this group is a peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer chain, small molecule therapeutic chemical linker that attaches the polypeptide to a substrate, a chemical linker that acts as a sensing moiety, or It is a reactive linker that attaches the polypeptide to a large molecule such as a protein, polysaccharide or polynucleotide.

In a preferred embodiment, the Nα-allyloxycarbonyl-amino acid amide is a zero-valent nickel complex LNi (1,5-cyclooctadiene) (L = 2,2′-bipyridine (bpy), 1,10-phene). Reacting with nanthroline (phen), 1,2-bis (dimethylphosphino) ethane (dmpe), and 1,2-bis (diethylphosphino) ethane (depe), the general formula LNiNHC (R ′) HC (
O) NR "amino-amidate metallacycle
(See Table 1 below).

As shown in Table 2 (below), these complexes initiate the polymerization of α-amino acid-N-carboxylic acid anhydrides (NCAs), have a defined molecular weight, a narrow molecular weight distribution, and , Block copolypeptides with quantitative uptake of initiating ligands as end groups. These initiators provide an easy way to synthesize complex copolypeptides where the carboxy terminus of the polymer chain can be quantitatively functionalized with a wide range of substituents. These substituents can include, but are not limited to, polymers (polystyrene, poly (ethylene oxide)), peptides, oligosaccharides, oligonucleotides, or other organic moieties.

An important feature utilized in linking these substituents to the polypeptide chain, and the only requirement for this substituent, is a primary amine group. This feature allows the preparation of complex polypeptide biological materials with great potential applications in medicine (drug delivery, tissue engineering). Specifically, chain end functional groups (eg, other polymers, fluorescent or radioactive labels, or biological molecules (
The ability to incorporate peptide sequences, oligonucleotides, or oligosaccharides) confers on these materials a highly desirable quality for many biotechnological applications. For example, this method can be used to label polypeptide chains and analyze chain mobility / in vivo / in vitro location. In addition, signal transduction or incorporation onto terminally functional polypeptide chains such as receptor groups is
It is essential for targeting delivery complexes as well as substrate-specific anchoring of these materials.

We have used Nα-Al for the formation of the desired amide-amidate nickelacycle.
A reaction (equation 7) was performed in which the loc-amino acid allylamide was used as a substrate.

[0078]

[Chemical 14]

The oxidative addition of allyl amides to nickel was unprecedented and therefore the reaction was not expected to be very successful (Collman, JP; Hegedus, L.
.S .; Norton, JR; Finke, RG Principles a
nd Applications of Organotransition
Metal Chemistry Second Edition, University Science
Ce, Mill Valley, 1987). Therefore, surprising results were obtained when it was found that the reaction of bpyNi (COD) with Nα-Alloc-L-leucine allylamide produced the amide-amidate species in good yield (isolated at 60%). Was given. However, this product was not expected, as evidenced by the lack of the by-product 1,5-hexadiene. This product Nickera cycle was found to be the result of an initial addition by the Alloc C—O bond, followed by a secondary addition by the N—H bond of the amide rather than the allylic N—C bond. (Equation 7). As a result, the product metallacycle 1 retains the allyl substituent on the nitrogen, which was studied by FAB / MS, ¹ H NMR of the hydrolysis product by reaction with HCl, and ¹³ C labeling. Was measured by. The N—H addition was also demonstrated by using Nα-2-hexenyloxycarbonyl-amino acid allylamide in a reaction that resulted in the formation of the by-product hexene identified by ¹³ C { ¹ H} NMR.

The reaction of easily synthesized Nα-Alloc-amino acid allylamides with zero-valent nickel was found to be common to different substituents (R ′ and R ″) and donor ligands, which , Allows the use of many combinations of amino acids and primary amines in the construction of the initiator complex (Table 1). This method thus provides a wide variety of end group functionalities on the polypeptide via amide bonds. It can be implemented by taking in sex.

[0081]

[Table 1]

At this point, it was necessary to determine whether these functional groups, once attached to the initiating complex, would then be quantitatively incorporated as end groups on the polypeptide chain. Using a nickel complex containing different bidentate donor ligands, γ
Polymerization of -benzyl-L-glutamate NCA (Glu NCA) revealed that the alkylphosphine ligands (dmpe and depe) promoted the most effective initiation. These initiators were able to prepare block copolypeptides of defined sequence and composition (Table 2).

[0083]

[Table 2]

It has also been found that the initiator can be used in the polymerization without isolation from the crude reaction mixture. This property greatly simplifies the use of these complexes, they are almost quantitative yields, but their isolation from the reaction solvent is tedious. Therefore, polymerizations were performed either isolated or with in situ initiators and the results were not noticeably different.

With respect to the degree of functionalization of the polymer, initiator 4 (see Table 1) was added with 1 equivalent of cis-
Reacted with 5-norbornene-endo-2,3-dicarboxylic acid anhydride, which should add to the initiator like the NCA monomer but does not form a polymer, and subsequent hydrolysis of the product with HCl 4 was completely consumed, yielding the addition product (Formula 8). Anhydrous IR stretch of starting material (1780 cm ^-1 )
Was observed to completely disappear over the course of the reaction. The only amino acid-containing compound present after hydrolysis was the coupled product (FAB MS: MH ⁺ :
Calculated 323.8, found 323). No unreacted monopeptide was detected from the hydrolysis of unreacted 4, indicating that all metal centers are active.

[0086]

[Chemical 15]

In addition, polymerization studies with Initiator 3 (see Table 1) produced polypeptides with 1-naphthyl end groups. These end groups were then quantified using fluorescence spectroscopy, showing that the number of end groups increased with the number of polymer chains. These fluorescent labels are useful for monitoring polypeptide position and mobility,
Desirable for applications such as in vitro drug delivery complex monitoring (Singhal, A .; Huang, L., Gene Therapeut.
ics: Methods and Applications of Dire
ct Gene Transfer, edited by JA Wolff, Birkhause
r, Boston, 1994).

Finally, MALDI-MS analysis of phenylglycine oligomers prepared with nickel complexes containing leucine isoamyl amide initiation groups revealed that almost all chains were end-functionalized with leucine residues of the initiator. (See Figure 7). For the non-functionalized oligo (phenylglycine) only very small peaks were observed, suggesting that the degree of chain functionalization was greater than 98%.

[0089] Another aspect of the amide-containing metallacycle present invention have the general formula:

[0090]

[Chemical 16]

[Wherein “M” is a low-valent transition metal capable of undergoing an oxidative addition reaction,
"Is an electron donor such as a Lewis base, and" R # "does not carry a free amine, hydroxyl, carboxylic acid, sulfhydryl, isocyanate, imidazole, or other hyperprotic or nucleophilic functionality 5 or 6-membered amide-containing metallacycles containing molecules containing any organic substituents], but these functionalities, if properly chemically protected, can be used to render them protic sources or nucleophiles. The structurally effective R substituents exhibit a number of properties, provided they are non-reactive as an agent, eg, as disclosed in the Examples below, The R substituent of is typically included in the structure of the side chain substituent of the amino acid or its derivative, in particular, in most cases R1 (and R in the central structure).
4) is a proton. Each of R2 and R3 (and R5 and R6) is independently and typically selected from the side chain substituents of amino acids. Typically, (
One of the substituents (such as R1) can be a proton (H) and the other can be a different side chain of a particular amino acid. The proton configuration (as either R2 or R3) is determined by the amino acids that are in the L or D conformation. The side chains may be from the family of naturally occurring L- or D-amino acids, or synthetic amino acids or one of their derivatives. Naturally occurring L- or D-amino acids (eg alanine, arginine, asparagine, aspartic acid, g-carboxyglutamate, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline. , Serine, threonine, tryptophan, tyrosine and valine)
And synthetic amino acids or derivatives thereof are well known in the art. The side chains of amino acids bearing polar functional groups (eg NH ₂ , COOH, SH, imidazole) can be blocked with standard peptide protecting groups.

In a preferred embodiment, the metal is a Group VIII transition metal and the donor ligand (s) can be any of those shown in Table 7. In another preferred embodiment, the metal is nickel and the donor ligand is a 2,2'-bipyridyl (bipy) moiety.
In another preferred embodiment, the R2 or R3 group consists of a side chain protected NCA formed from arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, serine, threonine, tryptophan and tyrosine. An amino acid side chain or alanine selected from the group:
It comprises an amino acid side chain selected from the group consisting of the side chain NCA formed from glycine, isoleucine, leucine, phenylalanine, proline and valine.

Addition of NCA Monomer A related aspect of the invention comprises a method of adding an amino acid-N-carboxylic acid anhydride (NCA) to a polyamino acid chain having an amide-containing metallacycle end group, the method comprising: With the polyamino acid chain so that the NCA is added to the polyamino acid chain. In a preferred embodiment of the method, the amide-containing metallacycle end group has the formula:

[0094]

[Chemical 17]

[Wherein M is a low-valent transition metal; L is a Lewis base ligand; R1 is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine , A component found in the side chain of an amino acid selected from the group consisting of, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine (for example hydrogen for glycine or methyl for alanine. R2 is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, Contains components found in the side chains of amino acids selected from the group consisting of serine, threonine, tryptophan, tyrosine and valine; R3 is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine , Isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine comprising a component found in the side chain of an amino acid; and R4 is a polyamino acid chain There is].

In a preferred embodiment of the method of adding an amino acid-N-carboxylic acid anhydride (NCA) to a polyamino acid chain having an amide-containing metallacycle end group, the metal group of the amide-containing metallacycle is nickel, palladium, platinum. , Cobalt, rhodium,
A transition metal selected from the group consisting of iridium and iron. Lewis base ligands include pyridyl ligands, diimine ligands, bisoxazoline ligands,
It is selected from the group consisting of alkylphosphine ligands, arylphosphine ligands, tertiary amine ligands, isocyanide ligands, and cyanide ligands. In a particular aspect of the invention, NCA is alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and It is an a-amino acid-N-carboxylic acid anhydride selected from the group consisting of valine.

Polymerization Reaction Another aspect of the invention disclosed herein is to combine the amino acid-N- by combining an NCA monomer with an initiator molecular complex composed of a low valent transition metal-Lewis base ligand. A method of polymerizing a carboxylic acid anhydride monomer is included. Specific embodiments of the invention disclosed herein includes a method of polymerizing a bearing amino -N- carboxylic acid anhydride monomers a ring having O-C ₅ and O-C ₂ anhydride linkage. The method comprises combining a first NCA monomer with an initiator molecular complex. The complex is composed of a low valent metal capable of undergoing an oxidative addition reaction, where:
The oxidative addition reaction clearly increases the oxidation state by two electrons and an electron donor containing a Lewis base. Initiator molecules are then opening the ring of the 1NCA via O-C ₅ or over any of the anhydride linkages in the O-C ₂ (across) oxidative addition, combines with the 2NCA monomer, an amide-containing Metara Form a cycle. The third NCA monomer then combines with the amide-containing metallacycle and the amide nitrogen of the amide-containing metallacycle attacks the NCA carbonyl carbon. This {any one? } NCA is subsequently added to the polyamino acid chain and the amide-containing metallacycle is regenerated for further polymerization.

In a preferred aspect of the invention, the efficiency of the initiator is controlled by proceeding the reaction in a solvent selected for its ability to influence the reaction. In a specific embodiment of the present invention, the solvent is ethyl acetate, toluene, dioxane, acetonitrile,
It is selected from the group consisting of THF and DMF.

As illustrated in Example 5 below, the efficiency of various initiators can be analyzed by polymerization experiments with Glu-NCA. The resulting polymer PBLG is α-helical in many solvents, has been extensively studied and is easily characterized.
H. Block, Poly (g-benzyl-L-glutamate) a
nd Other Glutamic Acid Continuing Po
lymers, Gordon and Break, New York, (
1983). The number average molecular weight of PBLG samples formed with bipyNi (COD) in DMF was found to increase linearly as a function of the ratio of initial monomer to initiator, indicating the absence of chain scission reactions. Show. LJ Fetter
s, "Monodisperse Polymers", Encycloped
ia of Polymer Science and Engineerin
g, Second Edition, Wiley-Interscience, New York, 10
: 19-25 (1987); O. Webster, "Living Polym.
orientation Methods "Science, 251: 887-89.
3 (1991). Such control over polypeptide molecular weight has been demonstrated by conventional NCA
This is a substantial improvement over the polymerization system (see Figure 1). The polymer had a narrow molecular weight distribution (Mw / Mn = 1.05 to 1.15) and was obtained in good yield (95-99% isolated). The kinetic analysis also showed that the polymerization behavior was good. The polymerization was carried out in DMF for the first half of the monomer concentration over 4 half-lives (kobs = 2.7 (1) × 10 ⁻⁴ s ^{−1 at} 298 K; [bipyNi (COD) = 0.67 mM).
], Which does not show any of the complexity of traditional NCA polymerization. Our starting system is
All features of the living chain growth process for Glu-NCA are shown. Analysis of other NCA monomers (e.g., e-carbobenzyloxy-L-lysine-N-carboxylic acid anhydride, Lys-NCA) also showed controlled polymerization, revealing distinct block copolypeptides with various structures. For preparation, we show that our starting system is of total utility.

Preparation of Block Copolypeptides Block copolymers have been able to play an important role in materials science and technology, as they allow essentially different properties to be effectively combined in a single material. For example, styrene and diene block copolymers are rubbery at room temperature (a characteristic of the polydiene phase) but can be molded at temperatures above the glass transition of the polystyrene phase. This distinguishes most conventional rubbers from block copolymers, which must be chemically crosslinked (vulcanized) to withstand the stresses encountered in use. Since chemical cross-linking is irreversible, it must be done while making the final part; cross-linked rubber is difficult to reprocess. The "crosslinking" step for styrene-diene block copolymers is instead the physical association of chains in glassy polystyrene domains: 2
The tendency of chain sections of different species to gather together with similar things. This association is strong enough to withstand the load at room temperature, but is easily reversible upon heating. Well-defined block copolymers spontaneously assemble into a variety of complex nanostructures, and other aligned nanostructure arrays can be produced using fluid flow or other fields. ZR Chen et al., Science, 277: 124.
8-1253 (1997). Therefore, block copolymers have had great commercial success and have attracted great interest from polymer physicists. However, little block copolymers of amino acids have been investigated, the reason being that our synthetic methods do not have sufficient control to create well-defined structures.
F. Cardinauz et al., Biopolymers, 16: 2005-202.
8 (1977). This is also true for the synthesis of block copolypeptides for use as biological materials or as selective membranes-the potential advantages of proteinaceous structures remain unrealized due to the lack of suitable synthetic tools. is there. The disclosed methods and ingredients have the potential to change it. Treating the monomer of interest with an initiator such as the zero-valent nickel complex bipyNi (COD) where bipy is 2,2′-bipyridyl and COD is 1,5-cyclooctadiene, and NCA To give an intermediate molecule that is isolatable and remains active for further ring-opening polymerization, and the target polypeptide is
It can be prepared in essentially 100% yield.

One aspect of the invention is to combine a quantity of a first amino acid-N-carboxylic anhydride monomer with an initiator molecule comprising a low valent transition metal-Lewis base ligand complex to form a polyamino acid chain. Producing and then combining an amount of a second amino acid-N-carboxylic acid anhydride monomer with the polyamino acid chain to add a second amino acid-N-carboxylic acid anhydride to the polyamino acid chain. , A block copolypeptide production method is provided. In a preferred embodiment of this method, the initiator molecule is combined with a first amino acid-N-carboxylic anhydride monomer and has the general formula:

[0102]

[Chemical 18]

Wherein M is a low-valent transition metal; L is a Lewis base ligand; and each of R 1 and R 2 and R 3 is independently alanine, arginine, asparagine, aspartic acid, Consisting of a side chain of an amino acid selected from the group consisting of cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; and R4 is poly Is an amino acid chain] to form an amide-containing metallacycle intermediate.

In a highly preferred embodiment of this method for producing a block copolypeptide, the low valent transition metal is nickel, palladium, platinum, cobalt, rhodium,
It is selected from the group consisting of iridium and iron. In another preferred embodiment of this method, the Lewis base ligand is from the group consisting of a pyridyl ligand, a diimine ligand, a bisoxazoline ligand, an alkylphosphine ligand, an arylphosphine ligand, a tertiary amine ligand, an isocyanide ligand and a cyanide ligand. To be selected.

In yet another preferred embodiment of the method, the 1a-amino acid-N-carboxylic acid anhydride monomer is arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, lysine, methionine, serine, threonine. , A side chain protected NCA formed from tryptophan and tyrosine
NCA selected from the group consisting of: a-amino acid-N-carboxylic acid anhydride, or selected from the group consisting of a side chain NCA formed from alanine, glycine, isoleucine, leucine, phenylalanine, proline and valine. It is a side chain of an amino acid.

Blocked Copolypeptides Another aspect of the invention is to provide an overall length of the blocked copolypeptides.
) The number of monomer units (residues) is greater than about 100; and the chain length distribution in the block copolymer composition is at least about 1.01 <Mw / Mn <1.25 [where Mw / Mn = number. Weight average molecular weight divided by the average molecular weight]. In one aspect, the block copolypeptide has 10 consecutive identical amino acids per block. In a preferred embodiment, the block
The copolypeptide is composed of the amino acid components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine. In another preferred embodiment, the copolypeptide is poly (e-benzyloxycarbonyl-L-lysine-block-g-benzyl-L-glutamate), PZLL-b-PBLG, diblock copolymer. In yet another preferred embodiment, the copolypeptide is a poly (g-benzyl-L-glutamate-block-e-benzyloxycarbonyl-L-lysine-block-g-benzyl-L-glutamate) triblock copolymer. . In a related aspect, the number of consecutive monomer units (residues) in the block copolypeptide is about 50 or 100 or 500 or 1000.
(See, eg, the examples disclosed in Tables 1 and 4). In a further related aspect, the total number of full-length monomeric units (residues) in the block copolypeptide is greater than about 200, or greater than about 500, or greater than about 1000 (eg, Table 1 and Table 1 See the example disclosed in 4).

An exemplary embodiment of the invention disclosed in the Examples below comprises a diblock copolymer composed of the amino acid components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine. The polymer is bipyNi (C
Living poly (e-carbobenzyloxy-L-lysine), PZL, prepared by adding Lys-NCA to OD) and further having chain-growth organometallic end groups.
Produces an L chain. Glu-NCA was added to these polymers to give PBL
This produces a G-PZLL block copolypeptide. The evolution of molecular weight with each step of monomer addition was analyzed using gel filtration chromatography (GPC) and the data are shown in Table 1 in Example 3 below. The molecular weight was found to increase as expected as each block of the copolymer grew, while the polydispersity remained low, suggesting successful copolymer formation. A. Noshay et al., Blo
ck Copolymers, Academic Press, New Yo
rk, (1977).

The chromatogram of the block copolypeptide shows a single sharp peak, showing a narrow chain length distribution (see Figure 2). The copolypeptide composition was easily prepared by varying the monomer feed composition, both equivalents. Reverse sequence copolypeptides (ie, PZLL-PBLG) and triblock constructs (eg, PBLG _0.39- b-PZLL _0.22- b-PBLG _0.39 ; Mn = 256,0).
00, Mw / Mn = 1.15) The successful preparation exemplifies sequence control with nickel initiators.

Block copolymerization was not restricted to the highly soluble polypeptides PBLG and PZLL. Copolypeptides were also prepared which both formed homopolymers insoluble in most organic solvents (eg DMF) and which contained L-leucine and L-proline. Data for these copolymerizations are shown in Table 1 in Example 2 below. PB
Due to the soluble effect of the LG and PZLL blocks, it is shown that all of the products are soluble in the reaction medium and the absence of any homopolymer impurities. The L-leucine containing block copolymer was found to associate tightly in 0.1M LiBr in DMF which is a good solvent for PBLG and PZLL. Due to the assembly properties of these materials, once deprotected, they are expected to be useful as tissue engineering scaffolds, drug carriers, and morphology-indicating components in biomimetic complex formation.

Aspects of the invention, as previously mentioned, provide a number of novel methods and compositions for the production of polypeptides with modified material properties. The description and illustrative examples disclosed herein provide many illustrative aspects of the invention. In a specific embodiment of the present invention, NCA is exposed to a solution containing a polyamino acid chain having an amidoamidate metallacycle end group to cause NCA to react with the amidoamidate metallacycle end group, the NCA being treated with the polyamino acid. Amino acid-N-carboxylic acid anhydrides (NCAs) are added to the polyamino acid chain by adding to the chain. The additional embodiment, O-C ₅ or O is reacted with an initiator molecule NCAs
By oxidative addition over -C ₂ anhydride bonds, it is to allow the initiator complex that is opening the ring of NCAs to regioselective, due to providing controlled polypeptide polymer, amino -N- carboxylic acid Methods of controlling anhydride polymerization are included.
Other embodiments include methods of making amide-containing metallacycles and are disclosed herein. Other aspects of the invention include compositions for use in peptide synthesis and design, including 5- and 6-membered amide-containing metallacycles and block copolypeptides.

In addition to block copolypeptides, a variety of other related polypeptides can be generated utilizing the methods disclosed herein, where the initiator molecule is the first amino acid- Combined with N-carboxylic acid anhydride monomer to form the 5- and 6-membered cyclic amide-containing metallacycle intermediates disclosed herein. For example, the domain is a repeat (2 or more) of the same amino acid, or a repeat (2 or more) of a mixture of distinct amino acids, or said 2
Polypeptides can be produced that can be combinations of all. The number of these domains,
Length, order, and composition can be varied to include all possible amino acids in any number of repeats. Preferably, the total number of full-length monomer units (residues) in these polypeptides with segregated domains of mixed monomers is
Greater than 100, the chain length distribution in the polypeptide is about 1.01 <Mw / Mn <
1.25 [where Mw / Mn = weight average molecular weight divided by number average molecular weight].

An illustrative example of such a polypeptide is, for example, 50 leucine residues in one domain, followed by a statistical mixture of 20 valine and 20 glycine as the second domain, followed by Furthermore, it could contain one sequence, finally followed by a third domain of 40 phenylalanines. Such polypeptides differ substantially from "statistically random" copolymers in which the entire polypeptide is made up of a statistical mixture of chained amino acids and there is no exact block domain. These polypeptides have segregated domains, with one difference that one statistical mixture is separated from the other. In statistical copolymers, for example, the amino acids will be distributed statistically (essentially randomly) along the entire polypeptide chain. In contrast, using the methods disclosed herein, 1
There can be a statistical mixture of leucine and glycine in each domain, followed by a polypeptide chain that is followed by a second domain consisting of a statistical mixture of glycine and valine. Since both copolymers have a statistical mixture of residues along the chain, these polypeptides differ in that the valine and leucine residues are segregated in separate domains.

[The method of living polymerization of NCAs disclosed herein will lead to various polypeptides and block copolypeptides with various new and useful properties. The disclosure set forth herein demonstrates in this connection the successful synthesis of such materials, a combination of acidic, basic and hydrophobic domains in which all molecular structures are well controlled. To create a new family of polypeptides. The potential for applications in biomedical engineering, drug delivery, and selective separation is good. In particular, these properties allow the preparation of complex polypeptide biological materials with potential applications in biology, chemistry, physics, and materials engineering. Potential applications include in medicine (drug delivery, tissue engineering), □ smart □ ₂ hydrogels (environmentally-reactive organic materials), and organic / inorganic biomimetic composites (artificial bones, high performance coatings) Including things.

Self-Assembled Amphiphilic Block Copolypeptides for Biomedical Applications Yet another aspect of the invention is at least one water-soluble block attached to a water-insoluble polypeptide domain (“insoluble block”). Includes the synthesis of amphipathic block copolypeptides, including polypeptides (“soluble blocks”). The soluble block contains oligo (ethylene glycol) terminating amino acid (EG-aa) residues and the insoluble block is mainly composed of nonionic amino acid residues. The amphipathic nature of polymers does not distinguish them from the assembly of small molecule lipids and surfactants, but gives them the ability to self-assemble into various well-defined structures in aqueous solution. The amphipathic block copolypeptides of the invention include one or more "soluble blocks." The amino acid composition of these soluble blocks comprises one or more oligo (ethylene glycol) functionalized amino acid residues. Preferred oligo (ethylene glycol) functionalized amino acid residues include EG-Lys, EG-Ser, EG-Cys, and EG-Tyr. The most preferred soluble block consists of oligo (ethylene glycol) terminated poly (lysine). The amphipathic block copolypeptides of the invention also include at least one "insoluble block" covalently attached to a soluble block. The insoluble block can include various amino acid residues or mixtures thereof, including natural amino acids, ornithine, or blocks entirely composed of one or more D-isomers of amino acids. However, insoluble blocks are typically composed primarily of non-ionic amino acid residues, but they generally form insoluble high molecular weight homopolypeptides. Such nonionic amino acids include phenylalanine, leucine, valine, isoleucine, alanine, serine, threonine, and glutamine,
It is not limited to them. In a preferred embodiment, any given insoluble block typically comprises 2-3 different amino acid components in a statistically random sequence.

Vesicle Formation The amphipathic block copolypeptides of the invention are included in compositions and methods of agents that form vesicular structures in aqueous solution. The method consists of suspending the amphipathic block copolypeptide in an aqueous solution and allowing the copolypeptide to spontaneously self-assemble into vesicles. Thus, the vesicle-containing composition is composed of the amphipathic block copolypeptide of the present invention and water. Vesicles resemble much more robust liposomes and are in a controllable size range from micron to less than 100 nanometers in diameter for biomedical / biotechnology applications (ie, drug delivery). To make them potentially valuable. In one embodiment, small vesicles having a diameter of about 50 nm to about 500 nm can be formed by sonicating a suspension of large vesicles. In addition, vesicle diameter can be controlled by adjusting the length and amino acid composition of the amphipathic block copolypeptide (see, eg, Examples below).

Examples The following examples are given by way of illustration only and not by way of limitation. The disclosures of all citations herein are incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

[0117] The method used in Example 1-amino acid-derived metallacycle Intermediate General experimental protocols and reagents infrared spectrum of the metal-mediated polypeptide synthesis was calibrated using polystyrene film Perkin
Recorded on an Elmer 1605 FTIR Spectrophotometer. Tandem gel filtration chromatography / light dispersion (GPC / LS), Wy
att DAWN DSP Light Dispersion Detector and Wyatt Optilab D
Performed on a Spectra Physics Isochrom liquid chromatographic pump with SP. Separation was performed with 0.1M LiBr in DMF eluent at 60 ° C.
With 10 ⁵ Å and 10 ³ Å Phenome
It was carried out with a next 5μ column. The optical rotation is a 1 mL capacity cell (length 1d
m) with the Perkin Elmer Model 141 Polari.
It was measured with a meter. NMR spectrum and bulk magnetic sensitivity measurement (Eva
ns method) was measured by a Bruker AMX 500 MHz spectrometer. [
DF Evans, J. Chem. Soc., 2003-2009 (1959);
J. K. Becconsal, J. Mol. Phys., 15: 129-135 (1
968)]. C, H, N elemental analysis is performed by Microanalytical Lab.
orientation of the University of California
nia, Berkeley Chemistry Department. Chemicals were obtained from commercial suppliers and used without purification unless otherwise noted. (COD) ₂ Ni was obtained from Strem Chemical Co. and ¹³ C ₁ -L-leucine and ¹³ C-phosgene were obtained from Cambridge Iso.
Obtained from top Labs. 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 o.
f Peptide Synthesis, 2nd Edition, Springer, Ber
lin / Heidelberg (1994); ER Blout et al., J. Am. C.
hem Soc., 78: 941-950 (1956); H. Kanazawa et al., Bull. Chem Soc. Jpn., 51: 2205-2208 (197).
8). Hexane, THF and THF-d ₈ was purified by distillation from sodium benzophenone ketyl. DMF and DMF-d ₇ were purified by drying on a 4 Å molecular sieve followed by vacuum distillation.

[0118]   (S)-[NiNHC (H) RC (O) NCH ₂ R] x, R = -CH ₂ CH ₂ C (
O) OCH ₂ C ₆ H ₅ ; 1   Glu NCA (15 mg, 0.058 mmol) was added to TH in a dry box.
Dissolve in F (0.5 mL) and PPh in THF (1.5 mL)₃(31 mg, 0.1
2 mmol) and (COD)₂Ni (16 mg, 0.058 mmol) stirred
Added to the homogeneous mixture. Stir this red / brown solution for 24 hours, then
The solvent was removed in vacuo, leaving a dark red oily solid. Add this to hexane (3 x 5 m
Extraction with L) gave a red / brown hexane solution and a yellow solid. This hexane
When the solution was evaporated, (PPh₃)₂Ni (CO)₂[IR (THF): 2000
, 1939cm^-1  (NCO, vs); 18 mg; Reference: IR (CH₂ClCH₂ Cl): 1994, 1933 cm^-1] To give a red oil and dry the solid
And a yellow powder product was obtained (10 mg, 75% yield). J. Chatt
Et al., J. Chem. Soc., 1378-1389 (1960).¹H NMR spectrum
Kuturu is THF-d₈Could not be obtained, but it may be due to the paramagnetism of the complex.
Is high (only a broad line for the benzyl ester group was observed). m_eff(T
HF, 293K) = 1.08m_B. THF (vs. ferrocene; about 7 mg / mL)
The osmotic molecular weight in the was 910 g / mol, which corresponds to an aggregation degree of 1.94.
. IR (THF): 3281 cm^-1(NNH, s br), 1734 cm^-1(NC
O, ester, vs), 1577 cm^-1(NCO, amidate, vs), NiC _{twenty three} H₂₆O_FiveCalculated value for: 58.87%, 5.59% H, 5.96% N;
Values: 59.07% C, 5.67% H, 5.56% N. [A]_D ²⁰(THF, c =
0.0034) =-71.

(S) -Cl- ⁺ H ₃ NC (H) RC (O) NHCH ₂ R, R = -CH ₂ CH (CH ₃ ) ₂ L-leucine NCA (9.2 mg, 0.058 mmol), Dissolve in THF (0.5 mL) in a dry box and PPh ₃ (31 mg, 31 mL in THF (1.5 mL).
0.12 mmol) and (COD) ₂ Ni (16 mg, 0.058 mmol) were added to a stirred homogeneous mixture. The red / brown solution was stirred for 24 hours, after which the solvent was removed in vacuo leaving a dark red oily solid. This was extracted with cold hexane (0 ° C., 3 × 2 mL) to give a red / brown hexane solution and a pale orange solid. When this hexane solution was evaporated, (PPh ₃ ) ₂ Ni (CO) ₂ [IR
(THF): 2000, 1939 cm ^-1 (nCO, vs); 17 mg] was obtained and a solid was dried to give an orange powder, which was purified by precipitation from THF / hexane to give a yellow powder. As (S)-[NiNHC (H) RC (O
) NCH ₂ R] _x , R = -CH ₂ CH (CH ₃ ) ₂ was obtained (6 mg, yield 80%). ¹
1 H NMR spectra were not obtained in THF-d ₈ but are likely due to the paramagnetism of the complex. IR (THF): 3290 cm ^-1 (nNH, s br), 1580 cm ^-1 (nC)
O, Amidate, vs). ^{_{[A] D 20 (THF,}} c = 0.001) = - 185. This product was dissolved in THF (5 mL) in a round bottom Schlenk flask in a dry box. The flask was placed on a Schlenk line under N ₂ atmosphere, then HCl (90 mL of a 1.0 M solution in Et ₂ O) was added. The yellow solution turned orange and then became hazy as it slowly turned green. After 2 hours the solvent was removed in vacuo leaving a green gummy solid.
This was extracted with D ₂ O to isolate the amino acid containing product. A single isolated compound (4 mg, 73%) was found to be identical to a reliable sample of L-leucine isoamylamide hydrochloride (Figure 1). _{^{1 H NMR (D 2 O)}} ; d 3.94 (t, NH 3 C H (CH 2 CH (CH 3) 2)
C (O)-, 1H, J = 7.5 Hz, 3.33, 3.14 (dm, -C (O
) NHC H ₂ CH ₂ CH (CH ₃ ) ₂ , 2H, J _gem = 107 Hz, J _mult = 6
Hz, 13Hz), 1.72 (dd , NH 3 CH (C H 2 CH (CH 3) 2) C (O
) -, 2H, J = 6 Hz, 7 Hz), 1.68 (m, NH 3 CH (CH 2 C H (CH 3) 2) C (O) -, 1H, J = 7 Hz), 1. 63 (m, -C (O) N
_{HCH 2 CH 2 C H (CH} 3) 2, 1H, J = 7 Hz), 1.43 (ddd, -C
_{(O) NHCH 2 C H 2} CH (CH 3) 2, 2H, J = 7 Hz), 0.98,0.
_{96 (dd, NH 3 CH (} CH 2 CH (C H 3) 2) C (O) -, 6H, J = 6 H
z). 0.92,0.90 (dd, -C (O) NHCH 2 CH 2 C H (C H 3) 2,
6H, J = 6 Hz). [A] _D ²⁰ (THF, c = 0.0033) = + 10.3
. Reliable sample: [a] _D ²⁰ (THF, c = 0.0033) = + 10.
5.

[0120]   Reaction of (PPh ₃ ) ₂ Ni (COD) with ¹³ C ₂ -L-leucine NCA   ¹³C₂-L-leucine NCA [L-leucine and O¹³CCl₂Prepared from
IR (CHCl₃): 3299 cm^-1(NNH, s br), 1836, 174
5 cm^-1(NCO, anhydride, vs);¹³C [¹H] NMR (THF-d₈); D1
52 (s, -NCUnlabeled L-leucine NCA except for the substitution of (O) O-)]
The procedure described above for the reaction used was followed exactly. Cold hexane
Extract with (0- ° C, 3 x 2 mL), red / brown hexane solution and pale orange solid
Got When the hexane solution was evaporated, (PPh₃)₂Ni (CO)₂[IR
(THF): 2000, 1939 cm^-1  (NCO, vs)] containing red oil
To give an orange powder which was precipitated from THF / hexane.
And purify (S)-[NiNHC (H) RC (O) NCH₂R]_x, R = -CH ₂ CH (CH₃)₂(5 mg, yield 66%) was obtained. IR (THF): 3288 cm^-1(NNH, s br), 1580 cm^-1(NC
O, Amidate, vs).

[0121]   Reaction of (PPh ₃ ) ₂ Ni (COD) with ¹³ C ₅ -L-leucine NCA   ¹³C_Five-L-leucine NCA [¹³C₁-L-leucine and OCCl₂Prepared from
Done; IR (KBr): 3308 cm^-1(NNH, s br), 1818, 17
63 cm^-1(NCO, anhydride, vs);¹³C [¹H] NMR (THF-d₈); D
171 (s, -CHRCUnlabeled L-leucine N, except for the substitution of (O) O-)]
The procedure described above for the reaction with CA was followed exactly. Cool the product
Extracted with xane (0- ° C, 3 x 2 mL), red / brown hexane solution and pale orange
To give a solid. When the hexane solution was evaporated, (PPh₃)₂Ni (¹³CO) ₂ [¹³C [¹H] NMR (THF-d₈): D202 (t, Ni (¹³ CO)₂, J _PC = 15 Hz); IR (THF): 1954, 1895 cm^-1(N¹³CO, v
s)] is obtained, and the solid is dried to give an orange powder, which is
Purified by precipitation from THF / hexane, (S)-[NiNHC (H) R¹³C (
O) NCH₂R]_x, R = -CH₂CH (CH₃)₂(6 mg, 80% yield) was obtained
It was IR (THF): 3290 cm^-1(NNH, s br), 1536 cm^-1(N¹³ CO, amidate, vs).¹³C {¹H} NMR (THF-d₈): D 182
(S, [NiNHC (H) R¹³ C(O) NCH₂R]_x).

[0122]   (S)-(2,2′-Bipyridyl) NiNHC (H) RC (O) NCH ₂ R, R
_{= -CH 2 CH 2 C (O} ) OCH 2 C 6 H 5; 2   (S)-[NiNHC (H) RC (O) NCH₂R]_x, R = -CH₂CH₂C (O
) OCH₂C₆H_Five(40 mg, 0.085 mmol) in DMF (0.5 mL) yellow
The color solution was dried in a dry box with 2,2'-bipyridyl (54 mg, 0.35 mmo.
l) was added to a solution of DMF (0.5 mL). Stir homogeneous mixture at 50- ° C for 2 days
During that time, the color changed from yellow to blood red. THF (1 mL) and torr
The ene (5 mL) layered on this solution resulting in the precipitation of a red powder. this
The powder was reprecipitated from DMF / THF / toluene (1: 2: 10) two more times,
(S)-(2,2'-Bipyridyl) NiNHC (H) RC (O) NCH₂R, R =-
CH₂CH₂C (O) OCH₂C₆H_FiveWas obtained as a red powder (49 mg, yield 92)
%).¹1 H NMR spectrum is THF-d₈Was not obtained in the
Most likely due to sex (only wide line for benzyl ester group observed
Was done). IR (THF): 3281 cm^-1(NNH, s br), 1732 cm^-1(NC
O, ester, vs), 1597 cm^-1(NCO, amidate, vs). NiC ₃₃ H₃₄N_FourO_FiveCalculated for: 63.37% C, 5.49% H, 8.95% N;
Found: 63.72% C, 5.49% H, 8.86% N.V. [A]_D ²⁰(THF,
c = 0.001) =-135.

[0123]   (S)-(2,2′-Bipyridyl) NiNHC (H) RC (O) NCH ₂ R, R
= -CH ₂ CH ₂ C (O) OCH ₂ C ₆ H ₅ Polymerization of Glu-NCA   Glu NCA (50 mg, 0.2 mmol) was added to DMF (in a dry box).
25 mL test tube that can be dissolved in 0.5 mL) and sealed with a Teflon (registered trademark) stopper.
I put it in. (S)-(2,2'-Bipyridyl) NiNHC (H) RC (O) NCH ₂ R, R = -CH₂CH₂C (O) OCH₂C₆H_FiveAliquot of 40 mM in DMF
The solution (50 mL) was then added to the flask via syringe. Stir bar
Add, seal the flask, remove from the drying box, 16 o'clock in a constant temperature bath of 25 ° C.
It was stirred for a while. The reaction mixture was added to HCl containing methanol (1 mM) to obtain poly.
The polymer was isolated by precipitating the mer. Polymer, then THF
It was dissolved in and reprecipitated by adding to methanol. This polymer
Was dried in vacuum to obtain a white solid PBLG (39 mg, yield 93%). this
Material¹³C [¹H] NMR,¹1 H NMR and FTIR spectra are PBLG
The data was identical to that seen in the authoritative sample of. H.Block, Po
ly (g-benzyl-L-glutamate) and Other Gl
uamic   Acid Continuing Polymers, Gordon and   Break, New York, (1983). 0.1 in DMF at 60- ° C
GPC of the polymer in M LiBr: Mn = 21,600; Mw / Mn =
1.09.

Of (2,2′-bipyridyl) Ni (COD) with ¹³ C ₂ -L-leucine NCA
In a reaction drying box, bipyNi (COD) (5.9 mg in THF (1 mL).
, 0.018 mmol) solution, 5 equivalents of ¹³ C ₂ -L-leucine NCA (1
4.5 mg, 0.091 mmol) was added. The mixture slowly changed color from purple to red and was stirred for 16 hours. The crude product was isolated by solvent evaporation to give a red oily solid. FTIR analysis of the crude reaction mixture showed (2,2'-bipyridyl) Ni (C
O) ₂ [IR (THF): 1978, 1904 cm ⁻¹ (nCO), vs; Literature:
IR (diethyl ether): 1983, 1914 cm ^-1 ], polyleucine [IR
(THF): 1653 cm ^-1 (n amide I, vs); 1546 cm ^-1 (n amide II, vs)] and ¹² C-amidate end group [IR (THF):-n (CO
) = 1577 cm ⁻¹ ]. RS Nyholm et al., J. Chem.
Soc., 2670 (1953). The reaction was carried out under the same conditions except for DMF-d ₇ (
0.5 mL). ¹³ C [ ¹ H] NMR (DMF-d ₇ ): d126 (
s, ¹³ C O ₂ ).

Reaction of ¹³ C ₅ -L-leucine NCA with (2,2′-bipyridyl) Ni (COD)
In a dry-box, bipyNi (COD) (5.9 mg in THF (1 mL).
, 0.018 mmol) solution, 5 equivalents of ¹³ C ₅ -L-leucine NCA (1
4.5 mg, 0.091 mmol) was added. The mixture slowly changed color from purple to red and was stirred for 16 hours. The crude product was isolated by solvent evaporation to give a red oily solid. FTIR analysis of the crude reaction mixture showed that (2,2′-bipyridyl) Ni ( ¹³ CO) ₂ [IR (THF): 1933, 1862 cm ⁻¹ (nCO, vs)] and ¹³ C-labeled polyleucine [IR (THF). : 1613 cm ^-1 (n amide I, v
s); 1537 cm ^-1 (n amide II, vs)] was confirmed. The reaction is another condition is performed DMF-d ₇ (0.5mL) among others are equivalent. ¹³ C { ¹ H} NMR (DMF-d ₇ ): d 198 (s, bipyNi ( ¹³ CO
) ₂ ; 177 (s, bipyNiN (H) C (H) R ¹³ C (O) N [CH (R) ^{1 3} C (O) NH] _n CH ₂ R), 174 (s, bipyNiN (H) C ^{(H) -R 1 3 C (} O) n [CH (R) 13 C (O) NH] n CH 2 R).

Polymerization of Glu-NCA and (2,2′-bipyridyl) Ni (COD) In a dry box, DMF (0.5 mL) was added to Glu NCA (50 mg, 0.2
mmol) was dissolved and placed in a 25 mL test tube that can be sealed with a Teflon plug. An aliquot of bipyNi (COD) (50 mM solution in DMF at 50 mM
mL) was then added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and stirred in a 25 ° C. constant temperature bath for 16 hours. The polymer was isolated by adding the reaction mixture to HCl containing methanol (1 mM) to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by adding to methanol. Drying the polymer in a vacuum,
White solid PBLG (41 mg, yield 98%) was obtained. ¹³ C [ ¹ H] N of this substance
The MR, ¹ H NMR and FTIR spectra were identical to the data found in the authentic sample of PBLG. H. Block, Poly (g-benz
yl-L-glutamate) and Other Glutamic Ac
id Continging Polymers, Gordon and Br
each, New York, (1983). 0.1ML in DMF at 60_ ° C
GPC of polymer in iBr: Mn = 22,100; Mw / Mn = 1.15
.

[0127]   As described above, a-amino acid-N-carboxylic acid anhydrides (NCAs) are
L₂Metallacycle by reacting with Ni (COD) type zero-valent nickel complex
A oxidative addition product was obtained. These oxidative addition reactions are based on the O--C of NCAs._FiveWell
Or OC₂Addition over any of the bonds results in keying after the addition of the second equivalent NCA.
This gave rise to the Ralamido-Amidate Nickera cycle. The origin and origin of these complexes
And structure selectively¹³Clarified by using C-labeled NCA reagent
It was A stable metallacycle is L = PPh₃Got at the time. Other Donor Riga
, The metallacycle intermediate reacts rapidly with other NCA molecules,
Was found to form a polypeptide with a large molecular weight distribution in quantitative yield
. These reactions show unusually stable metallacycle amide-containing nickel intermediates.
Provides a quick route to the body.   2 equivalents of PPh₃And 1 Ni (COD)₂As 1 equivalent of g-ben in THF
Reaction with dil-L-glutamate-N-carboxylic acid anhydride (Glu-NCA) at room temperature
Upon reaction, the NCA was observed to be rapidly consumed. From a golden brown solution, brown
A colored alkane soluble brown oil and a THF soluble yellow powder were isolated.
The oil was analyzed by decarbonylation of the intermediate 6-membered metallacycle and (P
Ph₃)₂Produced by trapping carbon monoxide with Ni (COD) (PP
h₃)₂Ni (CO)₂[IR (THF):-n (CO) = 2000, 1939 cm ^-1 ] Was confirmed. H. Kanazawa et al., Bull. Chem Soc.
Jpn., 51: 2205-2208 (1978). Infrared analysis of this yellow powder
Is the side chain benzyl ester and amine of the chiral nickelacycle, respectively.
1734 and 1577 cm attributed to the group^-1Shows the carbonyl stretch of
(See Scheme 2 in Figure 3).

The structure and origin of these products were elucidated when ¹³ C ₅ -L-leucine-N-carboxylic acid anhydride was reacted with (PPh ₃ ) ₂ Ni (COD) in THF.
The infrared spectrum of the crude reaction mixture is 1536 cm ^-1 stretched to ¹³ C-amidate groups [ ¹³ C [ ¹ H] NMR (THF-d ₈ ): 182 ppm] and (
PPh ₃ ) ₂ Ni ( ¹³ CO) ₂ stretch was applied at 1954 and 1895 cm ^-1 [ ¹³ C
[ ¹ H] NMR (THF-d ₈ ): 202 ppm], and isotope shifted from the unlabeled compound (see Formula 1 in FIG. 4). ^{When 13} C ₂ -L-leucine-N-carboxylic acid anhydride reacted with (PPh ₃ ) ₂ Ni (COD) in THF, analysis of the product showed (PPh ₃ ) ₂ Ni ( ¹² CO) ₂ [IR (THF): n (CO) = 200
0, 1939 cm ⁻¹ ], and ¹² C amidate [IR (THF): n (CO) = 1
580 cm ⁻¹ ]. (See equation 2 in FIG. 4). ^{Since no 13} C / ¹² C mixed product was observed, oxidative addition occurred at either the C ₅ -O or C ₂ -O bond, followed by decarbonylation and addition of the second NCA molecule, It was concluded that an amide containing nickela cycle was obtained (see Scheme 2 in Figure 3). Without being bound to a particular mechanism or theory, the conversion of the initially formed 6-membered amide-alkyl Nickera cycle to a 5-membered amide-amidate Nickera cycle was induced by the addition of additional NCA monomers. It may occur via migration-mediated ring contraction (see equation in Figure 4). Such a conversion was also observed for related nickel complexes. Furthermore, the sedation of the polymerization reaction with DCl and HCl was conclusively shown to indicate that the 6-membered metallacycle nickel-alkyl bond was not present after the addition of the NCA molecule to the initial metallacycle, leading to an amide-amidate structure. Provides additional supporting evidence for the ring contraction of.

The structure of these metallacyclic products was further confirmed by elemental analysis and acidolysis of the complex. The product metallacycle contains no phosphine according to elemental analysis and has the empirical formula [NiNHC (H) RC (O)
It was found to consist of NCH ₂ CHR] _x . Permeability molecular weight measurement in THF (
Approximately 7 mg / mL) showed that this complex aggregates as a dimer. Treatment of the L-leucine NCA-derived metallacyclic complex with HCl in THF yielded only one organic product. Analysis of this product by ¹ H NMR spectroscopy and polarimeter and comparison of its data with a reliable sample showed that it was optically pure L-leucine isoamylamide-HCl (Figure Three
(See Scheme 2).

[0130]   When the donor ligand bound to the nickel (0) precursor is changed (eg,
Alkylphosphine, a, a'-diimine), chemistry with Glu-NCA in THF
The only products that can be isolated from the stoichiometric reaction are certain starting nickel (0) compounds and
And poly (g-benzyl-L-glutamate), PBLG. 100 equivalents
When Glu-NCA was added to bipyNi (COD) in DMF, before nickel
All the precursor is consumed and PBLG has a narrow molecular weight distribution (> 95%) with good yield (> 95%).
(Mn = 22,100, Mw / Mn = 1.15). L₂= Donner Rigan
COD = 1,5-cyclooctadiene; bipy = 2,2'-bipyr
Jill. bipyNi (COD) is shown to initiate living polymerization of NCAs
It was TJ Deming, Nature, 390: 386-389 (1997).
). bipyNi (COD) oxidatively adds Glu-NCA,
u forms an active polymerization initiator, which rapidly consumes the rest of the monomer.
Was suspected. Select bipyNi (COD) to identify this active initiator
Target¹³A series of experiments were performed in which the C-labeled NCA monomer was reacted.   To completely consume all bipyNi (COD) in the reaction with NCAs
, At least a 5-fold excess of NCA monomer was used. BipyNi (COD)
, 5 equivalents in THF¹³C_FiveBy reacting with -L-leucine-N-carboxylic acid anhydride
It was IR of crude product and¹³C [¹[H] NMR analysis is performed using bipyNi (¹³CO)₂ [IR (THF): n (¹³CO) = 1933, 1862 cm^-1;¹³C [1H] N
MR (DMF-d₇): 198 ppm],¹³C-labeled poly-L-leucine [IR (
THF): 1613 cm^-1(N amide 1, vs); 1537 cm^-1(N amide I
I, vs);¹³C [¹H] NMR (DMF-d₇): 177 ppm (bipyNi
N (H) C (H) R¹³C (O) N [CH (R)¹³ C(O) -NH] nCH₂R)]
, And labeled nickel amidate end groups [¹³C [¹H] NMR (DMF-d₇):
174ppm (bipyNiN (H) C (H) R¹³ C(O) N- [CH (R) ¹³ C (O) NH]_nCH₂R)].¹³C₂-L-leucine-N-
The reaction with carboxylic acid anhydride is¹³A similar product was produced except for the position of the C label.
bipyNi (¹²CO₂) [IR (THF): n (CO) = 1978, 1904.
cm^-1],¹²Poly-L-leucine [IR (THF): 1653 cm^-1(N amide
I, vs); 1546 cm^-1(N-amide II, vs)¹³And¹²C-Amiday
End group [IR (THF): n (CO) = 1575 cm^-1] Were identified. reaction
To DMF-d₇Liberated when carried out in¹³CO₂The existence of¹³C [¹H] NMR
Was confirmed using [126 ppm (s,¹³CO₂)].

All of these experiments were similar to the reaction with (PPh ₃ ) ₂ Ni (COD), consistent with the initial addition of NCA to bipyNi (COD) over the C ₅ —O bond. The main effect of the ligand was itself shown on the reactivity of the resulting product. Ligand-free complexes from the PPh ₃ reaction are inactive towards further reactivity with NCAs, while bipy complexes and other complexes formed with a, a′-diimines and alkylphosphines are less efficient. It was a typical NCA polymerization initiator. This phenomenon was directly demonstrated by synthesizing the reactive metallacycle intermediate formed in the bipyNi (COD) / NCA reaction. Stable metallacycle (S)-[NiNHC (H) RC (O) NCH ₂ R] _x , R = -CH ₂ C
H ₂ C (O) OCH ₂ C ₆ H ₅ ) was reacted with excess bipy in DMF to give the ligand adduct (S)-(2,2′-bipyridyl) NiNHC (H) RC (O) NCH ₂
R, to form a _{R = -CH 2 CH 2 C (} O) OCH 2 C 6 H 5 ( see Scheme 2 of FIG. 3). (S)-(2,2′-Bipyridyl) NiNHC (H) RC (O) NCH _{2
R, R = -CH 2 CH 2} C (O) OCH 2 C 6 H ₅ and DMF in 100 equivalents Glu-N
Reaction with CA resulted in rapid polymer formation. PB formed by this reaction
LG was otherwise identical to that formed with bipyNi (COD) under the same conditions (Mn = 21,600, Mw / Mn = 1.09). The bipyNi (COD) -mediated polymerization of NCAs is therefore believed to proceed with an amide-amidate nickelacycle active end group (see Formula 4 in Figure 4).

[0132] Initiator Nα- allyloxycarbonyl for Examples 2 chain end functionalized polypeptides and block the copolypeptide - amino acid amide, zero valent nickel complex LNi (1,5-cyclooctadiene) (L = 2,2'-bipyridine (bpy)
, 1,10-phenanthroline (phen), 1,2-bis (dimethylphosphino) ethane (dmpe), and 1,2-bis (diethylphosphino) ethane (
depe) to give an amide-amidate metallacycle of the general formula: LNiNHC (R ′) HC (O) NR ″. These complexes are of α-amino acid-N-carboxylic acid anhydrides (NCAs). It was found to initiate the polymerization, resulting in a polypeptide with a well-defined molecular weight, a narrow molecular weight distribution and a quantitatively incorporated initiator ligand as an end group, the naphthyl substituent being incorporated as a fluorescent end group. The feasibility of the method is demonstrated.

General Experimental Protocols and Reagents Infrared spectra were recorded on a calibrated Perkin Elmer 1605 FTIR Spectrophotometer using polystyrene film. Tandem gel filtration chromatography / light dispersion (GPC / LS)
yatt DAWN DSP Optical Dispersion Detector and Wyatt Optilab
Performed on an SSI Accuflow Series III liquid chromatograph pump equipped with a DSP. Separation was performed with 0.1M LiBr in DMF eluent at 60 ° C.
Was run on a 10 ⁵ Å, 10 ³ Å and 500 Å Phenomenex 5μ column. NMR spectra were measured on a Bruker AVANCE 200 MHz spectrometer. FAB Mass spectroscopy measurements were performed at the University of California, Santa Barbara, Department of Chemistry. The MALDI mass spectrum shows the TFA solution and TFA of the analyte.
A sample prepared by mixing a solution of 2,5-dihydroxybenzoic acid in the solution and air-drying the mixture allows Thermo BioAnalyses to operate in positive ion mode.
Obtained using an is DYNAMO mass spectrometer. Fluorescence measurement is SPEX Fluo
Performed on roMax-2. Chemicals were obtained from commercial suppliers and used without purification unless otherwise noted. Alloc-L-aminoamide, epsilon-CBZ
-L-lysine NSA, (S) -phenylglycine NCA and γ-benzyl-L
-Glutamate NCA was prepared according to literature procedures (Tirrell, J. et al.
G; Fournier, M.J .; Mason, TL; Tirrel, D.A.
Chem. & Engr. News 1994, 72, 40-51; Viney, C.
.; Case, ST; Waite, JH, Biomolecular Ma
Terals, Mater. Res. Soc. Proc. 292, 1992: and Deming, TJ, J. Am. Chem. Soc., 1998, 120, 42.
40-4241: incorporated herein by reference). Hexane, THF and TH
F-d ₈ was first bubbled with dry nitrogen and then purified through a column of activated alumina. DMF and DMF-d ₇ were purified by drying over 4 Å molecular sieves and vacuum distillation.

[0134]   Alloc-L-amino acid amide: Alloc-L-leucine-isoamylurea Example procedure for mid synthesis   Isoamylamine (1.4 mL, 12 mmol) was treated with Al in THF (5 mL).
loc-L-leucine-N-hydroxysuccinimidyl ester (2.5 g,
8.0 mmol) solution. The reaction was stirred for 1 hour and then the precipitate formed
After removing the precipitate by filtration, dilute the solution with ethyl acetate (100 mL).
did. This solution was sequentially diluted with dilute aqueous HCl (2 × 30 mL), saturated NaHCO 3.₃water
Solution (2 x 30 mL) followed by saturated aqueous NaCl (2 x 30 mL)
After washing, MgSO_FourDried on. Then the solvent is evaporated in a vacuum to produce
Left behind (1.7 g, 77%). IR (THF): 1724cm^-1(VCO, Alloc, s), 1674cm^-1 (VCO, amidate, s).¹HNMR (CDC1₃): (6.00 (br
s, (CH₃)₂-CHCH₂CH (NHC (O) OCH₂CH = CH₂) C (O)
NHCH₂CH₂CH (CH₃)₂, 2H), 5.85 (m, (CH₃)₂-CHCH ₂ CH (NHC (O) OCH₂CH= CH₂) C (O) NHCH₂CH₂CH (CH₃ )₂, 1H), 5.25 (t, (CH₃)₂-CHCH₂CH (NHC (O) OCH ₂ CH = CH ₂) C (O) NHCH₂CH₂CH (CH₃)₂, 2H), 4.57 (d
, CH₃CHCH₂CH (NHC (O) OCH ₂CH = CH₂) C (O) NHCH₂
CH₂CH (CH₃)₂, 2H), 4.12 (m, (CH₃)₂CHCH₂CH(NH
C (O) OCH₂CH = CH₂) C (O) NHCH₂CH₂CH (CH₃)₂, 1H)
3.24 (q, (CH₃)₂CHCH₂CH (NHC (O) OCH₂CH = CH₂
) C (O) NHCH ₂CH₂CH (CH₃)₂, 2H), 157 (m, (CH₃)₂C
HCH₂CH(NHC (O) OCH₂CH = CH₂) C (O) NHCH₂CH₂CH
(CH₃)₂, 2H), 1.40 (m, (CH₃)₂CHCH ₂CH (NHC (O)
OCH₂CH = CH₂) C (O) NHCH₂CH ₂CH (CH₃)₂, 4H), 0.9
2 (d, (CH ₃)₂CHCH₂CH (NHC (O) OCH₂CH = CH₂) C (O
) NHCH₂CH₂CH (CH ₃)₂, 12H).

(S) -phenNiNHC (H) RC (O) OR == CH ₂ CH (CH ₃ ) ₂ 1,10-phenanthroline (phen) (13 mg, 0.073 mmol)
Was added to a suspension of Ni (COD) ₂ (20 mg, 0.073 mmol) in DMF (2 mL) and after standing at room temperature for 30 minutes, a Phen-Ni (COD) solution was formed. Alloc-L-leucine allyl ester (20 mg, 0.073 m
mol) was added to this purple solution, which then turned brown. 5 at room temperature
After standing for a time, the solution turned green, suggesting the formation of a single oxidative addition product. The green solution was heated at 80 ° C. for 20 hours to obtain a purple solution. The product was isolated from this solution by precipitation in diethyl ether (10 mL) and THF (2 x 10
mL) and dried in vacuo to give a purple powder (16 mg, 6
8%). IR (THF): 1620 cm ^-1 (vCO, carboxylate, s b
r). ¹ H NMR spectra could not be obtained in DMF-d, most likely due to the paramagnetism of the complex (only the broad line for the methyl group was observed). μ _{ef f} (296K) = 2.05 μ _B.

(S) -phenNiNHC (H) RC (O) NCH ₂ R, R = -CH ₂ CH (
CH ₃ ) ₂ , 2 Phen (13 mg, 0.073 mmol) in Ni (CO) in DMF (2 mL).
D) ₂ (20 mg, 0.073 mmol) was added to a suspension and allowed to stand at room temperature for 30 minutes, forming a solution of PhenNi (COD). After that, Alloc-L
-Leucine isoamylamide (20 mg, 0.073 mmol) was added to this purple solution which then turned brown. After standing at room temperature for 5 hours, the solution turned green, suggesting the formation of a single oxidative addition product. 20 this green solution at 80 ℃
Heated for hours to give a purple solution. The product was precipitated in diethyl ether (10 mL), isolated from this solution, washed with THF (2 x 10 mL) and dried in vacuo to give a purple powder (23 mg, 75%). ). IR (THF):
1578 cm ^-1 (vCO, amidate, s br). ¹ H NMR spectra could not be obtained in DMF-d, most likely due to the paramagnetism of the complex (only the broad line for the methyl group was observed). μ _eff (296K) = 2.34 μ _B.

(S) -phenNiNHC (H) R ¹³ C (O) NCH ₂ R, R = —CH ₂ CH
^{_{(CH 3) 2, 2- 13}} C Phen (13mg, 0.073mmol) and DMF (2 mL) Medium Ni (CO
D) ₂ (20 mg, 0.073 mmol) was added to a suspension and allowed to stand at room temperature for 30 minutes, forming a solution of PhenNi (COD). ¹³ C (amide) -all
oc-L-Leucine isoamylamide (20 mg, 0.073 mmol) was added to the purple solution which then turned brown. After standing at room temperature for 5 hours, the solution turned green, suggesting the formation of a single oxidative addition product. This green solution at 80 ° C for 2
After heating for 0 hour, a purple solution was obtained. The product was isolated from this solution by precipitation in diethyl ether (10 mL), washed with THF (2 × 10 mL) and dried in vacuo to give a purple powder (22 mg, 70%). . IR (THF):
1541 cm ^-1 (v ¹³ CO, amidate, s br).

(S) -depeNiNHC (H) R ¹ C (O) NCH ₂ R ² —, R ¹ = -CH ₂ (
^{_{CH 3) 2, R 2 =}} -CH 2 CH3,4 1,2- bis (diethylphosphino) ethane, depe (17μL, 0.07
3 mmol) in Ni (COD) ₂ (20 mg, 0.073 m in THF (1 mL).
mol)) and allowed to stand at room temperature for 5 minutes, depeNi (COD)
A solution of was formed. Then Alloc-L-valine n-propylamide (18.5 mg, 0.073 mmol) in DMF (1 mL) was added to the yellow solution which then turned orange yellow. The solution was heated at 80 ° C. for 20 hours to give an orange solution. The solvent was removed in vacuo, the residue was redissolved in THF and isolated from this solution by precipitation in hexane (10 mL). Dry the solid in vacuo to give 4 as a yellow powder (16 mg, 53%). IR (THF): 1578 cm ^-1 (vCO, amidate, s br). ¹ H NMR spectra could not be obtained in DMF-d and are likely due to the paramagnetic properties of the complex (only wide lines for alkyl groups were observed). μ _eff (296K) = 2.08 μ _B.

(S) -phenNiNHC (H) RC (O) O, R = CH ₂ CH (CH ₃ ) ₂
Isolation anhydrous 4M HCl in the L- leucine -HCl dioxane (1.0 mL) solution, CH ₂ Cl in ₂ (S) -
PhenNiNHC (H) RC (O) O, R = -CH 2 CH (CH 3) 2 (10m
g, 0.027 mmol). The solution immediately changed color from purple to orange. After stirring it for 2 hours, the solvent was removed in vacuo. The remaining solid was extracted with water and the insoluble nickel-containing residue was removed by filtration. The water was then removed by freeze drying to give the desired product. ^{FAB-MS: M-Cl -} : 132.
Calculated value 19, measured value 132.

[0140]   (S) -bpyNiNHC (H) R 1 C (O) NR 2, R 2 = -CH 2 CH (CH ₃ ) ₂ , R ² = -CH ₂ CH = CH ₂ , L-leucine allylamide-HCl from 1
Isolation of   Anhydrous 4M HCl in dioxane (1.0 mL) was added to CH₂Cl₂Medium 1 (10
mg, 0.025 mmol). The solution immediately changes color from purple to orange
changed. After stirring it for 2 hours, the solvent was removed in vacuo. The remaining solid is water
The residue containing insoluble nickel was removed by filtration. The water is then freeze dried
Removed by drying to give the desired product.¹ H NMR (D₂O): (5.91 (m, (CH₃)₂CHCH₂CH (NH₂)
C (O) -NHCH₂CH= CH₂, 1H), 5.73 (t, (CH₃)₂CHCH ₂ CH (NH₂) -C (O) NHCH₂CH = CH, 2H), 3.95 (m, (C
H₃)₂CHCH₂CH(NH₂) C (O) NHCH₂CH = CH₂, 1H), 3.8
1 (br s, (CH₃)₂CHCH₂CH (NH₂) -C (O) NHCH ₂CH =
CH₂, 2H), 1.74 (d, (CH₃)₂CHCH ₂CH (NH₂) C (O) N
HCH₂CH = CH₂, 3H), 0.96 (d, (CH ₃)₂CHCH₂CH (NH₂ ) C (O) NHCH₂CH = CH₂, 6H). FAB-MS: M-Cl^-:Calculated values
171.28, found 171.

[0141]   (S) -phenNiNHC (H) RC (O) -NCH 2 R, R = -CH 2 CH(CH ₃₎ isolation of _2, 2 from L- leucine isoamyl amide   Anhydrous 4M HCl in dioxane (1.0 mL) was added to CH₂Cl₂Medium 2 (10
mg, 0.024 mmol). The solution immediately changes color from purple to orange
changed. After stirring it for 2 hours, the solvent was removed in vacuo. The remaining solid is water
The residue containing insoluble nickel was removed by filtration. Then freeze the water
Removal by lyophilization gave the desired product.¹ H NMR (D₂O): (3.94 (t, NH₃CH(CH₂CH (CH₃)₂)
C (O)-, 1H, J = 7.5 Hz, 3.33, 3.14 (dm, -C (O
) NHCH ₂CH₂CH (CH₃)₂, 2H, J_gem= 10.7 Hz, J_mult= 6
  Hz, 13 Hz) 1.72 (dd, NH₃CH (CH ₂CH (CH₃)₂C (O
)-, 2H, J = 6 Hz, 7 Hz), 1.68 (m, NH₃CH- (CH₂C H (CH₃)₂) C (O)-, 1H, J = 7 Hz), 1.63 (m, -C (O)
NHCH₂CH₂CH(CH₃)₂, 1H, J = 7 Hz), 1.43 (ddd,-
C (O) NHCH₂CH₂CH (CH₃)₂, 2H, J = 7 Hz), 0.98 (d
, NH₃CH (CH₂CH (CH ₃)₂) -C (O)-, 3H, J = 6 Hz), 0
． 96 (d, NH₃CH (CH₂CH (CH ₃)₂) C (O)-, 3H, J = 6H
z), 0.92 (d, C (O) -NHCH₂CH₂CH (CH ₃)₂, 2H, 3H,
J = 6 Hz), 0.90 (d, -C (O) NHCH₂CH₂CH- (CH ₃)₂, 3
H, J = 6 Hz). FAB-MS: M-Cl^-: Calculated value 201.36, measured value
201.

Polymerization of Glu NCA with (S) -depeNiNHC (H) R ¹ C (O) NR ² , R ¹ = -CH ₂ CH (CH ₃ ) ₂ , R ² = -1-naphthyl, 3. γ-Ventyl-L-glutamate-N-carboxylic acid anhydride, Glu NCA (
50 mg, 0.2 mmol) was dissolved in DMF (1.0 mL) in a dry box and placed in a 25 mL test tube that could be sealed with a Teflon stopper. Three
Aliquot (140 μL of a 14 mM solution in DMF) was added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and stirred in a constant temperature 25 ° C. bath for 24 hours. The polymer was isolated by adding the reaction mixture to methanol containing HCl (1 mM) and precipitating the polymer. The polymer was dried in vacuum to give a white solid PBLG (19 mg, 90% yield). The ¹³ C [ ¹ H] NMR, ¹ H NMR and FTIR spectra of this material were identical to the data found in the authentic sample of PBLG. 60 ° C
GPC of polymer in 0.1M LiBr in DMF: Mn = 26,100; M
w / Mn = 1.15.

[0143]   Nα-trans 2-hexenyloxycarbonyl-L-leucine isoamylurea Isolation of mixed hexanes from the oxidative addition of amides to nickel.   Trans-2 hexenyloxycarbonyl-L-leucine isoamylamide (2
A solution of 31 mg, 0.015 mmol) in THF (0.5 mL) was added with THF (0.5 mL).
mL) in (PPh₃)_FourNi (741 mg, 0.015 mmol) was added to give a yellow-orange color.
A color solution resulted. The mixture was heated at 80 ° C for 2 days, during which time the color changed to dark brown.
Turned into Then the volatiles of this reaction were distilled in vacuum into an NMR tube and
The soluble nickel product was removed. 1-hexene, 2-hexene and 3-hexene
The presence in the distillate of a mixture of¹³Proved by C NMR. Hexenes
Is a mixture of intermediates η formed in the reaction³Rapid isomerization of hexenyl-nickel species
It seems that it was formed by embodying.¹³ C NMR (THF): (139.05 (CH₃CH₂CH₂CH₂CH =CH₂ ), 132.15 (trans-CH)₃CH₂ CH =CHCH₂CH₃), 131.
44 (CH₃CH₂CH₂CH =CHCH₃), 130.53 (trans- (CH ₃ CH₂CH₂CH =CHCH₃), 128.56 (cis-CH₃CH₂ CH =CH
CH₂CH₃), 125.92 (cis-CH₃CH₂CH₂ CH = CHCH₃) 1
24.79 (trans-CH₃CH₂CH₂ CH = CHCH₃), 114.10 (
CH₃CH₂CH₂CH₂ CH = CH₂), 34.95 (trans-CH)₃CH₂ C
H₂CH = CHCH₃), 33.73 (CH₃CH₂ CH₂CH₂CH = CH₂) 3
1.44 (CH₃CH₂CH₂ CH₂CH = CH₂), 29.71 (cis-CH)₃C
H₂ CH₂CH = CHCH₃), 22.89 (CH₃ CH₂CH₂CH₂CH = CH₂)
, 22.38 (trans-CH₃CH₂CH₂CH = CHCH₃), 18.05 (
trans-CH₃ CH₂CH₂CH = CHCH₃), 15.04 (cis-CH₃
CH₂CH₂CH = CHCH₃), 14.44 (trans-CH₃CH₂ CH =C
HCH₂CH₃), 13.79 (cis-CH₃ CH₂CH₂CH = CHCH₃) 1
3.60 (CH₃CH₂CH₂CH₂CH = CH₂), 13.32 (trans-C
H₃CH₂CH₂CH = CHCH₃), 13.20 (cis-CH₃CH₂CH₂CH
= CHCH₃).

Polymerization of Glu NCA with (S) -PhenNiNHC (H) RC (O) O, R = -CH ₂ CH (CH ₃ ) ₂ Glu NCA (50 mg, 0.2 mmol) was added to DMF (50 mg, 0.2 mmol) in a dry box.
1.0 mL) and placed in a 25 mL tube which can be sealed with a Teflon stopper. An aliquot of initiator (100 μL of 36 mM solution in DMF) was added to the flask via syringe. Add a stir bar, seal the flask,
It was removed from the drying box and stirred in a constant temperature bath of 25 ° C. for 24 hours. The reaction mixture is
The polymer was isolated by adding to HCl-containing methanol (1 mM) to precipitate the polymer. The polymer was dried in vacuo to give a white solid PBLG (18.1 mg,
Yield 87%) was obtained. ¹³ C [ ¹ H] NMR, ¹ H NMR and F of this material
The TIR spectrum was identical to the data found in a reliable sample of PBLG (Stevens, C; Watanabe, RJ Am. Chem. Soc.
., 1950, 72, 725-727). GPC of polymer in 0.1M LiBr in DMF at 60 ° C .: Mn = 45,500; Mw / Mn = 1.24.

Fluorescence measurement of 1-naphthyl functionalized PBLG A solution of 1-naphthyl functionalized PBLG (26.5 mg) in THF (2 mL) was placed in a cuvette. The sample was excited at a frequency of 324 nm, which was 390n
The maximum intensity of luminescence was produced at m. This emission was characteristic of the 1-naphthyl end group. When the molecular weight of the polymer was changed, the corresponding emission intensity was found to change inversely with chain length, suggesting that the number of end groups was proportional to the number of chains. Control experiments showed that the emission from the labeled polymer was an order of magnitude greater than that from the unlabeled PBLG (see Table 3 below).

[0146]

[Table 3]

Cis-5-Norbornene-endo-2-carboxylic acid-3-cal from reaction 4
Isolation of Voxyl L-valine n-propylamide A solution of cis-5-norbornene-endo-2,3-dicarboxylic anhydride (6.0 mg, 0.037 mmol) in THF (1 mL) was added to a solution of 4 in THF (1 mL). (1
6 mg, 0.037 mmol) was added. The yellow solution was heated at 40 ° C. for 2 days until the anhydride stretch at 1780 cm ⁻¹ was no longer detectable by FTIR. A diluted solution of HCl in water (0.5 mL) was added to the above reaction and the color immediately changed from yellow to orange. The mixture was stirred for 2 hours then the volatiles were removed in vacuo. The remaining solid was extracted with THF, filtered, and then E
Addition to t ₂ O precipitated the nickel-containing by-product. The soluble material was then condensed in vacuo to give the product (11 mg, 92%). FAB-MS: calculated value 323
.8, found 323.

(S) depeNiNHC (H) R ¹ -C (O) NR ² , R ¹ = -CH ₂ CH (CH ₃ ) ₂ , R ² = -CH ₂ CH ₂ CH (CH ₃ ) ₂ , 7 (S) -phenylglycol
Polymerization of Syn (S) -Phenylglycine NCA (50 mg, 0.28 mmol) was dissolved in THF (1.0 mL) in a dry box and placed in a 25 mL test tube that could be sealed with a Teflon plug. An aliquot of 7 (560 μL of a 50 mM solution in THF) was added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and stirred in a 25 ° C. constant temperature bath for 24 hours.
The polymer was observed to precipitate out of solution during this time. The reaction mixture is treated with HCl
The polymer was isolated by addition to methanol containing (1 mM) and centrifugation. The polymer was washed with excess water, methanol and then diethyl ether, then dried in vacuo to give the product as a white solid (35 mg, 93% yield). ¹ H NMR (TFA-d) and FTIR spectra of this material were identical to the data found in a reliable sample of poly (S) -phenylglycine. MALDI mass spectrometric analysis of the polymer shows about 1000 to about 4500 Da
And a separation between peaks equal to the mass of phenylglycine repeats (133.15 Da) was observed. Below 1000 Da, the spectrum was complicated by the presence of numerous matrix peaks. Absolute mass analysis of the peaks clearly showed that almost all chains were end-functionalized by the initiator leucine residue (FIG. 7). Some of the chains contain intact leucine isoamylamide end groups (b-series), while the rest contain leucine end groups that are hydrolytically cleaved after the C-terminal amide has been dissolved in wet TFA. Detached (a-series)
. As an example peak, 9a: predicted MH ⁺ : 1331.44 Da; measured MH ⁺ : 13
30.13 Da. Only very small peaks where the unfunctionalized oligo (phenylglycine) should appear are observed (c-series), these peaks may also contain adducts formed with functionalized chains. For example, 10c (1350.43 Da) is 9a
Had a mass almost equal to + O (1347.44 Da) [1347.53 Da
Observing peak]. Comparison of the peak intensities of the a- and b-series peaks (and adducts) to the c-series peaks determined that the degree of chain functionalization was greater than 98%. The above initiator is bis-1,5-cyclooctadiene nickel (Ni (COD
) ₂ ) as a nickel source and combined with various donor ligand components. We also have other sources of zero-valent nickel as well as other donor ligands (eg PR ₃ [R = Me, Et, Bu, cyclohexyl, phenyl],
R ₂ PCH ₂ CH ₂ PR ₂ [R = Me, phenyl], (α, α′-diimine ligand [1,10-phenanthroline, neocuproine]), diamine ligand [tetramethylethylenediamine], and isocyanate ligand [Tert-butyl isocyanide] has also been used successfully to generate these initiators. The use of other sources of zero-valent nickel, such as nickel-olefin complexes, nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorus donor ligand complexes, should be construed beyond the scope of this invention. Not a possible modification. Similarly, the use of other donor ligands (especially nitrogen or phosphorus based) or reaction solvents is a logical extension of this work. Finally, we
Other transition metals, especially palladium, platinum, cobalt, rhodium and iridium, can also be used in the reaction with alloc-amino acid amides to form amide-amidate metallacycle initiators, and thus another potential for the present invention. It was found to be an artificial modification.

Example 3-Rapid Synthesis of Well-Defined Block Copolypeptides General Experimental Protocols and Reagents Infrared spectra were calibrated using polystyrene film Perkin
Recorded on an Elmer 1605 FTIR Spectrophotometer. Tandem gel filtration chromatography / light dispersion (GPC / LS)
yatt DAWN DSP Optical Dispersion Detector and Wyatt Optilab
Performed on a Spectra Physics Isochrom liquid chromatograph pump equipped with a DSP. Separation was performed with 0.1M in DMF at 60 ° C as eluent.
Using LiBr, 10 ⁵ Å and 10 ³ Å P
Performed with a henomenex 5μ column. Optical rotation was performed using a 1 mL capacity cell (length 1 dm) using a Perkin Elmer Model 141.
It was measured with a Polarimeter. NMR spectra are from Bruker AM
Measured by X 500 MHz spectrometer. Chemicals were obtained from commercial suppliers and used without purification unless otherwise noted. (COD) ₂ Ni is Strem
Chemical Co., ¹³ C ₁ -L-leucine and ¹³ C-phosgene were obtained from Cambridge Isotope Labs. g-
Benzyl-L-glutamate NCA was prepared according to literature procedures. Hexane, THF and THF-d ₈ was purified by distillation from sodium benzophenone ketyl. DMF and DMF-d ₇ were purified by drying over 4 Å molecular sieves and vacuum distillation.

Reaction of ¹³ C ₂ -L-leucine NCA with (2,2′-bipyridyl) Ni (COD)
Response 5 equivalents of ¹³ C _2-L-leucine NCA (14.5 mg, 0.091 mmol) and in dry box THF (1 ml) Medium bipyNi (COD) (5.9mg,
0.018 mmol). This mixture slowly changed color from purple to red and was left stirring for 16 hours. The crude product was isolated by evaporating the solvent to give a red oily solid. By FTIR analysis of the crude reaction mixture, (2,2
'-Bipyridyl Ni (CO) ₂ [IR (THF): 1978, 1904 cm ^-1
(NCO, vs), polyleucine [IR (THF): 1653 cm ^-1 (n amide I, vs); 1546 cm ^-1 (n amide II, vs) and ¹² C-amidate end group [IR (THF): n ( CO) = 1577 cm ^-1 ].
Similarly, the reaction is otherwise the same conditions was performed in a DMF-d ₇ (0.5mL). ^{13 C [1 H] NMR (} DMF-d 7): d126 (s, 13 C O 2).

Reaction of ¹³ C ₅ -L-leucine NCA with (2,2′-bipyridyl) Ni (COD)
Response 5 equivalents of C _5-L-leucine NCA (14.5 mg, 0.091 mmol) and
BipyNi (COD) (5.9 mg, 0.1 ml) in THF (1 ml) in a dry box.
018 mmol). The mixture slowly changed color from purple to red and was left to stir for 16 hours. The crude product was isolated by evaporating the solvent to give a red oily solid. By FTIR analysis of the crude reaction mixture, (2,2'-
Bipyridyl) Ni ( ¹³ CO) ₂ [IR (THF): 1933, 1862 cm ^-1
(NCO, vs)] and ¹³ C-labeled polyleucine [IR (THF): 1613.
cm ^-1 (n amide ^{I, vs); 1537cm -1 (} n amide II, vs) confirmed the presence of. Similarly, this reaction was carried out under otherwise identical conditions under DMF-d ₇ (
0.5 mL). ^{13 C {1 H} NMR (} DMF-d 7): d 198 (s, bipyNi (13 C O
) ₂ ); 177 (s, bipyNiN (H) C (H) R ¹³ C (O) N [CH (R
) ¹³ C (O) -NH] _n CH ₂ R)), 174 (s, bipyNiN (H) C (H
^{) R 13 C (O) N} [CHC (R) 13 C (O) NH] n CH 2 R).

Polymerization of (2,2′-bipyridyl) Ni (COD) with Glu-NCA Glu NCA (50 mg, 0.2 mmol) was added to DMF (in a dry box).
25 mL which can be dissolved in 0.5 ml) and sealed with a Teflon stopper.
Put into a test tube. An aliquot of bipyNi (COD) (50 ml of 40 mM solution in DMF) was added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and placed in a constant temperature 25 ° C. bath for 16 hours.
The polymer was isolated by adding the reaction mixture to methanol containing HCl (1 mM) to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by adding to methanol. The polymer was dried in vacuum to give a white filamentous solid PBLG (41 mg, 98% yield). ¹³ C [ ^{1 of} this substance
¹ H] NMR, ¹ H NMR and FTIR spectra were identical to the data found in the authentic sample of PBLG. 0 of the polymer in DMF at 60 ° C
GPC in 0.1M LiBr is Mn = 22,000; Mw / Mn = 1.05.
Met.

As an exemplary embodiment of the invention, a diblock copolymer composed of the amino acid components g-benzyl-L-glutamate and e-carbobenzyloxy-L-lysine was synthesized. Polymer is Lys vs. bipyNi (COD) in DMF
A living poly (e-carbobenzyloxy-L-lysine) PZLL chain having an organometallic terminal group capable of chain growth was further prepared by adding -NCA. Gl
u-NCA was added to these polymers to give PBLG-PZLL block copolypeptides. The increase in molecular weight through each step of monomer addition was analyzed using gel filtration chromatography (GPC) and the data are shown in Table 1 below. The molecular weight increases as predicted by the growth of each block of the copolymer, while
It was found that the polydispersity remained low, indicating successful copolymer formation.
A. Noshay et al., Block Copolymers, Academic.
Press, New York (1977). The chromatogram of the block copolypeptide shows a single sharp peak, indicating a narrow chain length distribution (see Figure 2). The copolypeptide composition was easily adjusted by varying the monomer feed composition, both equivalents. Conversely sequence (i.e., PZLL-PBLG) and triblock structures _{(e.g., PBLG 0. 39 -b-PZLL 0.22} -b-PBLG 0.39; Mn = 256,000, Mw / Mn
= 1.15) successful preparation of the copolypeptide demonstrates the ability to control sequences with nickel initiators. Block copolymerization was not limited to the highly soluble polypeptides PBLG and PZLL. Copolypeptides were prepared containing L-leucine and L-proline which both form homopolymers that are insoluble in most organic solvents (eg DMF). Data for these copolymerizations are shown in Table 4 below. Due to the solubilizing effect of the PBLG and PZLL blocks, all of the products are soluble in the reaction medium, suggesting that there are no homopolymer impurities present. The L-leucine-containing block copolymer was found to associate strongly with 0.1M 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 morphology-indicating components in tissue engineering scaffolds, drug carriers, and biomimetic composite material formation.

[0154]

[Table 4]

The above-mentioned initiator is bis-1,5-cyclooctadiene nickel (Ni (COD
) ₂ ) as a nickel source and 2,2′-bipyridyl (bipy) as a donor ligand in tetrahydrofuran (THF) solvent. Other sources of zero-valent nickel (eg Ni (CO) ₄ ) as well as other donor ligands (eg PR ₃ [R = Me, Et, Bu, cyclohexyl, phenyl],
R ₂ PCH ₂ CH ₂ PR ₂ [R = Me, phenyl], a, a′-diimine ligand [
1,10-phenanthroline, neocuproine]), diamine ligands [tetramethylethylenediamine], and isocyanide ligands [tert-butylisocyanide] can also be used to initiate their polymerization. The use of other sources of zero-valent nickel, such as nickel-olefin complexes, nickel-carbonyl complexes, nickel-isocyanide or cyanide complexes, and nickel nitrogen or phosphorus donor ligand complexes, should be construed beyond the scope of this invention. But not a possible aspect. Similarly, the use of other donor ligands (especially nitrogen or phosphorus based) or polymerization solvents is a logical extension of this work. Finally, other transition metals, especially palladium, platinum, cobalt, rhodium, iridium and iron,
NCA monomers can be polymerized. Metals in "Group VIII" (ie Co, Rh, I
The use of r, Ni, Pd, Pt, Fe, Ru, Os) is thus an additional potential aspect of the invention.

Exemplary Diblock and Triblock Copolypeptides and Their Synthesis 1. Poly (e-benzyloxycarbonyl-L-lysine-block-g-ben
Dilu-L-glutamate), PZLL-b-PBLG, diblock copolymer Glu NCA (50 mg, 0.19 mmol) was dissolved in dimethylformamide (DMF) (0.5 ml) in a dry box and Teflon®. ) Placed in a 25 mL test tube that can be sealed by a stopper. 2,2'-bipyridyl and Ni (
(2,2′-bipyridyl) Ni (COD) prepared by mixing equal amounts of COD) _2.
Aliquot (50 ml of a 40 mM solution in DMF) was added to the flask via syringe. A stir bar was added, the flask was sealed and then stirred for 16 hours. An aliquot (50 mL) was removed from the polymerization and GPC analysis was performed (Mn = 2).
8,500; Mw / Mn = 1.12). Dimethylformamide (DMF) (0.5
e-benzyloxycarbonyl-L-lysine-N-carboxylic acid anhydride Lys-NCA (50 mg, 0.16 mmol) dissolved in (mL) was then added to the reaction mixture. After stirring for a further 16 hours, the reaction mixture was treated with HCl containing methanol.
The polymer was isolated by adding (1 mM) to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by adding to methanol. The polymer was dried in vacuum to give a white solid PZLL-b-PBLG (79 m
g, yield 93%) was obtained. ¹³ C [ ¹ H] NMR, ¹ H NMR and F of this material
The TIR spectrum was identical to the combined data found in the reliable samples of PBLG and PZLL, respectively. Block copolymer 6
GPC in 0.1 M LiBr in DMF at 0 ° C. was Mn = 52,700; M
It was w / Mn = 1.13.

2. Poly (M-benzyloxycarbonyl-L-lyl) using (PMe ₃ ) ₄ Co
Syn-block-K-benzyl-L-glutamate), PZLL-b-PBLG , diblock copolymer Glu NCA (50 mg, 0.19 mmol) in a dry box in DMF.
(0.5 mL) and placed in a 15 mL test tube that can be sealed with a TEFLON ^™ plug. (PMe ₃ ) ₄ ) Co aliquot (D
A 40 mM solution in MF: THF (1: 1) at 50 TL) was added to the flask via syringe. A stir bar was added, the flask was sealed and then stirred for 16 hours. An aliquot (50TL) was removed from the polymerization and GPC analysis was performed (Mn = 2).
1,500; Mw / Mn = 1.12). Lys dissolved in DMF (0.5 mL)
-NCA (50 mg, 0.16 mmol) was then added to the reaction mixture.
After stirring for a further 16 hours, the reaction mixture was added to HCl containing methanol (1 mM) to precipitate the polymer and isolate the polymer. Then, this polymer is
It was dissolved in F and reprecipitated by adding to methanol. The polymer was dried in vacuum to give a white solid PZLL-b-PBLG (82 mg, 97% yield).
%) Was obtained. The ¹³ C [ ¹ H] NMR, ¹ H NMR and FTIR spectra of this material were comparable to the combined data found in the authentic samples of PBLG and PZLL. Block copolymer 0.1 in DMF at 60 ° C
GPC in M LiBr was Mn-44,700; Mw / Mn = 1.13.

3. Poly (a-benzyl-L-glutamate-block-e-benzyloxy
Carbonyl -L- lysine - block -a- benzyl -L- glutamate) tributyl locking copolymer Glu NCA (250 mg, a 0.95 mmol), dissolved in dimethylformamide (DMF) (1.5 mL) in a dry box, Teflon (Registered trademark)
Placed in a 25 mL test tube that can be sealed by a stopper. Prepared by mixing equal amounts of 2,2'-bipyridyl and Ni (COD) ₂ (2,2'-bipyridyl)
An aliquot of Ni (COD) (50 mL of 40 mM solution in DMF) was added to the flask via syringe. Add a stir bar, seal the flask, then 1
Stir for 6 hours. An aliquot (50 mL) was removed from the polymerization and GPC analysis was performed (Mn = 100,100; Mw / Mn = 1.11). Dimethylformamide (
Lys-NCA (125 mg, 0.42 m) dissolved in DMF) (0.5 mL).
mol) was then added to the reaction mixture and it was stirred for 16 hours. The second aliquot (50 mL) was removed from the polymerization and GPC analysis was performed (Mn = 156,20).
0; Mw / Mn = 1.12). Finally, dimethylformamide (DMF) (1.
Glu-NCA (250 mg, 0.95 mmol) dissolved in 5 mL) was then added to the reaction mixture. After stirring for a further 16 hours, the reaction mixture was added to HCl containing methanol (1 mM) to precipitate the polymer and the polymer was isolated. The polymer was then dissolved in THF and reprecipitated by adding to methanol. The polymer was dried in vacuum to give a white solid PBLG-b
-PZLL-b-PBLG (505 mg, 96% yield) was obtained. ¹³ C of this substance
[ ¹ H] NMR, ¹ H NMR and FTIR spectra are PBLG and PZ
LL was identical to the combined data found in each reliable sample. The GPC of the block copolymer at 60 ° C. in 0.1 M LiBr in DMF was Mn = 256,300; Mw / Mn = 1.15.

4. General Preparation of Block Copolypeptides with Metal Initiators Other diblock and triblock copolymers except that either different monomers or different amounts of monomers were used in each polymerization reaction. hand,
Prepared by the same procedure as described above for either PZLL-b-PBLG and PBLG-b-PZLL-b-PBLG. Examples are shown in Table 4 (above) and Table 5 (below). It has been found that the nature of the amino acid monomer is not important in terms of limiting the effectiveness of these polymerizations. All amino acid NCAs tried
Were incorporated into the block copolypeptide in any sequence order determined by the order of addition of the initiators. Typical monomers include natural L-amino acids and natural D
-Amino acids, a-disubstituted-a-amino acids, racemic a-amino acids and synthetic a
-Including but not limited to amino acids. Block copolypeptides could be prepared using an initiator other than (2,2'-bipyridyl) Ni (COD). All of the initiators shown in Tables 7 and 8 below (except those which showed no polymer yield) were able to prepare block copolypeptides.

[0160]

[Table 5]

[0161] Features of Example 4 General initiator: effect of chemical structure on the evaluation and efficiency of multiple initiators: Effect of reaction conditions on the polymerization; and initiator mediated block copolypeptide synthesis general protocol and The infrared spectrum of the reaction reagent was calibrated using a polystyrene film.
Recorded on an Elmer 1605 FTIR Spectrophotometer. The optical rotation was measured using a 1 mL capacity cell (1 dm in length) using Perkin.
It measured with the Elmer Model 141 Polarimeter. NMR
Spectral and bulk magnetic susceptibility measurements (Evans method) are based on Bruker A
It was measured with an MX 500 MHz spectrometer. DF Evans, J. Chem. Soc
., 2003-2009 (1959); JK Becconsal, J. Mol.
Phys. 15: 129-135 (1968). C, H, N elemental analysis is Mic
roanalytical laboratory of the Uni
rsity of California, Berkeley Chemist
implemented by ry Department. For metal analysis, Thermo Jarr
It was carried out using an El Ash IRIS HR ICP analyzer. Chemicals were obtained from commercial suppliers and used without purification unless otherwise noted. (COD) ₂ N
i was obtained from Strem Chemical CO., and further ¹³ C ₁ -L-
Leucine and ¹³ C-phosgene are available from Cambridge Isotope La.
Obtained from bs. 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 Pepti.
de Synthesis, Second Edition, Springer, Berlin / Hei
delberg, (1994); ER Blout et al., J. Am. Chem So.
C., 78: 941-950 (1956); H. Kanazawa et al., Bull.
Chem Soc. Jpn., 51: 2205-2208 (1978). Hexane, THF and THF-d ₈ was purified by distillation from sodium benzophenone ketyl. DMF and DMF-d ₇ were purified by drying over 4 Å molecular sieves and vacuum distillation.

General Features of Active Metal Initiator Formation Efficient and controlled polymerization of NCAs with transition metal compounds has been shown to include amide containing 5
Requires the general formation of a six- or six-membered metallacycle (FIG. 6), which is the active intermediate in the polymerization. With respect to the results described in this disclosure, these metallacycles are formed by reacting 1 or 2 equivalents of NCA with a metal complex capable of undergoing an oxidative addition reaction whose valence increases by an apparent 2 . Various NCAs can be used in this reaction (ie, L-leucine NCA, Glu-N
CA and L-phenylalanine NCA), any NC of the general structure shown in FIG.
There is no reason that A would not work for this reaction. The metals most commonly undergoing a two-electron oxidative addition reaction are those of group VIII (ie, F
e, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt), and thus these
It is the most studied metal. Collman, JP; Roper,
WR, Adv. Orgmet., Chem., 1968, 7, 53-94. Amide-containing metallacycles can be formed by Fe, Co, Rh, Ir and Ni, and these complexes result in the controlled polymerization of NCAs. Pd and Pt complexes can also accelerate the polymerization of NCAs. Virtually any low-valent transition metal (ie,
Metals in the low oxidation state), when properly combined with electron donor ligand (s), can react with NCAs to produce amide-containing metallacyclic intermediates, which could act as active polymerization initiators. Other metals obviously belonging to this category are Au, Mn, Cr, Mo, W and V.

The range of substituents (R) that can be arranged on the amide-containing metallacycle was examined. These include side chain functional groups found in the amino acids themselves (e.g., phenylalanine derived _{R = CH 2 C 6 H 5} , g- benzyl glutamate-derived _{R = CH 2 CH (CH 3} ) 2 or R = CH ₂ CH ₂ CO a _{2 CH 2 C 6 H 5)} , therefore, it should include any organic moiety attached to the a- amino acids.

Determination of Initiator Efficiency The efficiency was quantified by measuring the polymer molecular weight and molecular weight distribution of the product, and measuring the polymerization kinetics. The polymer molecular weight and molecular weight distribution are Wy
att DAWN DSP Light Dispersion Detector and Wyatt Optilab D
Spectra Physics with SP Interferometer Refractometer
It was measured using tandem gel filtration chromatography / light dispersion (GPC / LS) performed on an Isochrom liquid chromatograph pump. Separations were performed on 10 ⁵ Å and 10 ³ Å Phenomenex 5μ columns with 0.1 M LiBr in DMF at 60 ° C. as eluent. The polymerization reaction rate was determined by periodically removing aliquots from the isothermal polymerization of Glu-NCA, diluting them with anhydrous chloroform to a known volume (10-fold), and measuring 1790 cm ⁻¹ in solution by FTIR spectroscopy to obtain the unreacted amount. Obtained from the kinetic data measured by recording the strength of the anhydrous stretch of. NCA concentration was determined by using an empirical calibration curve for Glu-NCA in chloroform (transmittance versus concentration). Pseudo log (concentration) vs. time plots gave pseudo first-order polymerization rates for various initiators.

[0165]   (S)-[NiNHC (H) RC (O) NCH ₂ R] _x , R = -CH ₂ CH ₂ C (O ) OCH ₂ C ₆ H ₅ ; NiGlu ₂   Glu NCA (15 mg, 0.058 mmol) was added to TH in a dry box.
Dissolve in F (0.5 mL) and PPh in THF (1.5 mL)₃(31 mg, 0
.12 mmol) and (COD)₂Ni (16 mg, 0.058 mmol) was stirred.
Add to the stirred homogeneous mixture. Stir the red / brown solution for 24 hours, then
The solvent was removed in vacuo, leaving a dark red oily solid. Add this to hexane (3 x 5 m
Extraction with L) gave a red / brown hexane solution and a yellow solid. Hexane solution
When evaporated, (PPh₃)₂Ni (CO)₂Containing red oily solid [IR (THF)
: 2000, 1939cm^-1  (NCO, vs); 18 mg, J. Chatt et al.
, J. Chem. Soc., 1378-1389 (1960). IR (CH₂ClC
H₂Cl): 1994, 1933 cm^-1], This solid is dried to give a yellow powder
The product was obtained (10 mg, yield 75%).¹1 H NMR spectrum is THF-
d₈However, it is highly possible that the paramagnetic property of the complex is the cause (benzyl
Only a broad line for ester groups was observed). m_eff= (THF, 293K
) = 1.08m_B. Permeation molecular weight in THF (vs. ferrocene; about 7 mg / mL)
910 g / mol; this corresponds to a degree of aggregation of 1.94. IR (THF): 3281 cm^-1(NNH, s br), 1734 cm^-1(NC
O, ester, vs), 1577 cm^-1(NCO, amidate, vs). NiC _{twenty three} H_{twenty five}N₂O_FiveCalculated for: 58.87% C, 5.59% H, 5.96% N;
Found: 59.07% C, 5.67% H, 5.56% N. [A]_D ²⁰(THF,
c = 0.0034) =-71.

(S)-[NiNHC (H) RC (O) NCH ₂ R] _x , R = -CH ₂ C ₆ H ₅ ; N
iPhe ₂ L-phenylalanine NCA (45 mg, 0.24 mmol) was dissolved in THF (0.5 mL) in a dry box and PPh ₃ (12 mL) in THF (1.5 mL).
4 mg, 0.48 mmol) and (COD) ₂ Ni (64 mg, 0.24 mmo
l) was added to the stirred homogeneous mixture. Stir the red / brown solution for 24 hours,
The solvent was then removed in vacuo leaving a dark red oily solid. This was extracted with cold hexane (0 ° C., 3 × 2 mL) to give a red / brown hexane solution and a pale orange solid. The hexane solution was evaporated to give a red oil containing [PPh ₃ ) ₂ Ni (CO) ₂ [I
R (THF): 2000, 1939 cm ⁻¹ (nCO, vs)] and drying the solid gave an orange powder which was purified by precipitation from THF / hexanes,
(S)-[NiNHC (H) RC (O) NCH ₂ R] _x , R = -CH ₂ C ₆ H ₅ was obtained as a yellow powder (31 mg, yield 80%). ¹ H NMR spectrum is THF-
It was not obtained in d _8, likely caused by paramagnetic complexes. IR (THF): 3290 cm ^-1 (nNH, s br), 1574 cm ^-1 (nC)
O, Amidate, vs). ^{_{[A] D 20 (THF,}} c = 0.001) = - 170.

[0167]   (S)-(2,2′-Bipyridyl) NiNHC (H) RC (O) NCH ₂ R, R
_{= -CH 2 CH 2 C (O} ) OCH 2 C 6 H 5; (2,2'- bipyridyl) NiGlu ₂   NiGlu₂Yellow (40 mg, 0.085 mmol) in DMF (0.5 mL)
The color solution was mixed with 2,2'-bipyridyl (54 mg, 0.35 mmo in a dry box.
l) was added to a solution of DMF (0.5 mL). Stir the homogeneous mixture at 50 ° C for 2 days
During that time, the color changed from yellow to blood red. THF (1 ml) and torr
Ene (5 mL) was layered onto this solution, resulting in the precipitation of a red powder. This powder
The powder was reprecipitated from DMF / THF / toluene (1: 2: 10) two more times,
(2,2'-Bipyridyl) NiGlu₂Was obtained as a red powder (49 mg, yield
92%).¹1 H NMR spectrum is THF-d₈Was not obtained in the complex
Most likely due to paramagnetism (only wide line for benzyl ester groups
Observed). IR (THF): 3281 cm^-1(NNH, s br), 1732 cm^-1(NC
O, ester, vs), 1597 cm^-1(NCO, amidate, vs). NiC ₃₃ H₃₄N_FourO_FiveCalculated for: 63.37% C, 5.49% H, 8.95% N;
Found: 63.72% C, 5.49% H, 8.86% N. [A]_D ²⁰(THF,
c = 0.001) =-135.

Preparation of Other L ₂ NiGlu ₂ and L ₂ NiPhe ₂ Initiators The synthetic procedure for these compounds was to substitute various ligands (L ₂ ) for NiGlu instead of (2,2′-bipyridyl). except for using NiPhe ₂ to ₂ instead, were identical to those described for the preparation of (2,2'-bipyridyl) NiGlu _2. The range of ligands included phen, LiCN and tmeda. All of the complexes gave good analysis.

1. (PM ₃ ) ₂ FePhe ₂ L-phenylalanine NCA (32 mg, 0.16 mmol) was dissolved in THF (0.5 mL) in a dry box and Et ₂ O (4 mL) in (PMe ₃ ). ₄ Fe
(30 mg, 0.083 mmol) was added to the stirred homogeneous solution. The pale orange solution was stirred for 24 hours, after which the resulting off-white precipitate was isolated by centrifugation. The solid was washed with Et ₂ O (3 x 5 mL) then dried to give an off-white powder. This powder was dissolved in THF, precipitated with hexane and purified (36 mg, 91%). ¹ H NMR spectra were not obtained in THF-d ₈ but are likely due to the paramagnetism of the complex (only a broad line for the benzyl ester group was observed). IR (THF): 3296 cm ^-1 (nNH, s br), 1603 cm ^-1 (nC)
O, Amidate, vs).

2. (tBuNC) ₂ FePhe ₂ (PMe ₃ ) ₂ FePhe ₂ (20 mg, 0.042 mmol) was dissolved in THF (2 mL) in a dry box and tBuNC (24 mL) in THF (2 mL).
, 0.252 mmol). The solution was stirred overnight, during which time it slowly turned from brown to yellow. The yellow powder is added to hexane by adding THF
The product was isolated by repeatedly precipitating from. Dry to give a yellow solid (19
mg, 94%). IR (THF): 3289 cm ^-1 (nNH, s br), 2150 cm ^-1 (nN)
C, tBuNC, vs), 1626 cm ^-1 (nCO, amidate, vs).

3. (2,2′-Bipyridyl) FePhe ₂ (PMe ₃ ) ₂ FePhe ₂ (20 mg, 0.042 mmol) was dissolved in THF (2 mL) in a dry box and tBuNC in THF (2 mL). (33 mg
, 0.168 mmol). The solution was stirred overnight, during which time it changed from brown to deep red. DM the red powder by adding it to hexane.
The product was isolated by repeated precipitation from F: THF (1: 1). Drying gave a red solid (18 mg, 89%). IR (THF): 3291 cm ^-1 (nNH, s br), 1600 cm ^-1 (nC)
O, Amidate, vs).

Polymerization of Glu-NCA with (2,2′-bipyridyl) Ni (COD) Glu NCA (50 mg, 0.2 mmol) was dissolved in tetrahydrofuran (THF) (0.5 mL) in a dry box, Placed in a 25 mL test tube that can be sealed by a Teflon plug. (2,2′-Bipyridyl) N prepared by mixing equal amounts of 2,2′-Bipyridyl and Ni (COD) _2.
An aliquot of i (COD) (50 mL of 40 mM solution in THF) was added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and stirred in a constant temperature 25 ° C. bath for 16 hours. The polymer was isolated by adding the reaction mixture to methanol containing HCl (1 mM) to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by adding to methanol. The polymer was dried in vacuum and white solid PBLG (41 m
g, yield 98%) was obtained. ¹³ C [ ¹ H] NMR, ¹ H NMR and F of this material
The TIR spectrum was identical to the data found in a reliable sample of PBLG. H. Block, Poly (g-benzyl-L-glutamat
e) and Other Glutamic Acid Containing
Polymers, Gordon and Break, New York
, (1983). GP of polymer in 0.1M LiBr in DMF at 60- ° C
C: Mn = 98,100; Mw / Mn = 1.15.

General Polymerization of Glu-NC A with (2,2′-Bipyridyl) Ni (COD) in Various Solvents The procedure used was the same except that various solvents were substituted for THF. Identical to that used for (2,2'-bipyridyl) Ni (COD) in THF. Other solvent ranges include toluene, dioxane, acetonitrile, ethyl acetate and D
MF was included. The results of these polymerizations are shown in Table 6 below. Initiator efficiency was determined by analyzing the polymer yield, the closeness of the measured molecular weight to the theoretical value, and the narrow molecular weight distribution.

[0174]

[Table 6]

General Polymerization of (L ₂ ) Ni (COD) Initiator with Glu-NCA The procedure used is that instead of 2,2′-bipyridyl, various ligand molecules (L ₂
Was the same as that used for the (2,2′-bipyridyl) Ni (COD) initiator, except that The range of ligands (L ₂ ) includes tricyclohexylphosphine (PCy ₃ , 1 and 2 equivalents / metal), tert-butyl isocyanide (tBuNC, 2 and 4 equivalents), lithium cyanide (2 equivalents), trimethylphosphine ( PMe ₃ , 2 eq), triethylphosphine (PEt ₃ , 2 eq)
, Tributylphosphine (PBu ₃ , 2 equivalents), triphenylphosphine (PP
h ₃ , 1 and 2 equivalents), 1,2-bis (diphenylphosphino) ethane (DI
PHOS), 1,2-bis (dimethylphosphino) ethane (dmpe), tetramethylethylenediamine (tmeda), (−)-sparteine, 1,10-phenanthroline (phen), neocuproine (ncp), and figures The compounds shown in 5 are included. The results of these polymerizations are shown in Table 7 below. The initiator efficiency is
It was determined by analyzing the polymer yield, the closeness of the measured molecular weight to the theoretical value, and the narrowness of the molecular weight distribution.

[0176]

[Table 7]

[0177]   General polymerization of Glu-NCA with other transition metal initiators   The procedure used was different from that of (2,2'-bipyridyl) Ni (COD).
Used for (2,2'-bipyridyl) Ni (COD) except that the metal complex was replaced.
It was the same as the one used. The range of metal complexes includes (2,2'-bipyridyl) Ni (CO)₂; (S)-[NiNHC (H) RC (
O) NCH₂R]_x, R = CH₂CH₂C (O) OCH₂C₆H_Five(NiGlu₂); (
S)-(2,2'-Bipyridyl) NiNHC (H) RC (O) NCH₂R, R =
-CH₂CH₂C (O) OCH₂C₆H_Five(2,2'-bipyridyl NiGlu₂); (
S) -Li₂(CH)₂NiNHC (H) RC (O) NCH₂R, R = -CH₂CH ₂ C (O) OCH₂C₆H_Five(Li₂(CH)₂NiGlu₂); (S)-(phen
) NiNHC (H) RC (O) NCH₂R, R = -CH₂C_FiveH_Five(PhenNiG
lu₂); (S)-(phen) NiNHC (H) RC (O) NCH₂R, R =-
CH₂C_FiveH_Five(PhenNiPhe₂); (S)-(tmeda) NiNHC (H
) RC (O) NCH₂R, R = -CH₂C₆H_Five(TmedaNiPHe₂); Dm
peCoPhe₂; (PMe₃)₂CoPhe₂; (PMe₃)_FourCo; dmpeRh
Cl; dmpeIrCl; h^Five-C_FiveH_FiveCo (CO)₂(CpCo (CO)₂);
(2,2'-bipyridyl Co (CO)₂)₂; ((PPh₃)₂Co (CO)₂)₂;
(Pme₃)_FourFe; (2,2'-bipyridyl)₂Fe; (S)-(PM
e₃)₂FeNHC (H) RC (O) NCH₂R, R = -CH₂C₆H_Five((PMe₃
)₂FePhe₂); (S)-(tBuNC)₂FeNHC (H) RC (O) NC
H₂R, R = -CH₂C₆H_Five((TBuNC)₂FePhe₂); (S)-(2,2
′ -Bipyridyl) FeNHC (H) RC (O) NCH₂R, R = -CH₂C₆H_Five(
(2,2'-Bipyridyl) FePhe₂); (PPh₃)_FourPd; Tris (Given
Tylidene acetate) Dipalladium (Pd₂(DBA)₃) Plus 4 equivalent PEt₃
; (PEt₃)₂Pt (COD); and (dmpe)₂Co was included. these
The results of the polymerization of are shown in Table 8 below. Initiator efficiency is related to polymer yield and theoretical value.
By analyzing the closeness of the measured molecular weight and the narrowness of the molecular weight distribution.
I have

Polymerization of Glu-NCA with (PMe ₃ ) ₄ Co Glu NCA (50 mg, 0.2 mmol) was added to DMF (in a dry box).
0.5 mL) and placed in a 15 mL tube that can be sealed with a TEFLON ^™ stopper. An aliquot of (PMe ₃ ) ₄ Co ( _{4 in} DMF: THF (1: 1)
50 mM of 0 mM solution) was added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and stirred in a constant temperature 25 ° C. bath for 16 hours. The polymer was isolated by adding the reaction mixture to methanol containing HCl (1 mM) to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by adding to methanol. The polymer was dried in vacuum to give a white solid PBLG (42 mg, 99% yield). The ¹³ C [ ¹ H] NMR, ¹ H NMR and FTIR spectra of this material were identical to the data found in the authentic sample of PBLG. Polymer in DMF at 60 ° C. 0
GPC in .1M LiBr is Mn = 21,600; Mw / Mn = 1.11.
.

(S)-[CoNHC (H) RC (O) NCH ₂ R] ₂₅ , R = CH ₂ C ₆ H ₆ ; C
oPhe ₂ Phe NCA (9.0 mg, 0.046 mmol) was added to TH in a dry box.
Dissolve in F (0.5 mL) and (PPh ₃ ) ₃ Co (N _{2 in} THF (1.5 mL).
) (40 mg, 0.046 mmol) to a stirred homogeneous solution. The red / brown solution was stirred for 24 hours, after which the solvent was removed in vacuo leaving a red / orange oily solid. This was extracted with hexane (3 x 5 mL) to give an orange hexane solution and a light brown solid. When the hexane solution was evaporated, [(PPh ₃
) ₃ Co (CO)] ₂ [IR (THF): 1909, 1875 cm ^-1 (nCO, v
s); 15 mg; literature: IR (KBr): 1904, 1877 cm ^-1 ] was obtained as a brown oil which was dried to give a light brown powder product (11).
mg, 74% yield). ¹ H NMR spectra were not obtained in THF-d ₈ but are likely due to the paramagnetism of the complex (only broad line observed for phenyl ring). IR (THF): 3310 cm ^-1 (nNH, s br), 1600 cm ^-1 (nC)
O, Amidate, vs).

(S)-(dmpe) CoNHC (H) RC (O) NCH ₂ R, R = -CH ₂ C ₆ H ₆ dmpeCoPhe ₂ 1 (40 mg, 0.12 mmol) light brown DMF (0.5 mL) ) Solution in a dry box with bis (dimethylphosphino) ethane, dmpe (35TL, 0.
21 mmol) in DMF (0.5 mL). 5 of this homogeneous mixture
After stirring for 2 days at 0 ° C., the color changed from yellow to orange / red during that time. THF
(1 mL) and toluene (5 mL) were layered onto this solution resulting in the separation of a brown oil. This oil was added to DMF / THF / toluene (1: 2: 10).
2), the product (49 mg, yield 86%) was obtained. ¹ H NM
Although R spectra were not obtained in THF-d _8, (observed only wide lines relating phenyl and methyl groups) paramagnetic is likely to be responsible for the complex.
IR (THF): 3295 cm ^-1 (nN11, s br), 1603 cm ^-1 (n
CO, amidate, vs).

Polymerization of Glu-NCA with dmpeCoPh ₂ Glu NCA (50 mg, 0.2 mmol) was added to DMF (in a dry box).
0.5 ml) and placed in a 15 mL tube that can be sealed with a TEFLON ^™ stopper. Aliquot of dmpeCoPhe ₂ (50 of 40 mM solution in DMF
TL) was added to the flask via syringe. A stir bar was added, the flask was sealed, removed from the dry box and stirred in a constant temperature 25 ° C. bath for 16 hours. The polymer was isolated by adding the reaction mixture to methanol containing HCl (1 mM) to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by adding to methanol. The polymer is dried in vacuum to give a white solid P
BLG (41 mg, yield 98%) was obtained. ¹³ C [ ¹ H] NMR of this substance, ¹ H
The NMR and FTIR spectra were identical to the data found in the PBLG authentic sample. GPC of the polymer for 0.1M LiBr in DMF at 60 ° C. was Mn = 20,900; Mw / Mn = 1.07.

[0182]

[Table 8]

[0183] Example 5 oligo (ethylene glycol) functionalized amino acid and methods for their preparation of the polymer over: novel water soluble biocompatible polypeptide following experiments, oligo (ethylene glycol) functionalized lysine, serine, Synthesis of cysteine and tyrosine NCA monomers and their subsequent oligos (
Polymerization to ethylene glycol) functionalized polypeptides is described. General items Tetrahydrofuran (THF), hexane, N, N-dimethylformamide,
And the diethyl ether was dried by passing through an alumina column under nitrogen before use. All reactions were performed under an anhydrous nitrogen atmosphere unless otherwise noted. The above chemicals were obtained from commercial vendors and used without purification.
Co (PMe ₃ ) ₄ is derived from Klein et al. [Klein et al.
kiss (trimethylphosphonate) kobalt und sei
ne Derivate, Chem Ber., 108: 944-955 (19).
75); incorporated herein by reference]. Infrared spectra are calibrated Perkin Elmer using polystyrene film
Recorded on RX1 FTIR Spectrophotometer. ¹ H N
MR spectra were recorded on a Bruker AVANCE 200 MHZ spectrometer and referenced to internal solvent resonance. Tandem Gel Filtration Chromatography / Light Dispersion (GPC / LS) is a Wyatt DAWN DSP Light Dispersion Detector and Wya
Performed on an SSI pump equipped with a tt Optilab DSP. Separation using DMF in 0.1 M LiBr in 60 ° C. as eluent 10 ⁵ Å, 10 ⁴ Å and 10 ³ Å Phenomene
x 5μ column. Circular dichroism is measured by Oris Rap at room temperature.
It was run on id Scanning Monochromator. The path length of the quartz cell was 1.0 mm and the peptide concentration was 0.5 mg / mL. MALD
The ITOF mass spectrum is Thermo BioA operating in positive ion mode.
Collected by a sample prepared by mixing the analyte in THF with a solution of 2,5-dihydroxybenzoic acid in THF using a nalysis DYNAMO mass spectrometer and air-drying the mixture.

N-Hydroxysuccinimidyl 2- [2-methoxyethoxy] ethoxy] acetic acid . 1 Nα-tert-Butyloxycarbonyl-O- (2- (2-methoxyethoxy) ethyl) -L-serine. Compound 1. Scheme II. Nα-tert-butyloxycarbonyl-L-serine (4.59 g, 22.3
mmol) was dissolved in N, N-dimethylformamide (100 mL) and the solution was then cooled to 0 ° C. and treated with sodium hydride (1.97 g, 49.2 mmol). 1-Bromo-2- (2-methoxyethoxy) ethane (10.0
g, 49.2 mmol) was added to this solution and the reaction mixture was stirred at room temperature for 3 hours. The solvent was then removed under reduced pressure at a bath temperature of 40 ° C. The residue was dissolved in water (75 mL) and washed twice with diethyl ether (30 mL each time). Water layer then 1
Acidified to pH 3 with M HCl and then extracted with ethyl acetate.
The organic layer was dried over anhydrous MgSO _4, the solvent was removed in vacuo to give the compound as a yellow oil (4.1g, 63%). The compound had the following characteristics: _{FTIR (CHCl 3): 1735 (} v CO, s), 1712 (v CO, s). ¹ H
NMR (CDCl ₃ ); δ5.63 (d, (CH ₃ ) ₃ COC (O) NH CH (
CH ₂ OCH ₂ CH ₂ OCH ₂ CH ₂ OCH ₃ ) C (O) OH, 1H), 4.38 (s)
_{, (CH 3) 3 COC (} O) NHC H (CH 2 OCH 2 CH 2 OCH 2 CH 2 OCH 3) C (O) OH, 1H), 4.14-3.53 (m, (CH 3 ) ₃ COC (O) N
_{HCH (C H 2 OCH 2 CH} 2 O-C H 2 C H 2 OCH 3) C (O) OH, 10H),
_{3.47 (s, (CH 3)} 3 COC (O) NHCH (CH 2 OCH 2 CH 2 OCH 2 -
_{CH 2 OC H 3) C (} O) OH, 3H), 1.52 (s, (C H 3) 3 COC (O)
_{NHCH (CH 2 OCH 2 CH 2} OCH 2 CH 2 -OCH 3) C (O) OH, 9H)
. MALDITOF-MS: MH ⁺ : calculated 307.34, found 309.19.

[0185]   Preparation of O- (2- (2-methoxyethoxy) ethyl) -L-serine. Compound 2 . Scheme II.   Serine derivative Nα-tert-butyloxycarbonyl-O- (2- (2-me
Toxy-ethoxy) ethyl) -L-serine was used without further purification
. This serine derivative (4.13 g, 16.9 mmol) was concentrated with acetic acid (50 mL).
Dissolved in. The solution was placed in an ice bath and 1M HCl (34 mL) was added.
The mixture was added and the mixture was stirred for 30 minutes. Stirring is continued for 2 hours at ambient temperature and the solution
Was then concentrated under reduced pressure to give a yellow oil. This oil is then Et₃To N
So neutralize, CH₃The amine salt was removed by extraction with CN. Insoluble formation
The material was collected as a white solid (2.4g, 58%).¹ H NMR (D₂O): δ3.87 (m, NH₂CH(CH ₂OCH₂CH₂OCH ₂ CH₂OCH₃) C (O) OH, 3H), 3.67-3.60 (m, NH₂CH (
CH₂OCH ₂CH ₂OCH ₂CH ₂OCH₃) C (O) OH, 8H), 3.34 (s
, NH₂CH (CH₂OCH₂CH₂OCH ₂CH ₂OCH ₃) C (O) OH, 3H
).¹³C {¹H} NMR (D₂O): (173.05 (NH₂CH (CH₂OCH₂ CH₂OCH₂-CH₂OCH ₃)C(O) OH), 71.97, 70.87, 70
． 64, 70.48, 69.80 (NH₂CH (CH₂OCH₂ CH₂OCH₂−C
H₂OCH₃) C (O) OH), 59.10 (NH₂CH (CH₂OCH₂CH₂OC
H₂-CH₂OCH₃) C (O) OH)), 55.67 (NH₂CH (CH₂OCH₂ CH₂OCH₂-CH₂OCH₃) C (O) OH).   MALDIOF-MS: MH⁺: Calculated value 207.22, measured value 208.48. [A]_D ^{twenty three}= -11.2 (c = 0.05, H₂O).

[0186]   Preparation of O- (2- (2-methoxyethoxy) ethyl) -L-serine NCA. Conversion Compound 3. Scheme II .   O- (2- (2-methoxyethoxy) ethyl) -L-serine (0.64 g, 2.
6 mmol) against THF (100 mL) and COCl₂(1.93M Torue
Solution (1.63 mL) was added and the mixture was stirred at room temperature for 5 hours. Generate
The solution was concentrated to give a yellow oil as a crude product (0.49 g, 80%).
This oil was mixed with tetrahydrofuran, toluene and hexane (1: 4: 4).
) At -30 ° C and the product was collected as a white solid (0.27g, 4
5%). FTIR (THF); 1858cm^-1(VCO, s), 1792cm^-1, (VC
O, s),¹1 H NMR (CDCl₃): Δ 7.53 (s, RC (H) C (O)
OC (O) NH, R = -CH₂OCH₂CH₂OCH₂CH₂OCH₃, 1H), 4.
40 (t, RC (H) C (O) OC (O) NH, R = -CH₂OCH₂CH₂OC
H₂CH₂OCH₃, 1H), 3.90 (d, RC (H) C (O) OC (O) NH
, R = -CH ₂OCH₂CH₂OCH₂-CH₂OCH₃, 2H), 3.70-3.5.
5 (m, RC (H) C (O) OC (O) NH, R = -CH₂OCH ₂CH ₂OCH ₂ CH ₂OCH₃, 8H), 3.41 (s, RC (H) C (O) OC (O) NH, R
= -CH₂OCH₂CH₂OCH₂-CH₂OCH ₃, 3H).¹³C {¹H} NMR (
CDC1₃): (169.82 (RC (H)C(O) OC (O) NH, R = -C
H₂OCH₂CH₂OCH₂-CH₂OCH₃), 153.63 (RC (H) C (O)
OC(O) NH, R = -CH₂OCH₂CH₂OCH₂-CH₂OCH₃), 72.7
5, 72.26, 71.76, 71.32, 71.07 (RC (H) C (O) O
C (O) NH, R =-CH₂OCH₂ CH₂OCH₂−CH₂OCH₃), 59.95
(RC(H) C (O) OC (O) NH, R = -CH₂OCH₂CH₂OCH₂-CH ₂ OCH₃), 59.78 (RC (H) C (O) OC (O) NH, R = -CH₂O
CH₂CH₂OCH₂-CH₂OCH₃) [A]_D ^{twenty three}= -37.6 (c = 0.017)
, THF).

[0187]   Preparation of poly (O- (2- (2-methoxyethoxy) ethyl) -L-serine). Compound 4. Scheme II.   O- (2- (2-methoxyethoxy) ethyl) -L-seri in DMF (2 mL)
NCA (120 mg, 0.52 mmol) in Co (P) in THF (0.5 mL).
Me₃)_Four(3.8 mg, 0.010 mmol) and stirred for 18 hours. Heki
The polymer was precipitated from this solution by adding sun (20 mL)
. This polymer is H₂Dissolved in O (5 mL), dialyzed to remove impurities, then
Lyophilized to give the product as a white solid (70 mg, 71%). FTIR (KBr); 1631 cm^-1(Amide 1, s br), 1523 cm ^-1 , (Amide 11l, s br).¹1 H NMR: δ 8.45 (d,-(NHC
H (CH₂OCH₂CH₂OCH₂CH₂OCH₃) -C (O))_n-, 1H), 4.6
1 (m,-(NHCH(CH₂OCH₂CH₂OCH₂CH₂OCH₃) C (O))_n- , 1H), 3.80 (br s,-(NHCH (CH ₂OCH₂CH₂OCH₂CH ₂ OCH₃) C (O))_n-, 2H), 3.67-3.61 (br m,-(NHC
H (CH₂OCH ₂CH ₂OCH ₂CH ₂OCH₃) C (O))_n-, 8H), 3.36.
(S,-(NHCH (CH₂OCH₂CH₂OCH₂CH₂OCH ₃) -C (O))_n- , 3H).¹³C {¹H} NMR (CDC1₃): Δ 174.23 (-(NHCH
(CH₂OCH₂CH₂OCH₂CH₂OCH₃)C(O))_n-), 72.06, 71
． 15, 70.75, 70.59 (-(NHCH (CH₂OCH₂ CH₂OCH₂ C H₂OCH₃) C (O))_n-), 59.15 (-(NHCH (CH₂OCH₂CH₂
O-CH₂CH₂OCH₃) C (O))_n-54.52 (-(NHCH (CH₂OC
H₂CH₂OCH₂CH₂OCH₃) C (O))_n-). [A]_D ^{twenty three}= -28.3 (c =
0.012, H₂O).

Preparation of (2- (2-methoxyethoxy) ethyl) chloroformate. Compound 5. Scheme II . Di (ethylene glycol) monomethyl ether (5.
0 g, 42 mmol) at 0 ° C. with COCl ₂ (32.3 of a 1.93 M solution in toluene).
ml) and stirred for 1 hour. Then, the mixture was stirred at 10 ° C. for 2 hours. The solvent and excess phosgene were removed under reduced pressure to give a clear oil (7.0 g, 9
2%). FTIR (CH ₂ Cl ₂ ): 1780 cm ⁻¹ . This compound was used without further purification.

O- (2- (2-methoxyethoxy) ethyl) carbonyl-Nα-Cbz-L
-Preparation of tyrosine. Compound 6. Scheme II . Na-Cbz-L-tyrosine (5.00 g, 15.9 mmol) was added to water (10
Dissolved in NaOH (0.634 g, 15.9 mmol) in 0 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 at this temperature for 1 h. The mixture was then stirred for another 3 hours at ambient temperature. The solution was acidified with 1M HCl and extracted with ethyl acetate. Organic layer is M
Dry over gSO ₄ and remove the solvent under reduced pressure to give the product as a yellow oil (
6.42 g, 91%). This compound was used without further purification. _{FTIR (CH 2 C1 2);} 1764,1725cm -1. _{^{1 H NMR (CDC1 3):}} δ 3.39 (s, 3H), 3.57-3.79 (in, 8H), 4.38
(M, 2H), 4.63 (s, 1H), 5.05 (s, 2H), 6.93-7.
57 (m, 9H).

Preparation of O- (2- (2-methoxyethoxy) ethyl) carbonyl-L-tyrosine NCA . Compound 7. Scheme II. O- (2- (2-methoxyethoxy) ethyl) carbonyl-Nα-Cbz-L
-Tyrosine (6.43 g, 14.3 mmol) was dissolved in CH ₂ Cl ₂ (150 mL) and further (α, α-dichloromethyl methyl ether (2.46 g, 21.5 m
mol) was added to the solution. The mixture was then refluxed for 40 hours, after which the solvent was removed to give the product as a yellow oil (3.42g, 70.1%).
FTIR (THF); 1790, 1855 cm ^-1 . ¹ H NMR (CDC1 _3):
δ 7.04 (s, 4H), 4.53 (m, 1H), 4.31 (m, 2H), 3
． 71 (m, 2H), 3.59 (m, 2H), 3.50 (m, 2H), 3.30
(S, 3H), 3.09-2.97 (m, 2H). ¹³ C NMR (CDC1 ₃₎
: Δ 169.64, 153.73, 152.07, 150.55, 132.3
0, 130.74, 121.60, 71.84, 70.52, 68.89, 67
． 92, 59.00, 58.81, 36.94.

[0191] Preparation of Poly (O- (2- (2- methoxyethoxy) ethyl) carbonyl -L- tyrosine down). Compound 8. Scheme II . O- (2- (2-methoxyethoxy) ethyl) carbonyl-L-tyrosine NC
A (3.42 g, 10.0 mmol) was dissolved in THF (10 mL). Sodium t-butoxide (9.6 mg, 0.10 mmol) was then added. The resulting solution was stirred overnight to give the polymer as an off-white precipitate, which was isolated by washing with diethyl ether (50 mL) (1.94 g, 65
%). FT-IR (THF): 1660, 1547 cm ^-1 .

Preparation of S- (2- (2-methoxyethoxy) ethyl) carbonyl-L-cysteine . Compound 9. Scheme II . L-Cysteine hydrochloride (2.00 g, 12.7 mmol) was dissolved in 1M aqueous sodium hydrogen carbonate solution (25.4 mL) and this solution was diluted with water (75 mL). The solution was cooled with an ice bath and then coated with ether (50 mL). (2- (2-methoxyethoxy) ethyl) chloroformate (2.31 g,
(12.7 mmol) was added at once and stirred vigorously at 0 ° C. for 1 hour. Temperature 10
The temperature was raised to 0 ° C, and the mixture was further stirred for 2 hours. The solvent was then removed under reduced pressure. The solid formed was washed with methanol and the methanol layer was evaporated to give the product as a white oil (2.21 g, 65%). FTIR: 1709 cm ^-1 . ¹ H NMR (D ₂ O): δ 4.43 (m, 1H)
4.24 (m, 2H), 3.83-3.47 (m, 8H), 3.41 (s, 3)
H).

Preparation of S- (2- (2-methoxyethoxy) ethyl) carbonyl-L-cysteine N CA. Compound 10 . Scheme II . S- (2- (2-Ethoxymethoxy) ethoxy) carbonyl-L-cysteine (2.21 g, 8.26 mmol) was added to THF (100 mL) and COCl ₂ (.
5.13 ml of a 1.93M solution in toluene). The mixture was stirred at ambient temperature for 5 hours, then the solvent was removed under reduced pressure to give the product as a yellow oil (1.94 g, 80.1%). FTIR: 1791, 1862 cm ^-1 .

[0194] Preparation of poly (S- (2- (2- methoxyethoxy) ethyl) carbonyl -L- system in). Compound 11. Scheme II. O- (2- (2-Ethoxymethoxy) ethoxy) carbonyl-L-cysteine NCA (1.94 g, 6.61 mmol) was dissolved in THF (10 mL) and then sodium t-butoxide (6.4 mg, 0. 067 mmol) was added. The initially homogeneous solution was stirred overnight to give the polymer as an off-white precipitate,
Isolated by washing with diethyl ether (50 mL) (1.17 g,
70.9%). FTIR: 1635, 1517 cm ^-1 . In a round bottom flask, 2- [2- (2-methoxyethoxy) ethoxy] acetic acid (1
0 g, 58 mmol) and N-hydroxysuccinimide (7.5 g, 64 m
A dissolved mixture of (mol) in THF (about 300 mL) was cooled using an ice water bath.
Then dicyclohexylcarbodiimide (12 g, 58 mmol) was added with stirring. A white precipitate was observed to form after 5 minutes and the reaction mixture was then left in the refrigerator (4 ° C.) for 16 hours. The white precipitate dicyclohexylurea was removed by filtration and the filtrate was concentrated in vacuo to give an oil. The crude product was then dissolved in a small amount of THF (about 10 mL) and the resulting suspension was filtered to remove the precipitate. This procedure was repeated until a clear solution was obtained by dissolving in THF. Removal of residual THF in vacuo gave the product oil (9.
0 g, 59%). _{^{1 H NMR (CDCl 3):}} δ 4.49 (s, -OC (O) C H 2 O-, 2H)
, 3.77 (m, -OC (O ) CH 2 OC H 2 C H 2 O-, 2H), 3.65 (m
_{, -OCH 2 C H 2 CH 2} -, 4H), 3.52 (m, -OCH 2 C H 2 OCH 3, 2
H), 3.34 (s, -OCH 2 OC H 3, 3H), 2.82 (s, -C (O) C H 2 C H 2 C (O) -, 4H).

Preparation of Nε-2- [2- (2-methoxyethoxy) ethoxy] acetyl-Nα-CB ZL- lysine. Compound 2. Scheme III. Nα-CBZ-L- lysine (4.9 g, 17 mmol) and (THF: H ₂ O (
75 mL: 75 mL)) with respect to the middle NaHCO ₃ (2.0g, 23mmol) mixture was added THF (10 mL) in 1 (3.2g, 12mmol). 20 ° C
After stirring at rt for 1 h, THF was removed in vacuo. The product was converted into ethyl acetate (2 x 5
(0 mL), the organic fractions were combined and the solvent was removed in vacuo leaving a white solid. The crude product was recrystallized from MeOH and diethyl ether to give 2 as white crystals (3.0 g, 59%). MP = 115-117 (C. ¹ H NMR (CDCl ₃ ): δ 7.25 (m, −C)
_{H 2 C 5 H 5, 5H} ), 5.18 (s, -C H 2 C 5 H 5, 2H), 4.65 (t, -
NHC H (R) C (O ) OH, 1H), 3.72 (m, -NHCH ((CH 2) 3 C H 2 C (O) R) C (O) - + - O (C H 2 CH _{2 O) 2 C H 3,} 15H)
, 1.70 (m, -NHCH (( C H 2) 3 CH 2 C (O) R) C (O) -, 6H
).

Preparation of Nε-2- [2- (2-methoxyethoxy) ethoxy] acetyl-L-lysine -N-carboxylic acid anhydride. Compound 3. Scheme III. To a solution of 2 (4.9 g, 11 mmol) in anhydrous CH ₂ Cl ₂ (125 mL) under nitrogen was added 1,1-dichlorodimethyl ether (1.5 mL, 17 mmol).
) Was added. The solution was then heated at reflux for 20 hours, then the solvent was removed in vacuo. The crude oil was crystallized from THF and hexane to give 3 as white crystals.
Was obtained (2.8 g, 75%). _{^{1 H NMR (CDCl 3):}} δ 7.68 (br s, -N H, 1H), 7.3
5 (br s, -N H, 1H), 4.30 (t, -NHC H (R) C (O) O-
, 1H), 3.15 (m, -NHCH ((CH 2) 3 C H 2 C (O) R) C (O)
_{- + - O (C H 2} C H 2 O) 2 C H 3, 15H), 1.70 (m, -NHCH
_{((C H 2) 3 C} H 2 C (O) R) C (O) -, 6H. FTIR (THF): 18
56cm ^-1 (vCO, anhydrides s), 1789cm ^-1 (vCO, anhydrides, vs)
, 1677 cm ^-1 (vCO, amide, s).

Poly (Nε-2- [2-methoxyethoxy) ethoxy] acetyl-L-lysine ). Compound 4. Scheme III. 3 (730 mg, 2.2 mmol) in a dry box with THF (15 mL).
And was placed in a 75 mL reaction tube which can be sealed with a Teflon stopper. An aliquot of 2,2′-bipyridyl) Ni (1,5-cyclooctadiene) (600 μL of 36 mM THF solution) was then added to the flask via syringe. Add stir bar, seal flask, remove from drying box, isothermal 25 ° C
The mixture was stirred in the bath for 24 hours. The polymer was isolated by adding the reaction mixture to diethyl ether to precipitate the polymer. The polymer was then dissolved in THF and reprecipitated by addition to diethyl ether. The polymer was dried in vacuum to give 4 as a white fibrous solid (550 mg, 87% yield). Polymer 6
GPC in 0.1M LiBr in DMF at 0 ° C. was Mn = 101,000;
Mw / Mn = 1.21. ^{FTIR (THF): 1672cm -1 (} vCO, ^{amides, s), 1650cm -1 (vCO} , amide ^{l, br vs), 1538cm -1} (vCO, amide ll
, Br s).

Example 6- Synthesis of Self-Assembled Amphiphilic Block Copolypeptides for Biomedical Applications In the following example, we describe the general structure: (EG-Lys) _x- (insoluble block) _y. And the (EG-Lys) _x- (insoluble block) _y- (EG-Lys) _z , where x, y, and z represent the number of amino acids in each domain, and the di- and triblock copolyesters. The peptide was prepared.

Sample synthesis of poly (Nε-2- [2- (2-methoxyethoxy) ethoxy] acetyl-L- lysine) -block- (L-leucine / L-valine) diblock copolymer Nε-2 [2 -(2-Methoxyethoxy) ethoxy] acetyl-L-lysine)
-N-carboxylic acid anhydride, EG-Lys NCA, 100 mg was dissolved in anhydrous THF (3 mL) in a dry box and sealed with a Teflon plug 15 m.
L was placed in a reaction tube. Add an aliquot of (PMe ₃ ) ₄ ) Co to a reaction tube containing a stir bar (
2.37 mg in 1 ml THF) was added. The flask was sealed and stirred overnight to form a poly (EG-Lys) block. L-Leucine-N-carboxylic acid anhydride Leu NCA (5 mg), L-valine-N-carboxylic acid anhydride Val NC
A mixture of A (1 mg) was dissolved in anhydrous THF (1 mL) and then added to the reaction flask. After stirring for an additional 16 hours, the block copolymer was removed from the reaction mixture by removing the solvent in vacuo and the residue was resuspended in double purified water to give a molecular weight cutoff of 1
This solution was isolated and purified by dialyzing this solution against 4 liters of double purified water for 8 hours using a 000 Spectrapore dialysis membrane. This dialysis was repeated twice, and the copolymer was isolated by freeze-drying the solution (yield: 76 mg.
). GPC analysis of this polymer: Mn = 39,000 and Mw / Mn = 1.2
. FTIR (THF): 1650 cm ^-1 (vCO, amide 1 vs), 1540
cm ⁻¹ (vCO, amide 11 s).

Poly (Nε-2- [2- (2-methoxyethoxy) ethoxy] acetyl-L- lysine) -block- (L-leucine / L-valine) -block- (Nε-2- [2- ( 2-Methoxyethoxy) ethoxy] acetyl-L-lysine triblock copolymer sample synthesis Nε-2 [2- (2-methoxyethoxy) ethoxy] acetyl-L-lysine)
-N-carboxylic anhydride, EG-Lys NCA, 50 mg dissolved in anhydrous THF (3 mL) in a dry box and sealed with Teflon stopper 15 mL
It was put in the reaction tube of. An aliquot of (PMe ₃ ) ₄ ) Co (T
2.37 mg in 1 ml HF) was added. The flask was sealed and stirred overnight to form a poly (EG-Lys) block. L-Leucine-N-carboxylic acid anhydride Leu NCA (5 mg) and L-valine-N-carboxylic acid anhydride Val N
A mixture of CA (1 mg) was dissolved in anhydrous THF (1 mL) and then added to the reaction flask to form a center block. After stirring for another 16 hours, EG-
Lys NCA (50 mg) was dissolved in anhydrous THF (1 mL), placed in the reaction tube, and stirred for additional 16 hours to obtain the final poly (EG-
Lys) blocks were formed. Remove the solvent from the reaction mixture in vacuo, resuspend the residue in double purified water and dialyz this solution against 4 liters of double purified water for 8 hours using a Spectrapore dialysis membrane with a molecular weight cutoff of 1000. The copolymer was isolated and purified by. This dialysis was repeated twice, and the copolymer was isolated by freeze-drying the solution (yield: 78 mg). GPC analysis of this polymer: Mn = 41,000 and Mw / Mn = 1.1. FTIR (THF): 1650 cm ^-1 (vCO, amide 1 vs), 1540
cm ⁻¹ (vCO, amide 11 s).

Vesicle formation Spontaneous vesicle formation from di- and triblock copolypeptides was observed by light microscopy after first dissolving in water to give a milky suspension. The size of the vesicles varied greatly, with diameters up to a few microns. Smaller vesicles were prepared by sonicating a solution of large vesicles, similar to the method of manipulating lipid vesicles. The polymer was typically dissolved in double purified water at a concentration of 5 mg / mL and then sonicated with a Via Cell sonicator for 9 minutes at a power of 13 watts. The sonication was repeated 3 times, after which the opalescent suspension became a clear solution.

[0202] Small胞特analysis optical microscopy was performed on Nikon Optiphot2-POL. The initial sample was approximately 5 mg / ml in double purified water and 50 μ of the sample was placed on a glass slide and covered with a coverslip for observation. The optical dispersion is a Brookhavev with a laser output of 30 mwatt and a wavelength of 546 nm.
en Instruments Dynamic Light Scatter
ing (DLS) system. Samples were sonicated as above and added to DLS cuvettes. The sonicated vesicles ranged from 50 nm to 500 n depending on the copolymer chain length and amino acid composition (see, eg, Table 9 below).
It had an average diameter of up to m.

[0203]

[Table 9]

Although the present invention has been described in considerable detail with reference to certain preferred embodiments, other embodiments are possible. For example, the length of each domain, and the composition of the insoluble block domain, can be altered synthetically to alter the self-assembly structure formed. In addition, D- or L- or stereomixtures of amino acids can be used in these block copolymers to alter the secondary structure of the polypeptide or alter biological stability and interactions. Accordingly, the spirit and scope of the present invention is
It should not be limited to the description of the preferred embodiments contained herein.

[Brief description of drawings]

FIG. 1 PB of various initiators as a function of initiator concentration in Glu-NCA polymerization.
Comparison of LG molecular weight control ability. A, phenethylamine initiator; B, bipyNi (
COD) initiator; C, sodium tert-butoxide initiator; D, [M] ₀ /
[L] Theoretical molecular weight calculated from ₀ . All polymerizations were carried out in anhydrous DMF at 25 ° C for 1 day in a sealed tube. The molecular weight (Mn) is 0.1M LiBr in DMF at 60 ° C.
Determined by tandem GPC / light dispersion inside.

FIG. 2: Lys-NCA and Glu-N with bipyNi (COD) initiator in DMF.
2 is a chromatogram of PBLG _0.78 -b-PZLL _0.22 diblock copolymer prepared by sequentially adding CA. The polymer was directly injected into GPC and eluted through a 10 ⁵ Å and 10 ³ Å Phenomenex 5 μm column with 0.1 M LiBr in DMF at 60 ° C., using a Wyatt DAWN DSP light dispersion detector and Wyatt Optil.
Detected by ab DSP.

FIG. 3 shows two chemical reaction schemes related to the amino acid-derived Nickela cycle, which is an intermediate in nickel initiator-mediated polypeptide synthesis.

FIG. 4 shows four chemical reaction schemes associated with the amino acid-derived Nickela cycle, which is an intermediate in nickel initiator-mediated polypeptide synthesis.

[Figure 5]   1 shows the chemical structures of some ligands used in NCA polymerization reactions.

FIG. 6 shows the formation of amide-containing metallacycles by reaction of NCAs with metal initiators.

FIG. 7: MAL of leucylisoamylamide-C-terminal oligo (phenylglycine) s
DI-MS is shown. The partial broadening of the spectrum is shown in the upper right. Mass series were observed in TFA for (a) leucine-OH termination oligomers resulting from hydrolysis of terminal amides, (b) leucine isoamylamide termination oligomers resulting from intact end-functionalized chains, and (c) unfunctionalized chains. . For example, 9a represents the similarly labeled MH ⁺ ion of series a, series b, and series c of nona (phenylglycine). The various series of ions also form adducts with O atoms and CO ₂ and are labeled as such.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] June 18, 2001 (2001.6.18)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Claims

[Correction method] Change

[Contents of correction]

[Claims]

[Chemical 1] [Wherein M is any low-valent transition metal capable of undergoing a two-electron oxidative addition reaction, L is a Lewis base ligand; one of R1 and R2 is an amino acid side group, The others are hydrogen; and R3 is any functional end group that can be attached to a primary amine group.

[Chemical 2] [Wherein M is a low-valent transition metal; L is a Lewis base ligand; one of R1 and R2 is an amino acid side group, the other is hydrogen; and R3 is a primary Any functional end group other than hydrogen that can be attached to an amine group, amino acid side groups or polyamino acid chains, wherein at least two consecutive amino acid side groups are the same] Amide-containing metallacycle.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C07F 17/02 C07F 17/02 (31) Priority claim number 60/187, 488 (32) Priority date 2000 March 7, 2000 (3.7.3) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 193,054 (32) Priority date March 29, 2000 (2000. 3.29) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K) E, LS, MW, SD, SL, Z, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, 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, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Yu, Miuer USA 95164 San Jose, California. Oh. Box 640657 (72) Inventor Carton, Scott, A .. United States 93101 Santa Barbara Laguna Street 1515 Apartment California 6 (72) Inventor Hwang, Junjung United States 93117 Goleta Cypress Walk, California 766 Number M (72) Inventor Vilasta, Michael, Dee. United States 93117 Goleta, California State Brego Road 6639 F Term (reference) 4H006 AA01 AA03 AB46 AB82 AB84 BP10 BV11 BV22 4H050 AA01 AA03 AB82 AB84 WB11 WB14 WB16 WB21 4J001 DA01 DB01 DB02 DB04 DC12 DD01 DD07 DD14 DD20 JEAB GA40

Claims (28)

[Claims] 1. A quantity of alloc-amino acid amide is added to a low valent transition metal-
A method of making an amide-containing metallacycle which is combined with an initiator molecule containing a Lewis base ligand complex to form an amide-amidate metallacycle. 2. The amide-amidate metallacycle has the general formula: Where M is a low valence transition metal and L is a Lewis base ligand; R1
And one of R2 is an amino acid side group and the other is hydrogen; and R3 is any functional end group capable of being attached to a primary amine group). Method. 3. The alloc-amino acid amide has the general formula Alloc-NH—CH (R ′) C in which R ′ is an amino acid side group and R ″ is a functional end group.
The method of claim 1 having (O) NHR ". 4. The side chain functionality (fun) of a naturally occurring L-amino acid having any R ′ group.
cationality), the side chain functionality of any corresponding D-amino acid, or a non-naturally occurring synthetic amino acid side chain, provided that any reactive functionality of said side chain groups eliminates their reactivity. The method of claim 3, wherein the method is properly protected. 5. The method of claim 3, wherein the R "group is a peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer chain, small molecule therapeutic or chemical linker that attaches the polypeptide to another molecule. 6. The low-valent transition metal is zero-valent nickel, and the zero-valent nickel-Lewis base complex is LNi (1,5-cyclooctadiene), wherein L is 2,2'-bipyridine (bpy), 1,10-
Phenanthroline (phen), 1,2-bis (dimethylphosphino) ethane (
2. The method of claim 1 selected from the group consisting of dmpe) and 1,2-bis (diethylphosphino) ethane (depe). 7. A general formula: Wherein M is a low valent transition metal; L is a Lewis base ligand; one of R1 and R2 is an amino acid side chain group and the other is hydrogen; and R3 is a primary amine group. A 5-membered amide-containing metallacycle containing a molecule of any functional end group that can be attached. 8. R1 or R2 is alanine, arginine, asparagine,
Claims containing side chains of amino acids selected from the group consisting of aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine. Item 7. The amide-containing metallacycle according to Item 7. 9. R1 or R2 is an oligo (ethylene glycol) functionalized (
An amide-containing metallacycle according to claim 7, comprising a side chain of an amino acid selected from the group consisting of EG-) cysteine, EG-lysine, EG-serine, and EG-tyrosine. 10. An amide-containing metallacycle according to claim 7, wherein R3 is a peptide, oligosaccharide, oligonucleotide, fluorescent molecule, polymer chain, small molecule therapeutic or chemical linker that connects the polypeptide to another molecule. . 11. A soluble block polypeptide is formed by combining (1) an amount of an oligo (ethylene glycol) functionalized amino acid-N-carboxylic acid anhydride (EG-aa-NCA) monomer with an initiator molecule. To do; and (2
) A method for producing an amphipathic block copolypeptide comprising the step of attaching an insoluble block by combining the soluble block with a composition comprising at least one other amino acid NCA monomer. 12. The method according to claim 11, wherein the amino acid component of EG-aa-NCA is lysine, serine, cysteine, or tyrosine. 13. The method of claim 11, wherein the insoluble block comprises a mixture of amino acids. 14. A method of adding an amino acid-N-carboxylic acid anhydride (NCA) monomer to a soluble block polypeptide, combining the NCA monomer with the soluble block polypeptide and adding the NCA monomer to the polypeptide. Wherein the soluble block has one or more oligo (ethylene glycol) functionalized amino acid residues. 15. An amphipathic composition comprising a soluble block polypeptide having one or more oligo (ethylene glycol) linked amino acid residues and an insoluble block consisting essentially of nonionic amino acid residues. Block copolypeptide. 16. An insoluble block containing (1) a soluble block polypeptide having EG-lysine residues; and (2) a mixture of 2-3 different types of amino acid components in a statistically random sequence. Amphiphilic block copolypeptides, including polypeptides. 17. An amphipathic block copolypeptide composed of at least three blocks wherein one or more of said blocks is a soluble block polypeptide and another block is an insoluble block polypeptide. 18. A method of forming vesicles, which comprises the step of suspending the amphipathic block copolypeptides of claim 15 in an aqueous solution and allowing the copolypeptides to spontaneously self-assemble into vesicles. 19. The suspension vesicles are sonicated to obtain a diameter of about 50 nm.
19. The method of claim 18, comprising forming small vesicles having a size of from about 500 nm. 20. Vesicle-containing compositions comprising the amphipathic block copolypeptides of claim 15 and water. 21. A method for producing EG-functionalized amino acid monomers, which comprises a step of combining an ethylene glycol (EG) derivative with an amino acid having a reactive side chain group. 22. The EG derivative is of the general formula (CH ₃ OCH ₂ CH ₂ ) _n X, where n is about 1 to 3, and X is chloroformate, N-hydroxysuccinimidyl. 22. The method according to claim 21, having (succidimydyl) acetic acid and a reactive group selected from the group consisting of halides. 23. The method of claim 21, wherein the amino acid is selected from the group consisting of lysine, serine, cysteine, and tyrosine. 24. The method of claim 21, further comprising converting the EG functionalized amino acid to an NCA monomer. 25. An amount of EG-functionalized amino acid-N-carboxylic acid anhydride (N
CA) A method for producing a soluble block polypeptide comprising the step of combining a monomer with a low valent transition metal-Lewis base ligand complex to produce an EG-functionalized polyamino acid chain. 26. The method of claim 25, wherein the low valent transition metal is selected from the group consisting of nickel, palladium, platinum, cobalt, rhodium, iridium and iron. 27. The Lewis base ligands are pyridyl ligands, diimine ligands, bisoxazoline ligands, alkylphosphine ligands,
26. The method of claim 25, selected from the group consisting of arylphosphine ligands, tertiary amine ligands, isocyanide ligands and cyanide ligands. 28. The method of claim 25, wherein said EG-functionalized amino acid-N-carboxylic acid anhydride monomer is selected from the group consisting of EG-cysteine, EG-lysine, EG-serine, and EG-tyrosine. .