ES2360203T3 - CHEMICAL BINDING "SEUDO" - NATURAL. - Google Patents

Abstract

Method for synthesizing a desired polypeptide of the formula: aaNH2-Q-aax-aay-W-aaCOOH in which Q and W each indicate the optional presence of one or more additional amino acid residues, aaNH2 indicates the amino acid residue with terminal N of the polypeptide; aax and aay indicate the internal adjacent amino acid residues, which have the x and y side chains, respectively, and in which aay is a ribosomically unspecified amino acid residue and aaCOOH indicates the C-terminal amino acid residue of the polypeptide; the method comprising: (A) linking a first peptide having the formula: aaNH2-Q-aax-COSR, in which R is any group compatible with the thioester group, to a second peptide having the formula: Cys-W- aaCOOH, to thereby form the polypeptide: aaNH2-Q-aaX-Cys-W-aaCOOH; and (B) incubating said polypeptide in the presence of a Raa-X reagent, in which Raa-X is selected from the group consisting of X-CH2COOH, X- (CH2) 2COOH, X- (CH2) -C (O ) NH2, X-CH2OH, X-CHOHCH3, X-CH2CHOHCH3, X-CH2CH2OH, X- (CH2) 2NH-GP, X-CH2NH-C (NH2) 2, X- (CH2) 2NH-C (NH2) 2 , X-CH2CH3, X-CH2φ, X-φ-OH (for), X-CH2φ-OH (for), X-CH2φ-OPO3 (for), X-CH2-φ- (OH) 2 (goal for) , X-CH (COOH) 2, X-CH2-IM-GP, X-CH- (CH3) 2, X-CH2CH- (CH3) 2, X-CH (CH3) -CH2-CH3, X-CH2- CH (CH3) -CH2-CH3, in which X is I or Br, and X-φ, in which X is F, and X-CH2-IN, in which X is F, I or Br, in the that φ indicates a benzyl group, IN indicates an indole group, IM indicates an imidazole group and GP indicates a protective group; said incubation being under conditions sufficient to form said desired polypeptide.

Description

Field of the Invention

The present invention relates to methods and compositions for extending the technique of natural chemical ligation in order to allow the binding of a wider range of peptides, polypeptides, through an amide bond. The procedures and uses for said proteins and derived proteins are disclosed.

Background of the invention

During the past 30 years, medical attention has increasingly focused on the possibility of using naturally produced proteins as therapeutic drugs for the treatment of diseases.

Recombinant DNA techniques have become the main procedure for the commercial production of many polypeptides and proteins due to the large amounts that can be produced in bacteria and other host cells. The production of recombinant proteins involves transfecting or transforming the host cells with DNA encoding the desired exogenous protein and the growth of the cells under conditions that favor the expression of the recombinant protein. E. coli and yeast are preferred as hosts because they can be made to produce recombinant proteins at high values (see, US Patent No. 5,756,672 (Builder et al.).

Numerous US patents have been published regarding the general bacterial expression of proteins encoded by recombinant DNA (see, for example, US patents 4,565,785; 4,673,641; 4,738,921; 4,795,706; 4,710 .473). Unfortunately, the use of recombinant DNA techniques has not been achieved in all cases. Under some conditions, certain heterologous proteins expressed in large quantities in bacterial hosts precipitate within the cells in dense aggregates, recognized as bright spots, visible within the cell membrane under a phase contrast microscope. These precipitated protein aggregates are called "refractive bodies", and can constitute a significant part of the total cell proteins (Brems et al., Biochemistry (1985) 24: 7662).

The recovery of the proteins of these bodies has presented numerous problems, such as the separation of the protein embedded within the cell from the cellular material and the proteins that house it, and how to recover the proteins of the inclusion body in biologically active For general magazine articles on refractive bodies see Marston, above, Mitraki and King, Bio / Technology, 7: 690 (1989); Marston and Hartley, Methods in Enzymol., 182: 264-276 (1990); Wetzel, "Protein Aggregation In Vivo: Bacterial Inclusion Bodies and Mammalian Amyloid," in Stability of Protein Pharmaceuticals: In Vivo Pathways of Degradation and Strategies for Protein Stabilization, Ahern and Manning (Eds.) (Plenum Press, 1991); and Wetzel, "Enhanced Folding and Stabilization of Proteins by Suppression of Aggregation In Vitro and In Vivo," in Protein Engineering-A Practical Approach; Rees, A. R. et al. (Eds.) (IRL Press at Oxford University Press, Oxford, 1991). An alternative route of bioactive protein production is therefore necessary.

An alternative to recombinant protein production involves the use of the principles of organic chemistry to synthesize proteins. Existing procedures for chemical protein synthesis include step-by-step solid phase synthesis, and condensation of the fragment either in solution or in solid phase. Merrifield's classic step-by-step solid phase synthesis involves the covalent bonding of an amino acid corresponding to the amino acid of the carboxy terminal of the desired peptide chain to a solid support and the extension of the polypeptide chain to the amino terminus by step-by-step coupling of the activated amino acid derivatives having activated carboxyl groups. After completion of assembly of the fully protected solid phase linked peptide chain, the solid phase covalent peptide bond is cleaved by appropriate chemistry and the protecting groups are removed to give the polypeptide product.

Unfortunately, there are many drawbacks to the step-by-step solid phase synthesis process, including the formation of the solid phase joined by products that come from the incomplete reaction in the coupling and deprotection stages in each cycle. The larger the peptide chain, the greater the challenge to obtain well-defined high purity products. The synthesis of proteins and large polypeptides by this route is an extensive and laborious task.

The solid phase fragment condensation method (also known as segment condensation) was designed to overcome the difficulties in obtaining long polypeptides by the solid phase step-by-step synthesis procedure. The segment condensation process involves the preparation of several peptide segments by the solid phase step-by-step synthesis procedure, followed by cleavage of the solid phase and purification of these protected segments to the maximum. The protected segments are condensed one by one to the first segment, which is attached to the solid phase. Frequently, however, technical difficulties are encountered in many of the stages of condensation of solid phase segments. See E. Atherton et al., "Solid Phase Fragment Condensation - The Problems", in Innovation and Perspectives in Solid Phase Synthesis 11-25 (R. Epton, et al., 1990). For example, the use of protecting groups in segments to block unwanted binding reactions can often make the poorly soluble protected segments interfere with the effective activation of the carboxyl group. The limited solubility of the protected segments can also interfere with the purification of the protected segments. See K. Akaji et al., Chem. Pharm. Bull. (Tokyo), 33: 184-102 (1985). Protected segments are difficult to characterize with respect to purity, covalent structure, and are not suitable for high resolution analytical ESMS (mass spectrometry with electroatomization) (based on charge). Racemization of the remainder of the C-terminal of each activated peptide segment is also a problem, except if the linkage is performed on glycine residues. On the other hand, cleavage of the polypeptide bound to the solid phase, completely assembled from the solid phase and the removal of the protective groups can often require angry chemical procedures and prolonged reaction times that cause degradation of the fully assembled polypeptide.

Condensation of segments can be carried out in solution instead of in solid phase. See H. Muramatsu et al., Biochem. and Biophys. Res. Commn. 203 (2): 1113-1139 (1994). However, the condensation of segments in solution requires the purification of segments before ligation as well as the use of protective groups in a range of different functional side chain groups to prevent multiple unwanted side reactions. In addition, solution ligation does not allow for easy purification and washing steps provided by solid phase linkages. In addition, the limitations regarding the solubility of protected peptide segments and intermediate reaction products of protected peptides worsen.

Chemical ligation of peptide segments has been explored in order to solve the solubility problems frequently encountered with maximally protected peptides. Chemical ligation involves the formation of a selective covalent bond between a first chemical component and a second chemical component. The exclusive, interreactive functional groups present in the first and second components can be used to perform the chemoselective ligation reaction. For example, chemical ligation of peptides and polypeptides involves the chemoselective reaction of segments of peptides or polypeptides that carry amino acid residues with C-terminal and N-terminal, exclusive, interreactive. Several different chemistries have been used for this purpose, the examples of which include natural chemical ligation (Dawson et al., Science (1994), 266: 776-779; Kent et al., WO 96/3478; Kent et al., WO 98/28434), oxime-forming chemical ligation (Rose, et al., J. Amer. Chem. Soc. (1994), 116: 30-34), thioester-forming ligation (Schnölzer, et al., Science (1992), 256: 221-225), thioether forming ligation (Englebretsen, et al., Tet. Letts. (1995), 36 (48): 8871-8874), hydrazone forming ligation (Gaertner, et al. , Bioconj. Chem. (1994), 5 (4): 333-338), and thiazolidine-forming ligation and oxazolidine-forming linkage (Zhang, et al., Proc. Natl. Acad. Sci. (1998), 95 ( 16): 9184-9189; Tam, et al., WO 95/00846; US Patent No. 5,589,356) or by other procedures (Yan, LZ and Dawson, PE, "Synthesis of Peptides and Proteins without Cysteine Residues by Native Chemical Ligation Combined with Desulfurization, "J. Am. Chem. Soc., 2001; 123, 52 6-533; Gieselnan, et al., Org. Lett., 2001, 3 (9): 1331-1334: Saxon, et al., "Traceless" Staudinger Ligation for the Chemoselective Synthesis of Amide Bonds., Org. Lett., 2000, 2, 2141-2143).

Of these procedures, only the natural chemical ligation method provides a ligation product with a natural amide bond (i.e., peptide) at the ligation point. The methodology of the original chemical ligation (Dawson, et al., Above and WO 96/34878) has demonstrated a strong methodology for generating a natural amide bond at the point of ligation. Natural chemical ligation involves a chemoselective reaction between a first peptide or polypeptide segment that has an α-carboxythioester moiety at terminal C and a second peptide or polypeptide that has a cysteine moiety at terminal N. A thiol exchange reaction provides an intermediate attached to the initial thioester, which is spontaneously reconfigured to provide a natural amide bond at the ligation point while regenerating the thiol of the cysteine side chain. The main drawback of the original natural chemical ligation approach is that it requires a cysteine in the N terminal, that is, it only allows the coupling of segments of peptides or polypeptides that possess a cysteine in the terminal

N.

Notwithstanding this drawback, the natural chemical ligation of peptides with other amino acids with N-terminal apart from cysteine has been reported (WO 98/28434). In this context, ligation is performed using a first peptide or polypeptide segment having an α-carboxy thioester at terminal C and a second peptide or polypeptide segment having a thiol-substituted N- {auxiliary group} at terminal N , represented by the formula HS-CH2-CH2-O-NH- [peptide]. After ligation, the thiol-substituted N- {auxiliary group} is separated by cleavage of the HS-CH2-CH2-O-auxiliary auxiliary group to generate a natural amide bond at the ligation point. A limitation of this procedure is that the use of a mercaptoethoxy auxiliary group can successfully lead to the formation of an amide group only in a glycine residue. This produces a ligation product that at the time of cleavage generates a glycine residue at the position of the N-substituted amino acid of the second peptide or polypeptide segment. As such, this embodiment of the process is only suitable if the reaction ligation product containing a glycine residue in this position is desired, and in any case can be problematic with respect to ligation yields, stability of the precursors and the ability to remove the auxiliary group attached to O. Although other auxiliary groups may be used, for example the HSCH2-CH2-NH- [peptide], without limiting the ligation reaction to a glycine residue, said groups Auxiliaries cannot be removed from the bound product.

Therefore, a widely applicable and strong chemical ligation system is necessary that extends natural chemical ligation to a wide variety of amino acid, peptide, polypeptide, polymer and other molecule residues by means of an auxiliary group containing easily separable thiol and effective, and that couples said molecules together with a natural amide bond at the ligation point. The present invention contemplates these and other needs.

Brief description of the figures

Figure 1 illustrates the general reactions of pseudonatural chemical ligation.

Figure 2 illustrates the general method of preparing the synthetic bioactive proteins of the present invention.

Figure 3 represents the basic structure of a preferred type of synthetic erythropoietic stimulating proteins.

Figure 4 shows the general structure of SEP analogs permutated in a circle that have no free amino or carboxy terminal. The binding points to pPEG ("precision PEG", as described herein) are in the same position relative to the SEP analogs. The SEP analogs are in a circle form by an oxime link between the new positions of the N / C terminals such as P126C and A125X. The total molecular weight of the SEP analogs will be between approximately 50 and 80 kDa, depending on the pPEG used for the modification of a given analogue, with the P.M. Hydrodynamic mediated by estimated pPEG that is greater than approximately 100 kDa.

Figure 5 presents the basic structure of bioactive GCSF synthetic proteins. In the figure, "J" designates an unnaturally encoded moiety having a hydrophobic natural chain.

Figure 6 shows the structure of analogs of preferred synthetic RANTES. In the figure, NNY = nonaoyl, X = unnaturally encoded amino acid having a hydrophobic side chain, pPEG and FA = fatty acid.

Figure 7 schematically represents the formation of a branched chain pPEG polymer.

Summary of the invention

The present invention relates to methods and compositions for extending the technique of natural chemical ligation that allows the ligation of a wider range of peptides, polypeptides through an amide bond. The procedures and uses for said proteins and modified proteins are disclosed. The invention is particularly suitable for use in the synthesis of synthetic bioactive proteins optionally modified by polymer, and of pharmaceutical compositions containing said proteins.

In detail, the process of the invention provides a synthetic protein (especially a protein having a monomer molecular weight greater than about 25 kDa) containing a pseudoamino acid residue whose side chain has the formula: -S-Raa, in which Raa is an optionally substituted terminal fragment of an amino acid side chain specified in the ribosome, or an analogue of the terminal fragment of an amino acid side chain specified in the ribosome.

Specifically, said synthetic protein has a biological activity possessed by a bioactive protein produced in the ribosome.

The process further provides such synthetic proteins in which a variety of amino acid residues of the protein are attached to adjacent amino acid residues by an amide bond.

Such synthetic proteins are disclosed in which the pseudoamino acid residue is an amino acid residue with D configuration or is an amino acid residue with L configuration, or in which said synthetic proteins contain both pseudoamino acid residue (s) in configuration D and rest (s) of pseudoamino acid (s) in configuration L.

The process also provides said synthetic proteins in which the pseudoamino acid moiety is selected from the group consisting of a pseudoarginine; a pseudoasparagine; a pseudo aspartate; a pseudodopamine; a pseudoglutamate; a pseudoglutamine; a pseudohistidine; a pseudoisoleucine; a pseudoleucine; a pseudolysin; a pseudomethionine; a pseudophenylalanine; a pseudo serine; a pseudotreonin; a pseudotryptophan; a pseudotyrosine and a pseudovaline.

The method further provides such synthetic proteins in which the bioactive protein specified in the ribosome is a mammalian protein (for example, a human, ape, bovine, murine, porcine, sheep protein

or equine).

The method further provides such synthetic proteins in which the protein has a bioactivity selected from a protein receptor or a fragment thereof, from a protein receptor ligand or a fragment thereof or from a cytokine (especially in which the cytokine from the group consisting of an interleukin, a lymphokine, a RANTES protein, an erythropoiesis stimulating protein, a tumor necrosis factor (TNF) an interferon, a growth factor and a single peptide hormone).

Synthetic proteins SEP-0, SEP-1 and SEP-3 are disclosed.

All synthetic proteins mentioned in which one or more of its amino acid residues are modified by one or more polymer adducts are disclosed, and particularly in which the synthetic protein comprises one or more amino acid residues that are modified by one or more polymer adducts in the amino acid residue (s) corresponding to at least one of the glycosylation points of a bioactive protein encoded by ribosomes.

A pharmaceutical composition with homogeneous molecules comprising a synthetic protein is disclosed, in which the protein possesses a biological activity that mimics a biological activity associated with a bioactive mammalian protein specified in the ribosome, and has a higher monomer molecular weight. at approximately 25 kDa, and contains a pseudoamino acid residue whose side chain has the formula: -S-Raa, in which Raa is selected from the group consisting of an optionally constituted terminal fragment of an amino acid side chain specified in the ribosome , or an analogue thereof.

A method of treating a human disease or condition is disclosed, which comprises administering to an individual in need of such treatment an effective amount of a pharmaceutical composition comprising one or more pharmaceutical compositions of homogeneous molecules each comprising a synthetic protein, wherein the synthetic protein has a molecular weight of the monomer greater than about 25 kDa and contains a pseudoamino acid residue whose side chain has the formula: -S-Raa, in which Raa is selected from the group consisting of a terminal fraction optionally substituted for an amino acid side chain specified in the ribosome, or an analogue thereof; the synthetic protein presenting a biological activity that mimics a biological activity of a bioactive human protein receptor specified in the ribosome or a fragment thereof, a protein receptor ligand or a fragment thereof, or a cytokine.

The invention provides a method for synthesizing a desired polypeptide, and the desired polypeptide is prepared by said method, wherein the desired polypeptide has the formula:

aaNH2-Q-aaX-aaY-W-aaCOOH

wherein Q and W indicate the optional presence of one or more additional amino acid residues, aaNH2 indicates the N-terminal amino acid residue of the polypeptide; aaX and aaY indicate internal adjacent amino acid residues having the x and y side chains, respectively, and in which aaY is a ribosomically unspecified amino acid residue and aaCOOH indicates the C-terminal amino acid residue of the polypeptide; Understanding the procedure:

(TO)

binding a first polypeptide having the formula: aaNH2-Q-aaX-COSR, in which R is any group compatible with the thioester group, which comprises, in a non-limiting manner, aryl, benzyl and alkyl groups, to a second peptide having the formula: Cys-W-aaCOOH, to thereby form the polypeptide: aaNH2-Q-aaX-Cys-W-aaCOOH; Y

(B)

incubate the polypeptide in the presence of a Raa-X reagent, in which Raa-X is selected from the group consisting of X-CH2COOH, X- (CH2) 2COOH, X- (CH2) -C (O) NH2, X -CH2OH, X-CHOHCH3, X-CH2CHOHCH3, X-CH2CH2OH, X- (CH2) 2NH-PG, X-CH2NH-C (NH2) 2, X- (CH2) 2NH-C (NH2) 2, X-CH2CH3 , X-CH2φ, X-φ-OH (for), X-CH-2φ-OH (for), X-CH2φ-OPO3 (for), X-CH2-φ- (OH) 2 (target for), X -CH (COOH) 2, X-CH2-IMPG, X-CH- (CH3) 2, X-CH2CH- (CH3) 2, X-CH (CH3) -CH2-CH3, X-CH2-CH (CH3) -CH2-CH3, in which X is I or Br, and X-φ, in which X is F, and X-CH2-IN, in which X is F, I or Br, in which φ indicates a benzyl group, IN indicates an indole group, IM indicates an imidazole group and PG indicates a protective group; the incubation being sufficient to form the desired polypeptide.

The invention further provides the embodiment of said process, in which the rest of amino acids aa and is selected from the group consisting of a pseudoasparagine; a pseudo aspartate; a pseudodopamine; a pseudoglutamate; a pseudoglutamine; a pseudohistidine; a pseudoisoleucine; a pseudoleucine; a pseudolysin; a pseudomethionine; a pseudophenylalanine; a pseudo serine; a pseudotreonin; a pseudotryptophan; a pseudotyrosine and a pseudovaline.

Description of the preferred embodiments

The present invention relates to methods and compositions for extending the technique of natural chemical ligation that allows the ligation of a wider range of peptides, polypeptides through an amide bond, thereby facilitating the chemical synthesis of said molecules. The procedures and uses for said proteins and modified proteins are disclosed. The invention is particularly suitable for use in the synthesis of synthetic bioactive proteins, optionally modified with polymer, preferably having a molecular weight greater than about 25 kDa, and of pharmaceutical compositions containing said proteins.

I. Chemical synthesis of peptides and bioactive proteins

In general, the synthesis of bioactive proteins and peptides uses the step-by-step solid phase peptide synthesis protocol of the "Merrifield" chemistry developed in the early 1960s, using conventional automated peptide synthesizers. These syntheses can use solid phase or solution ligation strategies. Although such chemistry can be easily used to produce many polypeptides, it is unsuitable for the production of proteins or large polypeptides due to the associated yield losses, byproduct production and incomplete reactions. To study these limitations, chemical ligation techniques have been developed that allow one to bind preformed peptide fragments in order to achieve the synthesis of major polypeptides and proteins.

Chemical ligation involves the formation of a selective covalent bond between a first chemical component and a second chemical component. The functional, interreactive, exclusive groups present in the first and second components can be used to make the ligation reaction chemoselective. For example, chemical ligation of peptides and polypeptides entails the chemoselective reaction of segments of peptides or polypeptides that carry amino acid residues with C-terminal and N-terminal, interreactive, exclusive and compatible. Different chemistries have been used for this purpose, the examples of which include natural chemical ligation (Dawson et al., Science (1994), 266: 776-779; Kent et al., WO 96/34878; Kent et al., WO document 98/28434), oxime-forming chemical ligation (Rose, et al., J. Amer. Chem. Soc. (1994), 116: 30-34), thioester-forming ligation (Schnölzer, et al., Science (1992 ), 256: 221-225), thioether forming ligation (Englebretsen, et al., Tet. Letts. (1995), 36 (48): 8871-8874), hydrazone forming ligation (Gaertner, et al., Bioconj Chem. (1994), 5 (4): 333-338), and thiazolidine-forming ligation and oxazolidine-forming ligation (Zhang, et al., Proc. Natl. Acad. Sci. (1998), 95 (16) : 9184-9189; Tam, et al., WO 95/00846; US Patent No. 5,589,356) or by other methods (Yan,

L. Z. and Dawson, P. E., "Synthesis of Peptides and Proteins without Cysteine Residues by Native Chemical Ligation Combined with Desulfurization," J. Am. Chem. Soc., 2001; 123, 526-533; Gieselnan, et al., Org. Lett., 2001, 3 (9): 1331-1334: Saxon, et al., "Traceless" Staudinger Ligation for the Chemoselective Synthesis of Amide Bonds., Org. Lett., 2000, 2, 2141-2143).

When ligation involves the coupling of a polypeptide that possesses an N-terminal cysteine residue, the natural chemical ligation method is preferably used (Dawson et al., Science (1994), 266: 776-779; Kent et al., WO 96/34878; Kent et al., WO 98/28434; US Patent Application Serial No. 09 / 097,094). Natural chemical ligation involves a chemoselective reaction between a first peptide or polypeptide segment that has an α-carboxythioester moiety at terminal C and a second peptide or polypeptide that has a cysteine moiety at terminal N. An exchange reaction of thiol provides an intermediate attached to the initial thioester, which is spontaneously reconfigured to provide a natural amide bond at the ligation point while regenerating the thiol of the cysteine side chain. In many cases, the natural protein sequence will comprise cysteine residues suitably placed so that polypeptide fragments having a cysteine residue at the N-terminal can be synthesized and used in a natural chemical ligation reaction.

Although this methodology has proven to be practical, robust and useful for the synthesis of long polypeptides and proteins, its requirements for a cysteine residue at the point of ligation limit its application. Cysteine is one of the least common amino acids. A typical protein domain comprises 150 to 200 amino acids; Many proteins contain regions of more than 60 amino acids in length that do not contain any cysteine residues. A solution to this problem involves designing an unnatural protein in which one or more of the protein's natural amino acid residues have been replaced with cysteine residues, thus allowing the natural chemical ligation procedure to be used. This solution is often complicated by the unpredictable effects that the introduction of a cysteine residue may have on the structure or function of the protein. Said cysteine residues can be used to immobilize the synthesized protein covalently.

Two alternative solutions for this problem are disclosed that can be used separately or together. The first of these solutions (referred to herein as "pseudonatural chemical ligation") involves the use of unnatural pseudoamino acid residues at preselected positions in the peptides used in protein synthesis. The structures of said pseudoamino acids mimic both the cysteine structures and the structures of the naturally occurring amino acids in said preselected positions in the protein being synthesized. The second such solution (referred to herein as "prolonged natural chemical ligation") is described in US Patent Application Serial No. 60 / 231,339 (Kent, et al., Filed September 8, 2000), and implies ligating a first component comprising a carboxyl thioester, and more preferably an α-carboxyl thioester with a second component comprising a stable N-substituted acid, and preferably, Nα-substituted, amino or aryl thiol alkyl with 2 or chain chain 3 carbons

A. Pseudonatural chemical ligation

Pseudonatural chemical ligation refers to the thioalkylation of the cysteine side chains generated at the link points from the natural chemical ligation. A preferred aspect is the thioalkylation of the cysteine ligation points in which at least one peptide contains a natural cysteine having its thiol side chain protected with a suitable protecting group.

The thiol moiety of a cysteine group is modified in a desired side chain, for example, in the side chain of an amino acid specified in the ribosome, an analogue of said amino acid, or in an amino acid not specified in the ribosome. As used herein, an amino acid specified in the ribosome is an amino acid that is recognized by ribosomes in the process of protein translation and can be incorporated into a protein produced in the ribosome. There is considerable published literature describing chemical modifications of the thiol moiety of the cysteine side chain (see, for example, "Current Protocols in Protein Science", edited by: John E. Colligan et al., John Wiley & Sons, NY (2000 )). Kaiser, E.T. has described the conversion of side chains with cysteine residue to mimic the chemical properties of a natural amino acid side chain (see, for example, Kaiser, ET et al., "Chemical Mutation of Enzime Active Sites", Science, 2 November 1984; 226 (4674): 505-11). In addition, the use of a cysteine side chain to introduce a marker into a peptide or protein has been described. Modifications in the cysteine side chain are studied in Chemistry of Protein Conjugation and Crosslinking, S.S. Wong, (1991, CRC Press); Chemical Modification of Proteins, Gary E; Means et al., (1971, Holden-Day), Chemical Modification of Proteins: Selected methods and analytical procedures, Glazer, A. N. et al. (1975, Elsevier); Chemical Reagents for Protein Modification, R. L. Lundblad (1991, CRC Press). Tam et al. (Biopolymers, (1998), 46: 319-327) have disclosed the use of homocysteine (-CH2-CH2-SH) for non-natural chemical ligation, followed by thioalkylation using methyl pnitrobenzenesulfonate (methylation reagent) for convert the homocysteine side chain into a natural methionine side chain (-CH2-CH2-S-CH3). According to the present invention it is necessary to use protecting groups to prevent the destruction of the natural cysteines involved in the disulfide pairing by peptides containing at least one natural cysteine that it is not desired to convert. Suitable protecting groups are described below.

Although the pseudonatural chemical ligation method does not facilitate the simulation of the side chains of certain amino acids specified in the ribosome (for example, the glycine, alanine, valine and proline side chains) (the alanine side chain can be formed, however, by a desulfurization reaction (Yan,

L. Z. and Dawson, P. E., "Synthesis of Peptides and Proteins without Cysteine Residues by Native Chemical Ligation Combined with Desulfurization", J. Am. Chem. Soc., 2001; 123, 526-533), can be used to form side chains that mimic many amino acids specified in the ribosome or not encoded. The amino acids produced according to the pseudonatural chemical ligation method of the present invention will contain a thioether bond, and will not present any branching in beta (because all will include a methyl group in the beta position, that is, aa-βCH2-S-. In this way, the slope of the side chain structure can be conferred to the pseudoamino acid versions of the branched amino acids in beta, isoleucine and threonine, without having the beta geometry and its inherent limitations.

Significantly, the methods of the present invention can be used to form side chains of amino acids that are the same length as that of the amino acids specified in the ribosome, or are longer or shorter than said length. Such alteration in the length of the side chain can be used to stabilize (or destabilize) the three-dimensional configuration to increase the stability of the protein (or to increase the ability of the protein to alter its configuration) and thus accept a different range of substrates, inhibitors, receptors, ligands, etc. In comparison with those accepted by the natural protein. For example, Cys-CH2-SH + Br-CH2-COOH gives Cys-CH2-S-CH2-COOH (said "glutamic pseudo acid" has an additional atom in the side chain, namely a group -S-; alternatively, if used instead of aspartic acid, it will have two additional atoms in the side chain, that is a group -CH2-S-). Other side chains have the same number of atoms in the side chain, but differ by the inclusion of the thioether bond (-S-). For example, Cys-CH2-SH + Br-CH2-CH2-NH-PG, followed by the removal of GP from Cys-CH2-S-CH2-CH2-NH2. The resulting structure has no additional atoms in the side chain, but a group -CH2- is replaced with -S-. Methionine is another example herein, Cys-CH2-SH + I-CH2-CH3 gives Cys-CH2-S-CH2-CH3 (versus the natural met-structure of Met-CH2-CH2-S-CH3) . In this way the thioether is relocated. Arginine also: Cys-CH2-SH + Br-CH2-NH-CH ((- NH2) (= NH2 +)) gives Cys-CH2-S-CH2-NH-CH ((- NH2) (= NH2 +)). Preferably, the protection of aminoreactive groups can be used, particularly to construct pseudolysin to avoid unwanted side reactions. Once the thioalkylation reaction is carried out, the protecting group can be removed.

In general, when it is desired to mimic a natural protein as closely as possible, it is more preferred to use a pseudoamino acid molecule that has a side chain length that is the same length as the amino acid specified in the ribosome normally present in said position in the protein; it is even less preferred to use a pseudoamino acid molecule that has a side chain length that is that of an atom longer than that of an amino acid specified in the ribosome, and it is even less preferred to use a pseudoamino acid molecule that has a length of the side chain that is two atoms longer than that of an amino acid specified in the ribosome. In addition, it is preferred to select a cysteine ligation point that is in a position where genetic changes are not likely to destroy function or where amino acids are conserved at this point in related proteins. These points can be identified by alanine exploration, homology modeling and other procedures.

In pseudonatural chemical ligation, a peptide that contains a cysteine residue in the amino terminal is linked to a peptide that has a thioester in the carboxy terminal, as in natural chemical ligation. The thiol side chain of the cysteine is then reacted with a compound of the formula Raa-X, in which X is a good leaving group, and Raa is a group whose structure mimics the terminal part of the side chain of an amino acid specified in the ribosome or synthetic.

Significantly, pseudonatural chemical ligation reactions act with the natural L-configuration of the side chain with cysteine, or with the D-configuration. The use of the D-configuration can provide protease resistance at the ligation point, and of This mode may be desirable when it is desired to increase proteolysis stability. However, when using D-cysteine, the structure of the central axis on this side can be altered. Such alteration, in addition to protease resistance, may be desired to alter bioactivity. However, to minimize the impact on bioactivity, it is preferred to place the D-cysteine at the point of high flexibility, such as a messy area, such as in the messy loop that will be located on the surface of the resulting folded molecule, in a messy terminal of the molecule. Desirably, the pseudonatural chemical ligation reactions can be used to place large loaded side chains (for example, the Lys, Arg, Asp or Glu side chains) on the surface of the synthesized molecule.

Examples of good suitable leaving groups, X, include halogens, especially iodine and bromine. Examples of Raa groups include PO4, COOH, COO, CONH2, guanidinium, amino, alkyl, substituted alkyl, aryl, substituted aryl, imidazole, alkylated imidazole, indole or indole groups.

For the selection for which Raa should be used, it will depend on the amino acid side chain that is desired to be present in a certain position. Thus, for example, a desired polypeptide or protein having the amino acid sequence:

aaNH2-Q-aax-aay-W-aaCOOH

wherein Q and W each indicate the presence or optional absence of additional amino acid residues, and aax and aay indicate adjacent internal residues (with the x and y side chains, respectively), and aaNH2 and respectively indicate the remainder of the amino terminal (N -) and the rest of the carboxy (C-) terminal of the polypeptide or protein and is synthesized by preparing two peptide fragments:

aaNH2-Q-aaX-COSR and Cys-W-aaCOOH

wherein Cys indicates the replacement of a and cysteine and R is any group compatible with the thioester group, which comprises, in a non-limiting manner, aryl, benzyl and alkyl groups. Examples of R include thioesters of 3-carboxy-4-nitrophenyl, benzyl esters and esters of mercaptopropionic acid and leucine (see, for example, Dawson et al., Science (1994), 266: 776-779; Canne et al., Tetrahedron Lett. (1995) 36: 1217-1220; Kent et al., WO 96/34878; Kent et al., WO 98/28434; Ingenito et al., JACS (1999), 121 (49): 11369- 11374 and Hackeng et al., Proc. Natl. Acad. Sci. USA (1999), 96: 10068-10073). Other examples include dithiothreitol, or alkyl or aryl thioesters, which can be produced by intein-mediated biological techniques, which are also well known (see, for example, Chong et al., Gene (1997), 192: 277-281; Chong et al., Nucl. Acids. Res. (1998), 26: 5109-5115; Evans et al., Protein. Science (1998), 7: 2256-2264 and Cotton et al., Chemistry & Biology (1999) , 6 (9): 247-256; and then linking the fragments to form:

aaNH2-Q-aax-Cys-W-aaCOOH.

The ligated fragment is then reacted with Raa-X, in which Raa is a side group that mimics the structure of the side chain and. The reaction is carried out under conditions sufficient to convert the thiol group of the cysteine into a "pseudo" side chain. For example, if Raa is selected to be CH2-COOH or (CH2) 2COOH, then the reaction will lead to the formation of an amino acid residue that mimics the structure and function of aspartic acid ("pseudoAsp") or glutamic acid ("pseudoGlu "). As will be appreciated from the above description, a more complicated synthesis can be performed using more than two peptide fragments.

Natural chemical ligation involving "pseudoamino acids" according to the present invention significantly extends the application of the natural chemical bonding procedure for chemical protein synthesis. Natural chemical ligation involving "pseudoamino acids" uses the same chemistry in the ligation stage, forming a peptide bond in the Cys moiety with a thiol-containing side chain functionality; In a single second stage, the Cys natural thiol-containing side chain at the ligation point is converted by chemoselective reaction to give an unnatural side chain that contains the same functional group as a genetically encoded amino acid, but a non-side chain natural.

For example, cysteine at the ligation point can be reacted with bromoacetic acid (or other haloacetic acid) at neutral pH in water; only the unprotected thiol of the Cys at the ligation point reacts under these conditions to provide a polypeptide containing a S-carboxymethylated moiety at the ligation point, ie:

image 1

10 This unnatural amino acid contains a carboxyl moiety in the side chain, the same functional group as a glutamic acid or aspartic acid moiety. The S-carboxymethylated Cys is referred to herein as a "pseudoglutamic acid" ("pseudoglutamate", "pseudoglu") (where one more atom of the side chain is present and is the group -S- that forms a thioether bond) ( It can also be called "pseudo aspartic acid" where two more atoms

15 of the side chain are present, one of which is the -S- group that forms a thioether bond). For many proteins, the simple preservation of the loaded carboxylate side chain, even in this "pseudoamino acid" will be sufficient to preserve or provide desired properties to the protein. A key difference is that it functions as a natural chemical ligation point in a position devoid of a suitable cysteine.

The use of other reagents to modify the thiol of the side chain in the Cys residues at the ligation point (s) may provide other "pseudoamino acid" residues. For example, the reaction of thiol with a haloacetamide, for example, bromoacetamide, will give a "pseudoglutamine" at the ligation point, that is:

image 1

Also, the reaction with an N-protected haloalkylamine, for example,

image 1

30 followed by the removal of the amino protecting group will give a "pseudolysin" residue, that is:

image 1

Reaction of the remainder or of the Cys residues at the ligation point (s) with a suitable haloalkane, for example,

image 1

will provide an amino acid "pseudoleucine", that is:

image 1

Other halides containing aryl suitably selected, in which R may be H, OH, aryl, etc., for example,

image 1

they can also be used to generate pseudoamino acids corresponding to Phe, Tyr or Trp residues, that is:

image 1

A significant feature of this method is that cysteine residues that are not desired to be modified in the reactant segments are protected in the side chain, for example, as Cys (Acm), to avoid chemical modification of other Cys residues other than one (s) at the ligation point (s), or that any other Cys residues in the reacting segments are proposed for the simultaneous formation of identical "pseudoamino acids" by the chemical modification reaction after ligation. It will be appreciated that the same principles apply to the use of homocysteine at the point of ligation. It will also be appreciated that selenocysteine can be used at the point of ligation although the natural chemical ligation mediated by selenocysteine, and

20 converted by similar alkylation reactions to generate pseudoamino acids of the invention, when a selenoether group replaces the thioether group.

As used herein, the symbol φ indicates a benzyl group; IM indicates an imidazole group and IN indicates an indole group; GP indicates a protective group. Below is a summary of Raa side groups that can be used to synthesize peptides containing pseudoamino acid residues according to the present invention where X is a halogen (I, Br, Cl, F, with I and Br preferred for most, and F preferred for coupling in φ):

Basic Amino Acids:

30 Lys (without extra atoms)

-CH2-SH + X-CH2-CH2-NH-PG, followed by deprotection of -CH2-S-CH2-CH2-NH2

Arg (without extra atoms) 35 -CH2-SH + X-CH2-NH-C ((NH2) (= NH2 +)) gives -CH2-S-CH2-NH-C ((NH2) (= NH2 +))

His (2 extra atoms)

40 -CH2-SH + X-CH2-IM-PG, followed by check-out da -CH2-S-CH2-IM

Acid Amino Acids:

Asp (2 extra atoms) 45 -CH2-SH + X-CH2-COOH gives -CH2-S-CH2-COOH

Glu (1 extra atom)

-CH2-SH + X-CH2-COOH gives -CH2-S-CH2-COOH Polar acid amino acids without charge: Tyr (none or 1 extra atom)

-CH2-SH + F-ϕ-pOH gives -CH2-S-ϕ-pOH) (no extra atom, same geometry)

-CH2-SH + Br / I-CH2-ϕ-pOH gives -CH2-S-CH2-ϕ-pOH (1 extra atom) Gln (1 extra atom) -CH2-SH + X-CH2-C (O) ( NH2) gives -CH2-S-CH2-C (O) (NH2) Asn (2 extra atoms) -CH2-SH + X-CH2-C (O) (NH2) gives -CH2-S-CH2-C (O ) (NH2) Ser (2 or 3 extra atoms) -CH2-SH + X-CH2OH) gives -CH2-S-CH2OH (2 extra atoms) -CH2-SH + X-CH2-CH2OH) gives -CH2-S- CH2-CH2OH (3 extra atoms) Thr (2 or 3 extra atoms, disappearing beta branching)

-CH2-SH + X-CH ((CH3) (O-PG)) followed by the removal of PG gives -CH2-S-CH ((CH3) (OH)) (2 extra atoms) -CH2-SH + X -CH2CH ((CH3) (O-PG)) followed by the removal of PG from -CH2-S-CH2-CH ((CH3) (OH)) (3

extra atoms). Apolar amino acids: Leu (1 or 2 extra atoms)

-CH2-SH + X-CH ((CH3) (CH3)) -CH2-S-CH ((CH3) (CH3)) (1 extra atom) -CH2-SH + X-CH2-CH ((CH3) (CH3)) gives -CH2-S-CH2-CH ((CH3) (CH3)) (2 extra atoms)

Ile (2 or 3 extra atoms, disappearing the branching in beta) -CH2-SH + X-CH (CH3) -CH2-CH3 da -CH2-S-CH (CH3) -CH2-CH3 (2 extra atoms) -CH2 -SH + X-CH2-CH (CH3) -CH2-CH3 da -CH2-S-CH2-CH (CH3) -CH2-CH3 (3 extra atoms)

Phe (1 or 2 extra atoms)

-CH2-SH + F-ϕ da -CH2-S-ϕ (1 extra atom) -CH2-SH + Br / I-CH2- (ϕ da -CH2-S-CH2-ϕ (2 extra atoms) Met (without extra atoms) -CH2-SH + I-CH2-CH3 gives -CH2-S-CH2-CH3 Trp (1 extra atom) -CH2-SH + F-IN gives -CH2-S-IN The general pseudochemical ligation reaction is illustrated in Figure 1. Raa and X groups can be used to form preferred pseudoamino acids as shown in Table I. The residues are presented unchanged, however, it will be appreciated that ionizable groups such as NH2 or COOH may be in their form charged or provided as salts All reactions except those used to form pseudolysin are aqueous and are

carried out at a pH between 8 and 9, where stoichiometry and reaction optimization can be adjusted following standard protocols. Pseudolysin is formed in an aqueous reaction carried out at pH between 8 and 9, followed by reaction in piperidine / H2O (trifluoroacetic acid (TFA)).

Table I

Raa-X reagent

Reaction product Pseudoamino acid  Simulations or substitutions

X-CH2COOH (X = I, Br)

image 1 PseudoAsp (+ 2 atoms) PseudoGlu (+ 1 atom) Aspartic acid Glutamic acid

X- (CH2) 2COOH (X = I, Br)

image 1 PseudoGlu (+ 2 atoms) Glutamic Acid

X- (CH2) -C (O) NH2 (X = I, Br)

PseudoGln (+ 1 atom) PseudoAsn (+ 2 atoms) Glutamine Asparagine

X-CH2OH (X = I, Br)

image 1 Pseudo Ser (+ 2 atoms) Serine

X-CHOHCH3 (X = I, Br)

image 1 PseudoThr (+ 2 atoms) Threonine

X-CH2CHOHCH3 (X = I, Br)

image 1 PseudoThr (+ 3 atoms) Threonine

X-CH2CH2OH (X = I, Br)

image 1 Pseudo Ser (+ 3 atoms) Serine

X- (CH2) 2NH-PG (X = I, Br)

image 1 PseudoLys Lysine

X-CH2NH-C (NH2) 2 (X = I, Br)

Pseudoarg  Arginine

X- (CH2) 2NH-C (NH2) 2 (X = I, Br)

image 1 PseudoArg (+ 1 atom) Arginine

X-CH2CH3 (X = I, BR)

image 1 PseudoMet Methionine

X-Φ (X = F)

image 1 PseudoPhe (+ 1 atom) Phenylalanine

X-CH2Φ (X = I, BR)

image 1 PseudoPhe (+ 2 atoms) Phenylalanine

X-Φ-OH (PARA) (X = I, BR)

image 1 PseudoTyr Tyrosine

X-CH2Φ-OH (PARA) (X = I, BR)

image 1 PseudoTyr (+ 1 atom) Tyrosine

X-CH2Φ-OPO3 (PARA) (X = I, BR)

image 1 Pseudo-Phospho-Tyr Phosphotyrosine

X-CH2Φ- (OH) 2 (GOAL FOR) (X = I, BR)

image 1 PseudoDopamine Dopa

X-CH (COOH) 2 (X = I, BR)

image 1 PseudoGla γ-carboxyglutamic Acid

X-CH2-IM-PG (X = I, BR)

image 1 PseudoHis Histidine

X-CH2 -IN (X = F, I, BR)

image 1 PseudoTrp Tryptophan

X-CH- (CH3) 2 (X = 1, BR)

image 1 PseudoLeu (+ 1 atom) PseudoVal (+ 2 atoms) Leucine Valine

X-CH2CH- (CH3) 2 (X = I, BR)

image 1 PseudoLeu (+ 2 atoms) Leucine

X-CH (CH3) -CH2-CH3 (X = I, BR)

image 1 PseudoIle (+ 2 atoms) Isoleucine

X-CH2-CH (CH3) -CH2-CH3 (X = I, BR)

image 1 PseudoIle (+ 3 atoms) Isoleucine

5 The thiol group of the cysteine residue can be oxidized (-SH → -SO3) or replaced with oxygen to form serine (-SH → -OH). Said replacement can be carried out by reacting the cysteine with tosyl-Cl (Tos-Cl) thereby converting the thiol group, -SH, into a -S-Tos group. In the presence of a strong base, OH replaces the Tos substituent, thereby forming the desired -OH group.

In addition, the thiol group of the cysteine can be modified by a Michael addition reaction, as shown below:

image 1

5 in which R is H, alkyl, aryl, alkylaryl, amino or is a polymer molecule, which can be branched or unbranched, randomized or blocked.

Another aspect concerns the use of a peptide fragment whose amino terminal cysteine residue has been modified to contain an alkylamino moiety (and preferably a methylamino):

image 1

Protecting the amino group from the alkylamino moiety, peptide A can be linked to a second peptide of formula

15 general: Peptide B-αCOSR. During the removal of the protective group, an additional peptide having the general formula: C-αCOSR peptide can be linked to the above-linked peptides to form a branched peptide complex:

image 1

20 Since the branched protein still contains the thiol group of the cysteine, it will react with a Raa-X molecule in the manner described above, to thereby allow further modifications of the side chain. In particular, using an alkylamide as the R substituent, an additional amino group can be introduced into the molecule, thus allowing more branching or more ligation reactions:

25

image 1

The cysteine side chain modification can be used to introduce any of a variety of markers or binding molecules. For example, markers detectable by electron spin resonance, fluorescence (eg, 4-chloro-7-sulfobenzo-furazan ammonium (SBF) Pierce Chemical Co.) or magnetic image detection can be used. Such labeling is useful in the diagnosis of diseases and conditions, as well as in the analysis of molecular interactions (for example, to measure distances in the proteins of the entire membrane). The methodology of the present invention has several salient advantages with respect to the use of markers and binding molecules. Such substituents can be incorporated at a specific point, not randomly. In addition, the rest of the marker only needs to be stable for subsequent chemical ligation stages (if they exist). Such reactions are generally performed under very mild conditions (such as

used in natural chemical ligation; removal of Acm or other protecting groups that may optionally be used to protect thiol groups from other cysteine residues (as with Hg (CH2COOH) 2); and purification by reverse phase HPLC). The protection of said additional thiol groups prevents oxidative polymerization of Cys-containing peptides and thus facilitates their purification and handling.

II.

 Bioactive peptides and proteins

TO.

 Synthetic bioactive proteins

The procedures described above can be used to synthesize peptides and synthetic bioactive proteins. As used herein, a bioactive protein is said to be "synthetic" if non-recombinant technology has been used to polymerize any, and even more preferably, all of its amino acid residues. The term "non-recombinant technology" means that it distinguishes technologies that involve organic chemistry and other synthetic polymerization methods from technologies that involve the translation of RNA into protein, either in vivo or in vitro. Synthetic bioactive proteins will be produced in vitro using the principles of organic chemistry, and in particular the "natural chemical ligation" procedures as set forth herein. Said chemical synthesis will preferably be performed using a convergent synthesis method in which the polypeptide fragments will be synthesized and then ligated with each other to form the synthetic bioactive protein. Alternatively, the synthetic bioactive proteins of the present invention can be synthesized in a single non-convergent synthesis.

As mentioned above, the synthetic bioactive protein has a considerable molecular weight, preferably being greater than about 25 kDa. In the context of the present invention, said molecular weight determinations should be made by denaturing electrophoresis in SDS polyacrylamide. The term "monomer molecular weight" refers to the molecular weight of a monomeric synthetic protein, which is distinguished from synthetic proteins that may have multiple copies of a protein or polypeptide.

As used herein, a synthetic protein is said to have "biological activity" if it possesses a discernible bioactivity that depends on the structure of the synthetic protein or amino acid sequence, said variation in said structure or sequence increases, modify or attenuate the bioactivity of the protein. Without limitation, said "biological activity" includes the ability of the protein to mediate a catalytic signaling or induce the reaction. As used herein, a protein is said to "mediate a catalytic reaction" by converting a substrate into a product without consuming it or permanently modifying it. A protein is said to "mediate a signaling or induce the reaction" if its presence produces an organism, or if its tissues or cells initiate, continue, increase, modify or attenuate gene expression, cell differentiation.

or cell proliferation. Examples of such signaling or induction reactions include inducing cytokine expression or a response to cytokine expression, inducing erythropoiesis, inducing or attenuating inflammation and / or an inflammatory process, initiating angiogenesis or vascularization, inducing apoptosis, affecting the cell cycle, etc.

The bioactivity of synthetic bioactive proteins also includes the ability of the protein to specifically bind to a receptor, ligand or antibody. As used herein, the term "specific binding" refers to a structurally based binding interaction, such as that between a hormone and its receptor, or an antibody and an antigenic epitope, which is distinguished from the bond "non-specific" based on load, solubility, hydrophobia, etc.

Synthetic bioactive proteins are synthesized by condensation of amino acid residues. Such amino acid residues can be the encoded nucleic acid, the amino acids installed in the ribosome: alanine, arginine, asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, Threonine, tryptophan, tyrosine and valine. In one embodiment, the selected amino acid sequence will be composed of, everything constructed from polypeptide fragments that may contain a cysteine residue at the N terminal instead of the amino acid residues present in the natural sequence of this protein . The inclusion of said moiety allows the polypeptide to be linked to another polypeptide (modified to contain a thioester carboxy group) using the principles of the natural chemical ligation described herein, and, as such, facilitates the use of convergent synthesis to produce synthetic bioactive proteins.

Synthetic bioactive proteins may contain "irregular" amino acid residues. As used herein, the term "irregular amino acid residues" is intended to refer to amino acids that are not encoded by RNA and are not installed in the ribosome. This allows wide selectivity and flexibility in the design and / or construction of synthetic bioactive proteins. Examples of amino acids not installed in the ribosome that can be used according to the present invention include: D-amino acids, β-amino acids, pseudoglutamate, γ-aminobutyrate, ornithine, homocysteine, N-substituted amino acids (R. Simon et al., Proc. Natl. Acad. Sci. USA (1992), 89: 9367-71; WO 91/19735 (Bartlett et al.), US Patent No. 5,646,285 (Baindur), α-aminomethylene xiacetic acids (isoster of the amino acid-Gly dipeptide ) and α-amino-oxyacids, etc. Peptide analogs containing thioamine, vinyl amide, hydrazino, methylene, thiomethylene, phosphonamides, oxiamide, hydroxyethylene, reduced amide and substituted reduced amide isosters and β-sulfonamide (s) may be used.

In particular, the use of pseudoglutamate has advantages since the modification of the R chain allows the coupling of adducts of the polymer to the synthesized protein. Likewise, alkyl with a chain of 2 to 3 carbon atoms or Nα-substituted arylthiol amino acids in the N-terminal can be used. Such moieties (when present in the final terminal or in the polypeptide) can be used advantageously to bind this polypeptide to a polypeptide having a carboxy thioester moiety, according to the prolonged natural chemical ligation methods described herein.

All amino acid residues of the synthetic bioactive protein can be linked by a peptide bond (ie, an amide bond). It is disclosed that two amino acid residues may be linked to each other by a non-amide linkage (such as a thioester bond, an oxime bond, a thioether bond, a direct disulfide bond, a thiozolidine bond, etc. .) (Schnölzer, M. and Kent, SBH, Science (1992), 256: 221-225; Rose, K., J. Amer. Chem. Soc. (1994), 116: 30-33; Englebretsen, DR et al., Tetrahedron Lett., 36: 8871-8874; Baca, M. et al., J. Amer. Chem. Soc. (1995), 117: 1881-1887; Liu, CF et al., J. Amer. Chem. Soc. (1994), 116: 4149-4153; Liu, CF et al., J. Amer. Chem. Soc. (1996), 118: 307-312; Dawson, PE et al., (1994), Science, 266: 776-779). This makes it possible to take advantage of a variety of peptide bond modifications, substituents and isomeric replacements in the preparation of bioactive proteins.

Synthetic bioactive proteins can be conceived to present a bioactivity that mimics a bioactivity associated with a mammalian protein (including the human species, apes, bovine, murine, swine, sheep, equine, etc.), avian or fish. As used herein, a first protein is said to mimic a second protein if the first protein has a bioactivity that is modified, increased or attenuated with respect to the bioactivity of the second protein. A bioactivity is said to be "associated" with the protein if the presence depends directly or indirectly on the amino acid sequence of the protein.

Bioactive proteins can be genetically modified totally or partially, without reference to any corresponding natural bioactive counterpart. Preferred synthetic bioactive proteins of the present invention include receptors, protein receptor ligands. Examples of such proteins include the adrenocorticotropic hormone receptor and its bioactive fragments, angiotensin receptor, atrial natriuretic receptor, bradykinin receptor, growth hormone receptor, chemotactic receptor, dinorphine receptor, endorphin receptor, β-receptor Lipotropin and its bioactive fragments, encephalin receptor, enzyme inhibitor receptors, fibronectin receptor and its bioactive fragments, peptide receptors that release growth hormone and gastrointestinal, receptor for luteinizing hormone release peptide, hormone receptor Melanocyte stimulant, neurotensin receptor, opioid receptor, oxytocin receptor, vasopressin receptor, vasotocin receptor, parathyroid hormone receptor and fragments, kinase protein receptor, somatostatin receptor and substance P receptor.

Synthetic bioactive proteins also include enzymes, and structural molecules such as chitin or collagen, etc.

Synthetic bioactive proteins include cytokines. Cytokines are small proteins or biological factors (in the range between 5 and 20 kDa) that are released by cells and mediate specific effects on cell-to-cell interaction, communication and the behavior of other cells. As used herein, the term "cytokine" is intended to include interleukins (IL) (such as IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, etc.); lymphokines and signaling molecules such as erythropoiesis stimulating proteins (for example, erythropoietin (EPO)), tumor necrosis factor (TNF), interferons, etc., growth factors, such as transforming growth factor, nerve growth factor, growth factor from the brain, neurotrophin-3, neurotrophin-4, hepatocyte growth factor, T lymphocyte growth factor (TGF, TGF-β1, TGF-β2, TGF-β3, etc. .), colony stimulating factors (G-CSF, GMCSF, M-CSF, etc.), epidermal growth factor (EGF, LIF, KGF, OSM, PDGF, IGF-1, etc.), fibroblast growth factor (αFGF, βFGF, etc.), and hormones, particularly individual peptide hormones, such as growth hormone).

Cytokines, and particularly chemotactic cytokines, also comprise particularly preferred examples of proteins whose amino acid sequences can be used to aid in the design of synthetic bioactive proteins. Cytokines participate in innate and acquired immune responses through direct or indirect mediation of leukocyte regeneration, or acting as important signals in the activation of leukocytes that have accumulated at specific points of microbial invasion. Proinflammatory cytokines, interleukin-1β (IL-1β) and interleukin-6 (IL-6) and mediators reflect neutrophilic regeneration and activation, including the soluble intercellular adhesion molecule, interleukin-8 (IL-8) (Kotecha S ., European Journal of Pediatrics (1996), 155 Suppl. 2: S14-17). Interleukin-6 is a multifunctional cytokine mainly involved in the acute phase response, B lymphocyte differentiation and antibody production. (Akira S. et al .: Interleukin-6 in biology and medicine. Adv. Immunol. (1993), 54: 1-78). This cytokine is synthesized and secreted by many different cells, including monocytes, macrophages, T and B lymphocytes, endothelial cells and fibroblasts. IL6 is frequently induced with FNT-alpha and IL-1 proinflammatory cytokines in many alarm conditions, and circulating IL-6 plays an important role in inducing acute phase reactions, (Xing Z. et al., J Clin. Invest., (1998), 101 (2): 311-20). Cytokines that directly or indirectly influence both regeneration and leukocyte activation include FNT-alpha, granulocyte colony stimulating factor (G-CSF), granulocyte and macrophage colony stimulation factor (GM-CSF) and members of the chemokine family. Cytokines that primarily regulate the activation status of leukocytes include interferon-γ (IFN-γ), interleukin-10 (IL-10) and interleukin-12 (IL-12): Cytokines may also influence microbial elimination by inducing or regulating the expression of other effector molecules, which can occur in an autocrine or paracrine manner.

Colony stimulating factors are cytokines produced by a variety of myeloid and stromal cells that are needed for the proliferation and maturation of hematopoietic stem cells. In addition, studies show that these factors increase the activities of effector cells in mature leukocyte populations. Specifically, G-CSF prolongs the survival of neutrophils in vitro, increases the phagocytic activity of neutrophils and increases the allergy of neutrophils. (Standiford, T.J., et al., J. Invest. Med. (1997), 45: 335-345). In contrast, GM-CSF favors the proliferation and maturation of both neutrophils and macrophages; The activation properties of this cytokine are mainly directed towards mature macrophage populations. (Cheng, G.H., et al., J. Immunol. (1994), 152: 724-734).

Six intimately related subfamilies of chemotactic cytokines, called chemokines, have now been characterized. Of these, members of at least two subfamilies contribute to the defense of the pulmonary antimicrobial host (Mandujano J., et al., Amer. J. Respir. Crit. Care Med. (1995), 155: 1233-1238; Baggiolini M., et al., Ann. Rev. Immunol. (1997) 15: 675-705). Members of the CXC chemokine family, which include IL-8, MIP (macrophage inflammatory protein) -2, KC, ENA-78 and NAP-2, have predominant neutrophil stimulating and chemotactic activities, while the CC family, which includes MCP (monocyte chemotactic protein) -1, MCP-2, MCP-3, RANTES, MIP-1 alpha and MIP-1 beta, exerts predominant chemotactic and / or activating effects on macrophages, lymphocytes and eosinophils. (Huffnagle G.B., et al., The Role of Chemokines in Pneumonia, in Koch A, Strieter R, eds., Chemokines in Disease. Texas. R. G. Landis Co .; 1996: 151-168).

Interferon-gamma is a cytokine produced by T lymphocytes (both alpha, beta and delta gamma T lymphocytes) and NK cells that is instrumental in cell-mediated immunity against a broad spectrum of lung pathogens. This cytokine activates several functions of macrophage effector cells, including allergy stimulation, the ability to present the antigen, priming of the release of FNT from macrophages and increased in vitro macrophage antimicrobial activity against bacterial, fungal and microbacterial

Interleukin-12 is a cytokine that favors Th1 type immune responses, while inhibiting Th2 type immune responses. Specifically, IL-12 stimulates the development of T, Th1 and NK cells and increases the cytolytic activity of CD8 + cells and NK cells. Even more important, IL-12 serves as the largest inducer of IFN-gamma from T lymphocytes and NK cells.

In contrast to IL-12, IL-10 favors the development of Th2 type immune responses, while inhibiting cell-mediated immunity (Th1 type). (Howard M., et al., J. Clin. Immunol. (1992), 12: 239-247). This cytokine was shown to exert potent inflammatory properties, partly by directly deactivating neutrophils and macrophages and decreasing the expression of TNF, IFN-gamma and members of both the C-X-C and C-C chemokine families. IL-10 is instrumental in attenuating unwanted production of proinflammatory cytokines in states of generalized activation of immune cells.

EPO is a particularly preferred example of a protein whose amino acid sequence can be used to aid in the design of a synthetic bioactive protein with an erythropoiesis stimulating activity. EPO is the main factor responsible for regulating red blood cell production during steady-state conditions and for accelerating the recovery of red blood cell mass after bleeding (Jelkmann, W. (1992) Physiol. Reviews 72: 449 ; Krantz, SB (1991) Blood 77: 419; Porter, DL and MA Goldberg (1993) Exp. Hematol., 21: 399; Nissenson, AR (1994) Blood Purif. 12: 6). The circulating form of human EPO is a glycoprotein of 165 amino acids (aa) with a molecular weight of approximately 30,000 (Sawyer, ST et al. (1994), Hematol. Oncol. Clinics NA 8: 895; Jacobs, KJ et al. , Nature (1985), 313: 806; Lin, FK et al. Proc. Natl. Acad. Sci. USA (1985) 82: 7580). Although the cDNA for EPO provides a molecule with 166 amino acid residues, the carboxy terminal arginine is removed in a modification after translation (12). There are three potential points for N-linked glycosylation and all are filled. A carbohydrate residue attached to O is also present (13). The effects of glycosylation are complex. Although EPO from non-glycosylated E. coli exhibits complete biological activity in vitro, glycosylation is apparently necessary for total activation in vivo. Thus, natural EPO, produced by E. coli and deglycosylated, has very little activity in animal studies (Krantz, SB (1991) Blood 77: 419; Sasaki, H. et al. (1987), J. Biol. Chem., 262: 12059; Lowy, PH et al. (1960), Nature, 182: 102; Wojchowski, DM et al. Biochem. Biophys. Acta, 910: 224; Dordal, MS et al. (1985) Endocrinology, 116: 2293).

In accordance with the above, the variable glycosylation models have variable effects. For example, desialiated EPO has both increased activity in vitro and decreased activity in vivo, an effect attributed to the exposure of galactose residues that are recognized, bound and eliminated by hepatocytes (Spivak, JL and BB Hogans (1989) Blood, 73v: 90; Goldwasser, E. et al. (1974) J. Biol. Chem. 249: 4202). The fully sialiated EPO branching model also presents a difference in biological activity. Mainly the tetra-antennial branched EPO has activity equivalent to the "conventional" EPO, while the mainly bi-anteranial branched EPO has three times more activity in vitro but only 15% of normal activity in vivo (Takeuchi, M. et al. (1989) Proc. Natl. Acad. Sci. USA., 86: 7819). Studies have indicated that only sugars linked by N and not linked by O, are important in the functioning of EPO (Higuchi, M. et al. (1992)

J. Biol. Chem., 267: 770319).

Colony stimulating factors and RANTES also comprise particularly preferred examples of proteins whose amino acid sequences can be used to aid in the design of a synthetic bioactive protein.

B. Polymer modified proteins and polypeptides

The protein or polypeptide can be modified to contain one or more structurally defined polymer adducts at the preselected positions. Although the polymer modified proteins have been described above, the nature and manner of the possible polymer modifications of the present invention differ markedly from said prior efforts.

Since synthetic bioactive proteins are chemically synthesized, in whole or in part, it is possible to synthesize said proteins in a way that allows polymers linked by covalent bonds (such as polymers or sugar, polyethylene glycol, glycols, hyaluronic acid, etc.). ) to one or more points selected by the user and defined by the user in a polypeptide a central axis of protein. In addition, the synthetic production of proteins makes it possible to ensure that said modifications are present at each of the points selected by the user and defined by the user of each molecule in a preparation. Said uniformity and synthesis control remarkably distinguishes the present invention from the random modifications allowed for the use of prior art procedures. Significantly, this allows one to design a synthetic protein in which any non-critical moiety can be modified to contain a polymer adduct. In addition, for each of these points selected by the user and defined by the user, the user can define the precise link (amide, thioester, thioether, oxime, etc.) whereby said adducts will be attached to the central axis of the polypeptide or protein. In addition, the specific polymer adducts that are desired to be present at a specific point can be varied and controlled, so that a final preparation is obtained in which each protein or polypeptide present contains exactly the same modified adducts precisely at the same modified sites. . Therefore, the present invention allows the formulation of homogeneous preparations of polypeptide and proteins modified by the polymer.

Furthermore, by providing a means to vary the position and number of the coupling points to which the polymer can be attached, the present invention allows one to vary the nature of the bound polymer. The polymer adducts that can be incorporated at any point selected by the particular user and defined by the user can be of any defined length. Also, according to the methods of the present invention, it is possible to use polymers of different lengths at different points. Therefore, synthetic bioactive proteins can be monomodified or polyimodified, with a polymer adduct. When more than one polymer adduct introduced into a specific polypeptide or protein, the use of polymers can be "monospecial", "polyspecialized", "uniformly spiced" or "spiced variously in". As used herein, the term "monospecific" refers to a polypeptide or protein that has been modified by a single species of polymer; instead, the term "polyspecies" refers to a polypeptide or protein that has been modified in more than a single polymer species. Such polypeptides or polyspeciated proteins are said to be "uniformly spiced." If at each modified point of the polypeptide or protein, the same, single species of polymer is present. In contrast, a polypeptide or polypeptide protein is said to be "spiced differently" if the modified points of the polypeptide or protein are modified with different polymer species.

In addition, it is possible to vary the extent of linearity or lack of branching of the polymer adduct in each of the users selected at the points defined by the user. Therefore the polymer adducts can be linear, branched or uniformly branched. The term "uniformly branched" refers to all branches of a polymer at a specific point having the same structure and length. As can be seen, it is possible to independently vary both the length of any individual branching, as well as the polymer structure present at said branching point.

In summary, it is possible to define (1) the position and (2) the frequency of the modified polymer, of the selected user and of the points defined by the user in a polypeptide or protein structure, as well as controlling (3) the length,

(4) the species and (5) the degree of branching present in each of these points. In addition, with respect to the polypeptide and the proteins with multiple polymer modifications, it is possible to independently define each of the five variables identified above for each point. In addition, with respect to polypeptides and proteins that have modifications of the branched polymer, it is possible to independently define each of the five variables identified above for each branch point. Therefore, there is considerable flexibility in polymer modification.

C. Administration of proteins and peptides

The synthetic bioactive polypeptides and proteins optionally modified by the polymer can be used as pharmaceutical agents to effect the treatment of diseases and conditions. More preferably, when administered to a patient or individual in need of therapy, said synthetic bioactive polypeptides and / or proteins will be administered using a drug delivery system. The uses of said system allow a drug to be present in the patient in a way that makes it acceptable to increase the efficacy of the desired bioactivity. Preferred drug delivery systems include systems capable of administering polypeptides or proteins by the oral, nasal, or inhalation routes, or intramuscularly, subcutaneously, transdermally, intravenously, intraurally or intraocularly.

Such drug delivery systems may include formulations that provide site-specific release, or that increase protection for the intestinal mucosa. Suitable formulations include: dry powder formulations, administration by viral particle encapsulation, liposome encapsulation, transdermal patches, electrically assisted transport (electroporation therapy) and polymer / niazone conjugation.

Preferably, said drug delivery devices will respond to changes in the biological environment and will administer (or stop administering) drugs based on these changes. A range of materials has been expanded to control the release of drugs and other active agents: poly (urethanes), poly (siloxanes), poly (methyl methyl acrylate), poly (vinyl alcohol) for hydrophilicity and resistance, poly (ethylene) , poly (vinyl pyrrolidone), poly (2-hydroxy ethyl methacrylate), poly (n-vinyl pyrrolidone), poly (methyl methacrylate), poly (vinyl alcohol), poly (acrylic acid), polyacrylamide, poly (ethylene acetate -co-vinyl), poly (ethylene glycol), poly (methacrylic acid), etc. Preferably, biodegradable polymers will be used to facilitate the administration of drugs. Such polymers include polylactides (PLA), polyglycolides (PGA), poly (lactide-co-glycolides) (PLGA), polyanhydrides and polyorthoesters. Drug delivery devices, and methods for their use are described in US Patent Nos. 6,072,041, 6,041,253, 6,018,678, 6,017,318, 6,002,961, 5,879,712, No. 5,849,331, No. 5,792,451, No. 5,783,212, No. 5,766,633, No. 5,759,566, No. 5,690,954, No. 5,681,811, No. 5,654,000, No. 5,641,511, No. 5,438,040, No. 4,810,499 and 4,659,558.

III. Production of bioactive synthetic proteins

The production of bioactive synthetic proteins can be assumed to have the following stages or components: design, synthesis, peptide ligation, protein folding, bioactivity evaluation, polymer modification of the central axis of the polypeptide (Figure 2).

A. Design of preferred bioactive synthetic proteins

Preferably, the synthetic bioactive protein will be an erythropoiesis stimulating protein. As used herein, an erythropoiesis stimulating protein is a protein that is involved in the production of red blood cells. Erythropoiesis stimulating bioactivity of a protein can be determined by any of several means, such as following erythropoiesis after in vivo administration, testing the ability of the protein to intervene in the proliferation of EPO-dependent cell lines, etc.

A preferred erythropoiesis stimulating protein is mammalian EPO, more preferably human, and its analogues. Erythropoietin is synthesized and secreted primarily by tubular and juxtatubular and interstitial capillary endothelial cells of the kidney. Chronic kidney disease causes the destruction of EPO producing cells in the kidney. The consequent lack of EPO frequently causes anemias. The main clinical use of EPO is therefore the treatment of patients with severe renal impairment (hematocrit less than 0.3) who usually also receive transfusions as well as the treatment of anemic patients after chemotherapy. In general, EPO is synthesized recombinantly in hamster ovary cells for clinical use. EPO is an acidic protein (pI = 4.5) stable at pH and relative heat of 165 amino acid residues in its mature form. EPO transports its activity through the EPO receptor. Approximately 40 percent of the molecular mass of EPO is due to its glycosylation. Glycosylation is an important factor that determines the pharmacokinetic behavior of EPO in vivo. Non-glycosylated EPO has an extremely short biological half-life. Even so, it binds to its receptor and may even have a greater specific activity in vitro. However, recent experiments have shown that EPO PEGylation significantly increases its life, but significantly decreases EPO activity in a cell-based assay system.

Synthetic erythropoiesis-stimulating proteins ("SEP") are preferably chemically modified synthetic analogs of human urinary EPO. They preferably contain one or more polymer moieties (even more preferably pPEG moieties) attached to one or more peptide moieties (s) via a thioether, oxime, amide or other bond. Preferably, said links will be in one or more of the positions that are naturally glycosylated in human urinary EPO (i.e., positions, 24, 38, 83 and 126). Even more preferably, SEP molecules will have pPEG residues in two or more of said positions. Alternatively, other protein residues may be modified by polymer residues. Modification positions for bioactive synthetic proteins include residues located in a disordered loop, region or domain of the protein, or at or near the points of potential protease cleavage. For example, polymeric modifications can be introduced in one or more positions of 9, 69 and / or 125 EPO. The total molecular weight of the SEP molecules may vary between about 25 and about 150 kDa, and more preferably between about 30 and about 80 kDa. The molecular weight can be controlled by increasing or decreasing the number and structure of the polymer (such as pPEG) used for the modification of a given analog. The P.M. Hydrodynamic mediated by pPEG for larger assemblies is greater than approximately 100 kDa. In vitro assays in cells expressing the human EPO receptor indicate that the SEPs disclosed herein have an ED50 that is equivalent to that of recombinantly produced glycosylated human EPO.

The oxime bound pPEG analogs are preferably constructed by linking a functionalized amino-oxy, ketone or aldehyde pPEG to the SEP protein in an unnaturally encoded amino acid that carries an amino-oxy, ketone or aldehyde functionality in the side chain . For example, positions 89 and 117 of SEP-0 and 1 contain pseudoglutamates (an unnaturally encoded amino acid bearing a side chain of the formula -CHα-CH2-S-COOH (compared to the glutamate side chain -CHα- CH2-CH2-COOH)). SEP analogs that use thioether linkages are preferably constructed to contain a thiol functionality provided by a cysteine or unnatural amino acid with a side chain carrying the thiol. Figure 3 represents the basic structure of a type of preferred synthetic erythropoiesis stimulating proteins.

In an alternative embodiment, the SEP molecules may comprise "circle permutated" EPO analogs in which the natural amino and carboxy terminal of EPO have been transferred. Even more preferably, said transfer will move the amino and carboxy terminals to positions of low structural limitation, such as messy loops, etc. The messy loop around positions 125 and 126 (in relation to the natural EPO residue numbering system) is an example of such a transfer point. Even more preferably, said SEPs will be free of disulfide, and will be chemically modified to contain polymer residues in the preselected residues.

Alternatively, the SEP molecules may have amino and carboxy terminals moved to a natural glycosylation point, or other points suitable for glycosylation, such as positions 126 and 125. The SEP molecules may also include modifications of the amino and carboxy terminals. to eliminate or modify the load (such as by carboxy amidation, etc.).

In a preferred example of said circularly permutated molecules, new terminals N and C are provided by positions 126 and 125, respectively. The natural disulfide-forming cysteines at positions 7, 29, 32 and 161 are preferably substituted by the naturally uncoded amino acid, L-α-N-butyric acid (Aba), which cannot form disulfide bridges. Residues R166, E37 and V82 are preferably substituted with alanines to improve production. In addition, an additional cysteine is preferably inserted between positions 1 and 166 with respect to the natural EPO number scheme, which is numbered below as "0". The resulting molecule contains four cysteines (at positions 126, 0, 38 and 83 (read in the direction of terminal N to C)), which are used as (1) ligation points and (2) forming pPEG coupling points Thioether Optionally, a cysteine can substitute A125 to provide an additional pPEG coupling point. The total molecular weight of said SEP molecules in the present invention may be between about 25 and about 150 kDa, and more preferably between about 30 and about 80 kDa. The molecular weight can be controlled by increasing or decreasing the number and structure of the polymer (such as pPEG) used for the modification of a given analog. The P.M. Hydrodynamic mediated by pPEG for larger assemblies is greater than 100 kDa. The optional pPEG coupling points are located at positions 125, 9 and 24 (read in the direction of terminal N to C). Additional SEP analog designs have alternative N and C terminals in the untidy area of the loop and / or truncated debris from the new N and / or C terminals. The basic structure of the preferred molecules permutated in a circle is shown in the figure Four.

Alternatively, the bioactive synthetic proteins are a mammalian G-CSF, more preferably human. G-CSF causes rapid proliferation and the release of neutrophil granulocytes into the bloodstream, and thus provides therapeutic effect in the fight against infection. The human GCSF protein has a length of 174 amino acids (patent: EP 0 459 630 (Camble, R. et al.), And variants of its sequence have been isolated. The protein has four cysteine residues and an O-glycosylation point in the threonine of position 133.

In said synthetic GCSF molecules, the amino acid sequence of the molecule can be altered, relative to the natural GCSF sequence, to contain natural or more preferably non-natural hydrophobic moieties encoded in one or more of positions 1, 2 or 3 Optionally, said molecules can be further modified to contain a natural hydrophobic moiety, or more preferably unnaturally encoded at position 5 of GCSF, and / or at positions 173 and / or 174.

Preferably, the synthetic GCSF molecules will be modified by the polymer (preferably pPEG) in one or more of positions 63, 64 and / or 133 and / or in one or more of positions 1 and 174. The pPEG polymers can be linear or branched, loaded, unloaded or mixed; and may have a molecular weight range between about 5 and about 80 kDa, and more preferably between 40 and about 70 kDa of contribution to P.M. Total pPEG depending on the number and structure of the pPEG used for modification. The hydrodynamic radius has an effect of P.M. higher in vivo (for example, a branched 10 kDa pPEG has an effective P.M. of approximately 55 kDa). The P.M. Hydrodynamic mediated by estimated pPEG is greater than 100 kDa.

For analogs that use oxime bonding, pPEG is preferably linked to non-naturally encoded moieties that carry amino-oxy, ketone or aldehyde functionality in the side chain. For analogs that use thioether linkage, pPEG is preferably linked to a cysteine or an unnatural amino acid with a natural chain that carries a thiol. For analogs that use amide bond, pPEG is linked to a natural or unnatural amino acid that carries an amine in the reactive side chain.

The basic structure of the bioactive synthetic GCSF proteins is shown in Figure 5. In the figure, "J" designates an unnaturally encoded moiety having a hydrophobic side chain.

The bioactivity of said bioactive synthetic GCSF proteins can be analyzed by conventional means, such as testing their ability to mediate the proliferation of a human or mouse cell line that depends on the factor (e.g., NFS60 cell line), or by measuring neutrophil stimulation, and immunogenicity in vivo, either with or without an administration system that directs> 20 days of half-life / release).

Alternatively, the bioactive synthetic proteins are a mammalian chemokine, even more preferably human, RANTES. RANTES blocks the entry of M-tropic HIV strains through the primary CCR5 receptor, as well as the decrease-modulated inflammation pathways involved in asthma, transplant rejection and wound healing (Kalinkovich, A. et al. , Immunol. Lett. (1999), 68: 281-287). Synthetic RANTES molecules preferably differ from the RANTES chemokine that is being chemically modified, so that (1) the N-terminal serine found at position 1 of RANTES 1-68 is replaced with a nnonanoyl group ("NNY") and (2) Tyrosine at position 3 is substituted with an unnaturally encoded amino acid that has a hydrophobic side chain. It has been found that said components are extremely potent, having ED50 in the pM range, compared to the mM range for recombinant "Met-RANTES 1-68".

Powerful analogues of RANTES have been created that present one or more additional changes to those indicated above, such as the replacement of proline at position 2 with an unnaturally encoded amino acid having a hydrophobic side chain, and by coupling a fatty acid at the C terminal of the molecule. The specificity of the receiver has been improved in combination with the power by modifications to the area of loop N corresponding to residues 12 to 20 of RANTES. In a more preferred embodiment, the RANTES synthetic molecules incorporate a polymer moiety (such as pPEG, or nonanoy ("NNY") or Y3X at its N-terminal

or C to improve among others the half-life in vivo. The remainder of pPEG is preferably linked by an oxime bond to an unnaturally encoded amino acid that is introduced at position 66, 67, 68 or linker at position 68, preferably at position 67. The fatty acid preferably binds by an oxime bond (preferably by a linker) to moiety 68, such as a di-or tryptophan-peptide linker with amino-oxi modified amino acids. Alternatively, other coupling chemicals can be used. The molecular weight of the larger assemblies of said synthetic RANTES analogs is between about 25 kDa to about 45 kDa depending on the nature and structure of the pPEG bond, and for larger mounts they have a P.M. hydrodynamic mediated by pPEG greater than 60 kDa. The structure of the preferred synthetic RANTES analogs is shown in Figure 6.

B.

 Defined and specific polymer modification of synthetic bioactive proteins and peptides

Chemical moieties and polymers, in particular water soluble polymers, and their use to modify proteins are disclosed in order to provide protein drugs with improved properties relative to the unmodified protein. The expected beneficial effects of such modification include in a non-limiting manner (a) improved potency (b) longer life of the protein drug in the circulation due to increased proteolytic stability and reduced elimination rates (c) reduced immunogenicity and ( d) possibly differential direction compared to the unmodified molecule.

As stated above, although polyethylene glycol has been used to modify proteins (see, for example, US Patent No. 6,027,720, Kuga et al., Which refers to the modification of G-CSF lysine reducers to allow for coupling of polyethylene glycol molecules by reactive amine groups), said modifications are associated with molecular heterogeneity and molecular diversity.

As used herein, the term "molecular heterogeneity" refers to a variation in the number of polymer molecules attached to each position of a protein preparation. The term "molecular diversity" refers to (a) a variation in the protein point (s) that are modified by the polymer, (b) a variation in the length of the polymer adducts of different points of the same protein, or of the same point (s) in different proteins of a protein preparation or (c) a variation in the extent and / or nature of any branching of the polymer adducts of different points of the same protein, or of the same point (s) in different proteins of a protein preparation. As used herein, the term "molecularly homogeneous" refers to a preparation of a protein in which all protein molecules contain the amino acid sequence and the same polymer modifications at the same positions. A mixture of two or more "homogenous molecule" protein preparations is referred to herein as a "molecularly defined" preparation.

A solution to the problems of heterogeneity, diversity and inadequacy of the polymer identified above involves the production of a new class of biocompatible polymers that combine the advantages of both polypeptides (exact length, convenient synthesis) and "pPEG" ("Precision PEG") , a flexible, amphiphilic, non-immunogenic polymer, non-protease sensitive polymer) Rose, K. et al. (US Patent Application Serial No. 09 / 379,297) This new class of biocompatible polymer has the formula:

- [CO-X-CO-NH-Y-NH] n-

n is an integer, preferably between 1 and 100, and more preferably between 2 and 100, in which X and Y are biocompatible repeating elements of exact structure joined by an amide bond. Preferably, X and Y will be divalent organic radicals that lack reactive functional groups or are absent and are the same or different, and may vary independently with each repeat unit (n). Preferably, when n = 2 at least one of X or Y will be selected from the group consisting of an aliphatic and aromatic group, branched and linear, substituted, unsubstituted. More preferably, at least one of X or Y the divalent organic radicals will be selected from the group consisting of phenyl, a C1-C10 alkylene moiety, a C1-C10 alkyl group, a heteroatom-containing phenyl, a C1-C10 alkylene moiety containing heteroatoms, a C1-C10 alkyl group containing heteroatoms and a combination thereof.

Particularly preferred pPEG residues have the formula:

- {CO- (CH2) 2-CO-NH- (CH2) 3- (OCH2CH2) 3-CH2-NH} n-

wherein n is preferably between 1 and 100 and more preferably between 2 and 100; or

{CO- (CH2) 2-CO-NH- (CH2) 6-NH-CO- (CH2) 2-CO-NH- (CH2) 3- (OCH2CH2) 3-CH2-NH-} n-,

wherein n preferably ranges from 1 to 50 and more preferably between 2 and 50.

Such pPEG residues can be synthesized in any of a variety of ways. Such moieties, however, are preferably produced using a solid phase step-by-step chain unit assembly, rather than a polymerization process. The use of said assembly process allows the remains of a preparation to have a defined homogeneous structure, in terms of its length, the nature of its substituents X and Y, the position or positions (if any) of the branching points, and the length, the substituents X and Y, and the position or positions of some branches.

Preferably, said moieties will be synthesized in stages such as:

(to)

acylating the amino or hydroxyl group of a compound of formula Z-Q-support with a molar excess of a derivative of a diacid having the formula, HOOC-X-COOH, wherein Z is H2N- or HO-; Q is a linker

or a target molecule; and the support is a solid phase, matrix or surface;

(b)

activating the free carboxyl group of the product of step (a);

(C)

aminolisating the product of step (b) with a molar excess of a diamine having the formula, NH2-YNH2; Y

(d)

optionally repeat steps (a) - (c) using HOOC-H-COOH and NH2-Y-NH2, wherein said X and Y radicals are divalent or missing organic radicals and are the same or different, and may vary independently with each one of the units optionally repeated, and are the same or different from the substituents X and Y used in any of the above acylation and aminolisation steps.

Preferably, polymers of 6, 12, 18 and 32 monomers of the above repeating unit are used. When desired, the repeat unit may be used, for example together with the lysine amino group to form branched pPEG structures. The pPEG can be bound to synthetic proteins by a variety of chemicals, including thioether, oxime and amide bond formation.

The assembly of the solid-phase chain of units may include:

Step 1: Couple the protected or unprotected diacid to amino on resin to generate the amide bond at the point of attachment (where GP is a protective group that is present or absent depending on the diacid used):

PG-OOC-X-COOH + NH2-Y-NH-Resin

Stage 2: Separate the protective group (GP) from the resin, if present

HOOC-X-CO-NH-Y-NH-Resin

Step 3: Couple the protected or unprotected diamino to carboxy on the resin to generate the amide bond at the point of attachment (in which GP is present or absent depending on the diamino used)

PG-NH-Y-NH + HOOC-X-CO-NH-Y-NH-Resin

Stage 4: Separate the protective group (GP) on the resin, if present

-NH-Y-NH-OC-X-CO-NH-Y-NH-Resin

Stage 5: Repeat steps 1 to 4 "n" times to add "n" units then split the resin

- [CO-X-CO-NH-Y-NH] - [CO-X-CO-NH-Y-NH] - [CO-X-CO-NH-Y-NH] - [CO-X-CO- NH-Y-NH]

As discussed, linear or branched pPEG assemblies, water soluble polymers are preferred for coupling to the bioactive synthetic molecules of the invention. The pPEGs used carry pending groups that are charged or neutral under physiological conditions, and can be prepared to vary in coupling chemistry, length, branching and solubility depending on the structure of the pPEG used. As indicated above, the preferred pPEGs of the invention comprise a water soluble polyamide having the repeating unit -CO-X-CO-NH-Y-NH-, wherein one of X and Y or both, comprises a water soluble repeat unit, and even more preferably a "PEG" based repeat unit. Although oligoureas are in principle accessible using a solid phase procedure in two simple steps, as shown below for the case of an amino resin, NH2-resin and a symmetric diamine, NH2-Y-NH2:

Activation with carbonyldiimidazole → im-CO-NH-resin Aminolysis with diamine NH2-Y-NH2 → NH2-Y-NH-CO-NH-resin

in which these two steps can be repeated numerous times to give an oligourea with the repeating unit -NH-Y-NH-CO-, this method is less preferred. This is because the yields of the previous stages may not be quantitative at room temperature, even with very large excess reagents and long reaction times. Therefore, it is preferred to use a three-stage solid phase process, shown below for the case of an amino resin, NH2-resin, to form a polyamide. HO2C-X-CO2H and NH2-Y-NH2 reagents should be symmetric to avoid isomeric products.

Acylation with diacid HO2C-X-CO2H → HO-CO-X-CO-NH-Resin Activation with carbonyldiimidazole → im-CO-OCO-X-CO-NH-Resin Aminolysis with diamine NH2-Y-NH2 → NH2-Y -NH-CO-X-CO-NH-Resin

These three steps can be repeated in the sequence numerous times to give a polyamide with repeating unit -NH-Y-NH-CO-X-CO-. The polymer contains an exact number of monomer units, X and Y can be varied independently at each stage and the terminal groups can be sectioned at will. For example, using succinic anhydride ("Succ") for the acylation stage and 4,7,10-trioxa-1,13-tridecanodiamide (referred to as "EDA" or "TTD") for the aminolysis stage, polyamides are formed at base of "PEG" in which X is -CH2CH2-, Y is -NH-CH2CH2CH2- (OCH2CH2) 3-CH2-NH- and the repeating unit is -NH-CH2CH2CH2- (OCH2CH2) 3-CH2-NHCOCH2CH2CO- . Despite the fact that the process involves divalent reagents without protecting groups, crosslinking is not a problem when resins are used for conventional commercial peptide synthesis (Rose et al. (US Patent Application Serial No. 09 / 379,297); and Rose et al., J. Am. Chem. Soc. (1999) 121: 7034), except as indicated below for the case of branched Lys nuclei for preparing branched mounts.

For example, similarly branched pPEG mounts can be prepared as for the "chemobody" mounts described in Rose et al. (US Patent Application Serial No. 09 / 379,297) and Rose, K. and Vizzavona, J.

(J. Am. Chem. Soc. (1999) 121: 7034), Therefore, branched pPEG assemblies having a branching core such as a lysine branching core can be prepared which is coupled by oxime linkers to a preferred water soluble polyamide such as - (COCH2CH2CO-NH-CH2CH2CH2- (OCH2CH2) 3-CH2-NH) n-. For example, oxime bonds can easily be formed between an amino-oxyacetyl group in a side chain of

5 Lys at the other end of the polyamide, and glyoxyl groups in a lysine core, for example the tetrameric core (O = CHCO) 4Lys2Lys-. Therefore, a linker oxime group outside each lysine branch point can be prepared as a structure -Lys (COCH2ON = CHCO-) amide-. An alternative assembly may place the oxime bond between the polyamide and the free pendant group of the polyamide, preferably using a monoprotected diamine and deprotection after coupling the polyamide, to generate a tetravalent branched assembly shown below:

[O = CHCO- (NH-CH2CH2CH2- [OCH2CH2] 3-CH2-NH-COCH2CH2CO-) n] 4Lys2Lys

Oxime chemistry can be used in the preparation not only of branched dimeric and tetrameric assemblies,

15 but is also suitable for assembling branched octamer assemblies (Rose, K. et al. J. Am. Chem. Soc. (1994) 116: 30; Rose, K. et al. Bioconj. Chem. (1996) 7: 552 ). It is of course possible to use other chemicals for such purposes when using said polyamides of pPEG (see, for example, Figure 7). In addition, the formation of polyamide can be incorporated into a synthesis scheme for the synthesis of peptides that the chemistry of Boc or Fmoc entails, but when elaborating such schemes it should be borne in mind that the aminolysis stage will eliminate the Fmoc group and eliminate protection with indole formyl if Boc chemistry is used.

Accordingly, said pPEGs can be prepared to have several branching nuclei (for example, lysine branching core) linked by a bond of choice (e.g., amide, thioether, oxime, etc.) to a linear water-soluble polyamide (for example, - (Succ-TTD) n-, etc.), where the free end of each polyamide

Water soluble can be protected with a desired pendant group (eg, carboxylate, amino, amide, etc.) to provide a desired charge. In addition, linear and branched pPEGs can be prepared to comprise a single functional group for coupling to a synthetic protein that carries one or more unique and interreactive functional groups, and the pending groups of the pPEGs will preferably be non-antigenic (see, for example , figure 7).

The disclosure of the background of the invention herein is included to explain the context of the invention. This should not be considered as an admission that any material cited, known or part of the prior art or general knowledge common in any part of the world was published as the priority date of any of the claims. Once the invention has been described in general, it will become more

35 clearly made reference to the following examples, which are provided by way of illustration and not limitation of the present invention.

Example 1

Synthesis of the synthetic SEP-0 erythropoiesis stimulating protein

A synthetic erythropoiesis stimulating protein (SEP) was synthesized. The sequence of the synthesized complete protein (called "SEP-0 (1-166)") is:

image 1

Four. Five

in which ψ indicates an unnatural amino acid residue consisting of a cysteine that is carboxymethylated in the sulfhydryl group. The SEP-0 protein was synthesized in solution from four polypeptide segments:

SEP-0: 1 segment (GRFN 1711; composed of residues 1-32 of SEQ ID No. 1): APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester

SEP-0: 2 segment (GRFN 1712; composed of residues 33-88 of SEQ ID No. 1): CSLNEKITVP DTKVNFYAWK RMEVGQQAVE VWQGLALLSE AVLRGQALLV KSSQPW-thioester (in which Cys33 is

55 protected by Acm)

Segment SEP-0: 3 (GRFN 1713, composed of residues 89-116 of SEQ ID NO: 1): CPLQLHVDKA VSGLRSLTTL LRALGAQK-thioester (in which Cys89 is protected by Acm)

Segment SEP-0: 4 (GRFN 1714; composed of residues 177-166 of SEQ ID No. 1): CAISPPDAAS AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR-carboxylate (in which the C-terminal cysteine (Cys161) carries a protective group picolil (peak)

Peptides SEP-0: 1, SEP-0: 2 and SEP-0: 3 were synthesized on a thioester generating resin by means of the in situ neutralization protocol for Boc (tert-butoxycarbonyl) chemistry and peptide synthesis in Step-by-step solid phase (SPPS) using proven SPPS, side chain protection and thioester resin strategies (Hackeng, et al., PNAS (1999) 96: 10068-10073 and Schnölzer, et al., J. Pept Prot. Res., (1992), 40: 180-193), in an ABI433A automatic peptide synthesizer or by manual chain assembly, or issued and purchased from commercial vendors. For example, a conventional series of Boc SPPS protecting groups was used, namely: Arg (Tos); Asp (cHex); Cys (4MeBzl) & Cys (Acm); Glu (cHex); His (DNP); Lys (CIZ); Be (Bzl); Thr (Bzl); Trp (formyl); Tyr (BrZ); Met, Asn, Gln were unprotected in the side chain. The SEP-0: 4 segment was synthesized analogously in an -OCH2-Pam-resin. Peptides were deprotected and simultaneously cleaved from the resin support using HF / p-crucible according to the conventional Boc chemistry procedure; however, for these peptides containing protective groups not removed in HF / p-crucible, the protecting groups were preserved. The peptides were purified by high pressure reverse phase liquid chromatography of preparation C4 (HPLC). Fractions containing pure peptides were identified using ES-MS (electrospray ionization mass spectrometry), mixed and lyophilized for subsequent ligation.

Stage 1: Ligation # 1 The SEP-0: 4 segment and the SEP-0: 3 segment were dissolved in TFE at a concentration of 15 mM. Saturated phosphate buffer (pH 7.9) containing 6M guanidinium chloride and 1% thiophenol was added, resulting in a clear solution of the peptide segments. The ligation mixture was added to a solution of 2 ml of TFE (trifluoroethanol), 6 ml of 6 M guanidinium chloride, 100 mM phosphate containing 25% β-mercaptoethanol and incubated for 20 minutes. The solution was acidified with a 15 mg / ml solution of TCEP (tris (2carboxyethyl) phosphine · HCl) in glacial acetic acid and loaded onto a reverse phase HPLC column of preparation C4 (1 inch in diameter). The peptides were then purified by reverse phase HPLC with preparation gradient. Fractions containing the desired bound product SEP-0: Acm + SEP-0: 3 + SEP-0: 4 were identified by ES-MS and mixed.

Stage 2: Elimination of Acm No. 1 For the removal of Acm, the aqueous acetonitrile solution containing the mixed fractions of SEP-0: Acm + SEP-0: 3 + SEP-0: 4 was diluted once with quality water HPLC, and solid urea was added for a final concentration of 2 molar. A threefold molar excess (relative to the expected total cysteine concentration) of a 30 mg / ml solution of Hg (acetate) 2 in 3% aqueous acetic acid was added and the solution was stirred for one hour. The solution was then prepared at 20% in β-mercaptoethanol, loaded onto a semi-preparation reverse phase HPLC column and purified with a step gradient. Fractions containing the desired product SEP-0: 3 + SEP-0: 4 were identified by ES-MS and lyophilized overnight.

Stage 3: Ligation # 2 Equal amounts of SEP-0: 3 + SEP-0: 4 and SEP-0: 2 were dissolved together in pure TFE at a concentration of 15 mM. 250 mM phosphate buffer (pH 7.5) containing 6 M guanidinium chloride and 1% thiophenol was added, resulting in a clear solution of the peptide segments. After one day of ligation, the ligation mixture was added to a solution of 10 ml of TFE, 10 ml of β-mercaptoethanol, 10 ml of piperidine and 20 ml of 6 M guanidinium chloride, pH 4, and incubated for 20 minutes to remove any remaining protective group. The solution was acidified with a solution of 15 mg / ml TCEP in 20% aqueous acetic acid, loaded onto an HPLC column in the reverse phase of preparation and purified with a linear gradient. Fractions containing the desired bound product SEP-0: Acm + SEP-0: 2 + SEP-0: 3 + SEP-0: 4 were identified by ES-MS and lyophilized overnight.

Stage 4: Carboxymethylation SEP-0: Acm + SEP-0: 2 + SEP-0: 3 + SEP-0: 4 was dissolved in TFE at a concentration of 15 mM. An excess of double (v / v) 200 mM phosphate buffer (pH 7.9) containing 6 M guanidinium chloride was added, resulting in a clear solution of the peptide segment. A 25-fold excess of bromoacetic acid dissolved in a minimum amount of methanol was added, and the solution was allowed to react for two hours. The solution was acidified with a solution of 15 mg / ml of TCEP in 20% aqueous acetic acid, loaded onto an HPLC column in the reverse phase of preparation and purified with a step gradient. Fractions containing the product SEP-0: Acm + SEP-0: 2 + SEP-0: 3 + SEP-0: 4 + Carboxymethylated Et were identified by ES-MS and mixed.

Stage 5: Elimination of the picolyl Zinc powder was activated in 2M HCl for 30 minutes. Peptide SEP-0: Acm + SEP-0: 2 + SEP-0: 3 + SEP-0: 4 + Et was dissolved in pure TFE at approximately a concentration of 10 mg / ml. The solution was diluted with 4 x (v / v with respect to TFE) 6 M guanidinium chloride, 100 mM acetate, pH 4, containing 35 mg / ml of L-methionine (freshly added) and 35 mg / ml of dodecyl sarcosine (i.e. sodium N-dodecanoylsarcosine). The solution was added to the activated Zn powder. The reaction was monitored at intervals of ~ 1 h and was terminated after five hours. After termination, the supernatant was removed and the remaining Zn powder was washed twice for five minutes with 6 M guanidinium chloride, pH 4, 100 mM acetate containing 35 mg / ml of L-methionine and 35 mg / ml of dodecyl sarcosine containing 20% TFE as well as once with the same solution containing 20% β-mercaptoethanol. The combined product was acidified with a 15 mg / ml solution of TCEP in 20% aqueous acetic acid, loaded onto an HPLC column in the reverse phase of preparation and purified with a gradual gradient. Fractions containing the product SEP-0: Acm + SEP-0: 2 + SEP-0: 3 + SEP-0: 4 + Modified Et-Peptide desired were identified by ES-MS and mixed.

Stage 6: Elimination of Acm No. 2 The mixed solution of SEP-0: Acm + SEP-0: 2 + SEP-0: 3 + SEP-0: 4 + Et-Pico was diluted 3 times with HPLC quality water, and solid urea was added for a final concentration of 2 molar. A triple molar excess (relative to the total expected cysteine concentration) of 30 mg / ml of Hg (acetate) 2 solution in 3% aqueous acetic acid was added and the solution was stirred for one hour. The solution was then prepared at 20% in β-mercaptoethanol, loaded onto a semi-preparation reverse phase HPLC column and purified with a gradual gradient. Fractions containing the desired product SEP-0: 2 + SEP-0: 3 + SEP-0: 4 + Et-Pico were identified by ES-MS, diluted 2 times (v / v) with water containing 2 x (w / w with respect to peptide mass) DPC (dodecylphosphocholine) and lyophilized overnight.

Stage 7: Ligation # 3 Equal amounts of SEP-0: 2 + SEP-0: 3 + SEP-0: 4 + Et-Pico and SEP-0: 1 were dissolved together in pure TFE at a concentration of 15 mM and He added 250 mM phosphate buffer (pH 7.5) containing 6 M guanidinium chloride. To the solution was added 1% thiophenol. After one day of ligation, the ligation mixture was added to a solution of 10 ml of TFE, 10 ml of β-mercaptoethanol, 10 ml of piperidine and 20 ml of 6 M guanidinium chloride, pH 4, and incubated for 20 minutes to remove some remaining protecting groups. The solution was acidified with a solution of 15 mg / ml of TCEP in 20% aqueous acetic acid, loaded onto an HPLC column in the reverse phase of preparation and purified with a linear gradient. The fractions containing the desired SEP-0 product (1-166) :( SEQ. ID. No. 1) bound ligand were identified by mass spectrometry with electroatomization, diluted twice (v / v) with water containing 2 x ( w / w with respect to the mass of peptide) dodecyl sarcosine and lyophilized.

Step 8: Folding: The SEP-0 peptide (1-166) bound whole was dissolved in 200 mM Tris buffer (pH 8.7) containing 6 M guanidinium chloride and 20% TFE and a tenfold molar excess ( with respect to Cys residues in proteins) of cysteine. This solution was dialyzed overnight against a solution of 200 mM Tris buffer (pH 8.7) containing 3M guanidinium chloride at room temperature. The solution was then dialyzed against a solution of 200 mM Tris buffer (pH 8.7) containing 1 M guanidinium chloride at room temperature for 4 hours at 4 ° C and finally against 10 mM phosphate buffer (pH 7.0 ) for 4 hours at 4 ° C to give the final folded product. Folding by ES-MS of electroatomization and DC spectrometry (circular dichroism) was verified.

Step 9 Purification: The folded polypeptide was concentrated 5 times in vials with centricon concentrator and loaded onto a 10 mM phosphate equilibrated Resource S cation exchange column, pH 7.0. The folded protein was eluted in a linear saline gradient to 500 mM NaCl in 10 minutes. Fractions containing the desired folded SEP-0 (1-166) product were identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and frozen and stored at -80 ° C. An analytical reverse phase HPLC chromatogram and an ES-MS spectrum of the folded protein product, as well as a DC spectrum demonstrated the presence of the folded protein.

Example 2

Synthesis of the synthetic protein SEP-1-L30 stimulating erythropoiesis

A second synthetic erythropoiesis stimulating protein (called SEP-1-L30) containing oxime-forming groups at positions 24 and 126 of SEP-0 was synthesized. These groups were then used to form SEP-1-L30, in which the linear carboxylate polymers of (EDA-Succ) 18 (EDA = (4,7,10) -trioxatridecane 1,13 diamine, also called TTD; Succ = - CO-CH2CH2CO-) have been attached to the central protein axis. The complete sequence of SEP-1 (1-166) is:

image 1

wherein ψ indicates an artificial amino acid residue consisting of a cysteine that is carboxymethylated in the sulfhydryl group, and in which Kox indicates an artificial lysine that is chemically modified in the ε-amino group with a linker oxime group coupled to the polymer Water soluble designed by an oxime bond.

A. Approximation of GRFN1776 and GRFN1711 with GRFNP32

The oxime formation was performed to couple the water soluble polymers that carry an aminooxyacetyl group to the peptides that carry a carbonyl ketone group. For its realization, the following peptide segments were synthesized:

SEP-1: 4 segment (GRFN 1776; composed of residues 117 to 166 of SEQ. ID. No. 2): CAISPPDAAK AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR-carboxylate (in which Lys126 is modified with a levulinic acid residue in the group ε-amino and in which Cys117 is protected with Acm)

SEP-1: 1 segment (GRFN 1711; composed of residues 1 to 32 of SEQ. ID. No. 2): APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester (in which Lys24 is modified with a levulinic acid residue)

The SEP-1: 1 segment (GRFN 1711) was synthesized in a thioester generating resin and the SEP-1: 4 segment (GRFN 1776) in a resin -OCH2-Pam- as in Example 1. Lysines 24 and 126 of these two peptide segments were initially protected with an Fmoc group in the ε-amino group. Once the chain assembly was finished, the amino groups carrying Fmoc were deprotected following the standard Fmoc deprotection procedures and modified by coupling levulinic acid to each of the peptide resins, respectively. The peptides were then deprotected and simultaneously cleaved into the resin support as described in Example 1. The peptides were purified by C4 reverse phase HPLC preparation. Fractions containing pure peptide were identified using ES-MS, mixed and lyophilized for subsequent ligation. GRFNP32 [- (EDA-Succ) 18 carboxylate] was assembled into the carboxyl-generating Sasrin resin following conventional protocols (Rose, K. et al. US Patent Application Serial No. 09 / 379,297; and Rose et al., J. Am. Chem. Soc. (1999) 121: 7034). An amino-oxyacetyl (AoA) moiety was coupled to the N-terminal amino group of the polymer by coupling a five-fold excess of activated Boc-amino-oxyacetic acid. The polymer chain was cleaved into the resin support using the classic Fmoc chemistry procedures. The polymer chain was purified by C4 reverse phase HPLC preparation. Fractions containing the pure polymer were identified using ES-MS, mixed and lyophilized for subsequent ligation.

Segment SEP-1: 4 and GRFNP32 were dissolved together in an equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA. The solution was then lyophilized. The anhydrous powder was dissolved and the polymer modified peptide was separated into an unmodified peptide and an unreacted polymer by C4 reverse phase HPLC with preparation gradient. Fractions containing the SEP-1: 4 + oxygenated GP32 product were identified by ES-MS and mixed and lyophilized.

Segment SEP-1: 1 and GRFNP32 were dissolved together at an equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA. The solution was then lyophilized. The dried powder was dissolved and the polymer modified peptide was separated from the unreacted polymer by C4 reverse phase HPLC with preparation gradient. Fractions having the desired SEP-1: 1 + GP32 oxygenated product were identified by ES-MS and mixed and lyophilized.

B. Synthesis of the synthetic protein SEP-1-L30 stimulating erythropoiesis

SEP-1-L30 was synthesized in solution from four polypeptide segments:

Segment SEP-1: 1 + GP32 (GRFN 1711+ GRFNP32, corresponding to residues 1 to 32 of SEQ ID No. 2): APPRLICDSR VLERYLLEAK EAEKoxITTGCA EH-thioester (in which Lys24 is modified in the εamino group with a oxime levulinic linker group that is coupled to GRFNP32 via an aminooxyacetyl-levulinum oxime bond (Lev-AoA) called Kox)

Segment SEP-1: 2 (GRFN 1712, corresponding to residues 32 to 38 of SEQ ID No. 2): CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW-thioester (where Cys33 is protected by Acm)

Segment SEP-1: 3 (GRFN 1713, corresponding to remains 89 to 116 of SEQ. ID. No. 2): CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys89 is protected by Acm)

SEP segment: 1: 4 + GP32 (GRFN 1776 + GRFNP32, corresponding to residues 117-166 of SEQ ID NO: 2): CAIS PPDAAKoxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate (in which Lys126 is modified in the ε-amino group with a linker levulinum oxime group that is coupled to GRFNP32 via an aminooxyacetyl-levulinum oxime bond (Lev-AoA) called Kox, and in which the C-terminal cysteine carries a picolyl (peak) protecting group.

The synthesis of additional peptides, ligation reactions, carboxymethylation, elimination reactions of the protective group, folding and purification were performed as described in Example 1 to give folded, complete SEP-1L30. An analytical reverse phase HPLC chromatogram and an ES-MS spectrum of the folded protein product as well as a DC spectrum demonstrated the presence of the folded protein.

Example 3

Synthesis of the synthetic protein SEP-1-L26 stimulating erythropoiesis

A third synthetic erythropoiesis stimulating protein (called SEP-1-L26) containing oxime-forming groups at positions 24 and 126 of SEP-0 was synthesized. These groups were then used to form SEP-1-L26, in which the linear carboxylate polymers of (EDA-Succ) 18 and (EDA-Succ) 6-amide have been attached to the central protein axis via the oxime bonds in positions 24 and 126, respectively. The complete sequence of SEP-1 (1-166) is:

image 1

5 in which ψ indicates an artificial amino acid residue consisting of a cysteine that is carboxymethylated in the sulfhydryl group, and in which Kox indicates an artificial lysine that is chemically modified in the ε-amino group with a linker oxime group coupled to a water soluble polymer designed by an oxime bond.

10 In contrast to SEP-1-L30, the assembly of SEP-1-L26 was designed to carry a smaller, no-load water soluble polymer bonded in position 126. The polymer bonded in position 24 was the same as in SEP-1L30. The assembly of the complete product was as described in Example 2.

A. Proximation of GRFN1776 with GRFNP6 and Oximation of GRFN1711 with GRFNP32

The oxime formation was performed to bind water soluble polymers that carry an amino-oxyacetyl group to peptides that carry a carbonyl ketone group. To accomplish this, the following peptide segments were synthesized:

20 Segment SEP-1: 4 (GRFN 1776; composed of residues 117 to 166 of SEQ. ID. No. 2): CAISPPDAAK AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR-carboxylate (in which Lys126 is modified with a levulinic acid residue in the ε-amino group and in which Cys117 is protected with Acm)

SEP-1: 1 segment (GRFN 1711; composed of residues 1 to 32 of SEQ. ID. No. 2): APPRLICDSR 25 VLERYLLEAK EAEKITTGCA EH- (in which Lys24 is modified with a levulinic acid residue)

The SEP-1: 1 segment (GRFN 1711) was synthesized in a thioester generating resin and the SEP-1: 4 segment (GRFN 1776) in a resin -OCH2-Pam- as in Example 1. Lysines 24 and 126 of these two peptide segments were initially protected with an Fmoc group in the ε-amino group. Once the chain assembly was completed, the 30 amino groups carrying Fmoc were deprotected following standard Fmoc deprotection procedures and modified by coupling levulinic acid to each of the peptide resins, respectively, following the conventional coupling protocols . The peptides were then deprotected and simultaneously cleaved into the resin support as described in Example 1. The peptides were purified by C4 reverse phase HPLC preparation. For each peptide, the fractions that

35 containing pure peptide were identified using ES-MS, mixed and lyophilized for subsequent ligation.

The water-soluble polymer (EDA-Succ carboxylate) 18 (GRFNP32) was assembled into the carboxyl-generating Sasrin resin following conventional protocols (Rose, K. et al. US Patent Application Serial No. 09 / 379,297; Rose et al., J. Am. Chem. Soc. (1999) 121: 7034). The water-soluble polymer (EDA-Succ) 6-amide 40 (GRFNP6) was assembled into the Sieber amide-generating resin following conventional protocols (Rose, K. et al. US Patent Application Serial No. 09 / 379,297; Rose et al., J. Am. Chem. Soc. (1999) 121: 7034). An amino-oxyacetyl (AoA) moiety was coupled to the N-terminal amino group of each polymer bound to the resin by coupling a five-fold excess of activated Boc-amino-oxyacetic acid. The two polymer chains were cleaved separately from the respective resin supports using the classic Fmoc chemistry procedures. Each string of

Polymers were purified by C4 reverse phase HPLC preparation. For each polymer, fractions containing pure polymer were identified using ES-MS, mixed and lyophilized for subsequent ligation.

Segment SEP-1: 4 and GRFNP6 were dissolved together in an equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA. The solution was then lyophilized. The anhydrous powder dissolved and the peptide

The polymer modified was separated into an unmodified peptide and an unreacted polymer by C4 reverse phase HPLC with preparation gradient. Fractions containing the SEP-1: 4 + oxygenated GP6 product were identified by ES-MS and mixed and lyophilized.

Segment SEP-1: 1 and GRFNP32 dissolved together in an equimolar ratio in aqueous acetonitrile at

50 50% containing 0.1% TFA. The solution was then lyophilized. The anhydrous powder was dissolved and the polymer modified peptide was separated into an unmodified peptide and an unreacted polymer by C4 reverse phase HPLC with preparation gradient. Fractions containing the SEP-1: 1 + oxygenated GP32 product were identified by ES-MS and mixed and lyophilized.

60 B. Synthesis of the synthetic protein SEP-1-L26 erythropoiesis stimulant

SEP-1-L26 was synthesized in solution from four polypeptide segments: Segment SEP-1: 1 + GP32 (GRFN 1711+ GRFNP32, corresponding to residues 1 to 32 of SEQ ID NO: 2): APPRLICDSR VLERYLLEAK EAEKoxITTGCA EH-thioester (in which Lys24 is modified in the εamino group with a linker levulinum oxime group that is coupled to GRFNP32 via an aminooxyacetyl-levulinum oxime bond (Lev-AoA) called Kox)

Segment SEP-1: 2 (GRFN 1712, corresponding to residues 33 to 38 of SEQ ID No. 2): CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW-thioester (where Cys33 is protected by Acm)

Segment SEP-1: 3 (GRFN 1713, corresponding to remains 89 to 116 of SEQ. ID. No. 2): CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys89 is protected by Acm)

Segment SEP-1: 4 + GP6 (GRFN 1776 + GRFNP6, corresponding to residues 117 to 166 of SEQ ID No. 2): CAIS PPDAAKoxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate (in which Lys126 is modified in group ε -amino with a levulinum oxime linker group that is coupled to GRFNP6 via an aminooxyacetyl-levulinum oxime bond (Lev-AoA) called Kox, and in which the C-terminal cysteine [ie, Cys161] carries a picolyl protecting group (peak ).

The synthesis of additional peptides, ligation reactions, carboxymethylation, elimination reactions of the protecting group, folding and purification were performed as described in Examples 1 and 2 to give folded, complete SEP-1-L26. An analytical C4 reverse phase HPLC chromatogram and an ES-MS spectrum of the folded protein product as well as a DC spectrum demonstrated the presence of the folded protein.

Example 4

Synthesis of the synthetic protein SEP-1-B50 stimulating erythropoiesis

A fourth synthetic erythropoiesis stimulating protein (called SEP-1-B50) was synthesized. The amino acid sequence of SEP-1-B50 is the same as that of SEP-1-L30:

image 1

wherein ψ indicates an artificial amino acid residue consisting of a cysteine that is carboxymethylated in the sulfhydryl group, and in which Kox indicates an artificial lysine that is chemically modified in the ε-amino group with a linker oxime group coupled to the polymer Water soluble designed by an oxime bond.

However, the protein was modified with a branched polymer assembly having four linear residues (SuccEDA) 12 instead of the linear polymer (Succ-EDA) 18 of SEP-1-L30. The modification was made by oxime links. The assembly of the complete product was as described in Example 2.

A. Summary of the GRFNP17 template that carries several thiol groups

The GRFNP17 template was manually synthesized in a (4-methyl) benzydrylamine (MBHA) resin that generates amide on a 0.4 mmol scale. Fmoc-Lys (Boc) -OH was coupled using conventional coupling protocols (Schnölzer, M., Int. J. Pept. Protein. Res., (1992), 40: 180-93). 2.1 mmol of amino acid, 10% DIEA in 3.8 ml of 0.5 M HBTU were used; that is, an excess of 5 times of amino acid. After removal of the Fmoc protecting group, Fmoc-Lys (Fmoc) -OH was coupled using conventional amino acid coupling protocols (2.1 mmol of amino acid, 10% DIEA in 3.8 ml of 0.5 M HBTU; that is an excess of five times of amino acid). After a second stage of Fmoc elimination, Fmoc-Lys (Fmoc) -OH was coupled using conventional amino acid coupling protocols (4.2 mmol of amino acid, 10% DIEA in 7.6 ml of 0.5 M HBTU; it is say an excess of 5 times of amino acid in relation to the free amine). After the final stage of deprotection of Fmoc, a 5-fold excess (relative to free amines) of S-acetyl thioglycolic acid pentanofluorophenyl ester (SAMA-oPfp) in DMF was coupled for 30 minutes. The Boc protecting group of the side chain of the lysyl moiety of terminal C was removed twice a minute of batch washes with pure TFA, followed by neutralization of the resin by washing with 10% DIEA in DMF. 2 mmol of Bocamino oxyacetic acid and 2 mmol of N-hydroxysuccinimide (NHS) were dissolved in 3 ml of DMF. After the addition of 2 mmol of DIC (di-isopropylcarbodiimide), the acid was activated for 30 to 60 minutes. The solution was added to the neutralized resin and coupled for 1 h. Finally, the S-linked acetyl groups were removed with 20% piperidine in DMF for 30 minutes. The template was deprotected and simultaneously excised from the resin support using HF / pcresol according to conventional Boc chemistry procedures in the presence of cysteine as an antioxidant for free aldehyde (Schnölzer, M., Int. J. Pept. Protein Res. , (1992), 40: 180-93). The polyamide recovered in 50% B [ie, 50% aqueous acetonitrile containing 0.1% TFA] (free aldehyde) was frozen and lyophilized. For purification, the crude product of the template was dissolved in 2 ml of 50% B, and 100 ml of 100% A [ie 0.1% TFA in water] was added to dilute the sample (avoid the addition of guanidinium chloride or acetate, since the addition of aldehyde is guaranteed). The template was loaded on a reverse phase HPLC column of preparation C4 equilibrated at T = 40 ° C in 3% B. The salts were eluted isocratically and the desired template, GRFNP17 was purified in a linear gradient. Fractions containing the desired product were identified by ES-MS, mixed and lyophilized.

B. Synthesis of branched water soluble GRFNP29 polymer

GRFNP29, a 15 kDa branched polymer (EDA-Succ) 12 of molecular weight per thioether linker of the GRFNP17 template containing purified thiol and a linear polymer GRFNP31, Br-acetyl carboxylate (EDA-Succ) 12, was synthesized. in which GRFNP31 was synthesized on a Sasrin carboxy-generating resin following conventional protocols (Rose, K. et al., US Patent Application Serial No. 09 / 379,297; Rose et al., J. Am. Chem. Soc. (1999) 121: 7034).

An excess of 1.3 molar times (over total thiols) of purified GRFNP31, Br-acetylated (EDA-Succ) 12, and the template containing purified thiol GRFNP17 were dissolved together in 0.1 M Tris-HCl / chloride 6 M guanidinium, pH 8.7 at a concentration ~ 10 mm. After dissolution, the solution was diluted triple (v / v) with 0.1 M Tris-HCl, pH 8.7 buffer. The ligation mixture was stirred at room temperature and the reaction was monitored by reverse phase HPLC and ES / MS. The traditional GRFNP31 reagent was added as needed until the desired reaction product was the main product. For the assay, 0.1 M acetate / 6 M acetate / 6 M guanidinium chloride, pH 4, was added 3 times (v / v), and the solution was loaded onto a C4 reverse phase HPLC column of preparation and prepared. purified with a linear gradient. Fractions containing the pure GRFNP29 assembly were identified using ES-MS, mixed and lyophilized.

C. Maximization of GRFN1776 and GRFN1711 with GRFNP29

Segments SEP-1: 4 and segment SEP-1: 1 were synthesized as described in Example 2. Segments SEP-1: 4 and GRFNP29 were dissolved together in an equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA. The solution was lyophilized. The dried powder was loaded on a reverse phase HPLC column (1 inch in diameter). The modified polymer peptide was separated from the unmodified peptide and the unreacted polymer by C4 reverse phase HPLC preparation gradient. Fractions containing the desired SEP-1: 4 + oxygenated GP29 product were identified by ES-MS and mixed.

Segment SEP-1: 1 and GRFNP29 were dissolved together in an equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA. The solution was frozen and lyophilized. The dried powder was dissolved in 50% aqueous acetonitrile containing 0.1% TFA and loaded onto a GPC preparation column (gel penetration chromatography) (1 inch in diameter). The polymer modified peptide was separated from the unmodified peptide and from the unreacted polymer by isocratic elution. Fractions containing the desired SEP-1: 1 + oxygenated GP29 product were identified by ES-MS and mixed.

D. Synthesis of the synthetic protein SEP-1-B50 erythropoiesis stimulant

SEP-1-B50 (SEQ ID No. 2) was synthesized in solution from four polypeptide segments:

Segment SEP-1: 1 + GP29 (GRFN 1711+ GRFNP29, corresponding to residues 1 to 32 of SEQ ID No. 2): APPRLICDSR VLERYLLEAK EAEKoxITTGCA EH-thioester (in which Lys24 is modified in the εamino group with a oxime levulinic linker group that is coupled to GRFNP29 via an aminooxyacetyl-levulinum oxime bond (Lev-AoA) called Kox)

Segment SEP-1: 2 (GRFN 1712, corresponding to residues 33 to 88 of SEQ ID No. 2): CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW-thioester (where Cys33 is protected by Acm)

Segment SEP-1: 3 (GRFN 1713, corresponding to remains 89 to 116 of SEQ. ID. No. 2): CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys89 is protected by Acm)

Segment SEP-1: 4 + GP29 (GRFN 1776 + GRFNP29, corresponding to residues 117 to 166 of SEQ ID No. 2): CAIS PPDAAKoxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate (in which Lys126 is modified in group ε -amino with a levulinum oxime linker group that is coupled to GRFNP29 via an aminooxyacetyl-levulinum oxime bond (Lev-AoA) called Kox, and in which the C-terminal cysteine carries a picolyl protecting group (peak).

The synthesis of additional peptides, ligation reactions, carboxymethylation, elimination reactions of the protecting group, folding and purification were performed as described in Examples 1 and 2, except that the purification was on a Resource Q column, to give SEP-1-B50 folded, complete. An analytical C4 reverse phase HPLC chromatogram and an ES-MS spectrum of the SEP-1-B50 folded protein product as well as a DC spectrum demonstrated the presence of the folded protein.

Example 5 Synthesis of the synthetic protein SEP-3-L42 stimulating erythropoiesis

A fifth synthetic erythropoiesis stimulating protein (called SEP-3-L42) was synthesized. The amino acid sequence of the complete SEP-3 protein is:

image 1

The cysteine residues at positions: 24, 38, 83 and 126 were modified with units of polymer (EDA-Succ) 18 (GRFNP32) functionalized with maleimide (by a Michael addition reaction) to form SEP-3-L42.

In detail, SEP-3 was synthesized in solution from four polypeptide segments:

SEP-3: 1 segment (GRFN 1747, corresponding to remains 1 to 37 of SEQ. ID. No. 3): APPRLICDSR VLERYLLEAK EAECITTGCA EHCSLNE-thioester (in which Cys7, Cys29 and Cys33 are protected by Acm)

Segment SEP-3: 2 (GRFN 1774, corresponding to remains 38 to 82 of SEQ. ID. No. 3): CIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LA-thioester (where Cys38 is protected by Acm)

Segment SEP-3: 3 (GRFN 1749, corresponding to residues 83 to 125 of SEQ ID No. 3): CSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS PPDAA-thioester (where Cys83 is in the side chain protected by Acm)

Segment SEP-3: 4 (GRFN 1750, corresponding to residues 126 to 166 of SEQ ID No. 3): CAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate (in which Cys161 is Pbo [ie, 4 (CH3S (O ) -) benzyl-] protected.

Peptide Synthesis SEP-3: 1 and SEP-3: 2 and SEP-3: 3 peptides were synthesized in a thioester generating resin using the in situ neutralization protocol for chemical SPPS BOC, using the side chain protection strategies established as described in example 1, with changes in the strategy of the protective group as seen in the specific peptides above. The SEP-3: 4 segment was synthesized analogously in a resin –OCH2-Pam. The peptides were deprotected and simultaneously cleaved from the resin support as described in example 1. The resulting peptides described above are purified by preparative HPLC-RP. Fractions containing the pure peptide were identified using ES-MS, accumulated and lyophilized for subsequent ligation.

Stage 1: Ligation # 1 The SEP-3: 4 segment and the SEP-3: 3 segment were dissolved in TFE at a concentration of 15 mM. Saturated phosphate buffer (pH 7.5) containing 6M guanidinium chloride and 1% thiophenol was added, resulting in a clear solution of the peptide segments. After ligation, the ligation mixture (defined as 1 volume) was added to 2 volumes of a solution of {2 ml of TFE, 6 ml of 6 M guanidinium chloride, 100 mM phosphate containing 25% β-mercaptoethanol } and incubated for 20 minutes. The solution was acidified with a 15 mg / ml solution of TCEP in glacial acetic acid and loaded onto a reverse phase HPLC column of preparation C4 with a linear gradient. Fractions containing the desired bound product SEP3: Acm + SEP-3: 3 + SEP-3: 4 were identified by ES-MS and mixed.

Stage 2: Elimination of Acm No. 1 For the removal of Acm, the aqueous acetonitrile solution containing the mixed fractions of SEP-3: Acm + SEP-3: 3 + SEP-3: 4 was diluted once with quality water HPLC, and solid urea was added for a final concentration of 2 molar. A threefold molar excess (relative to the expected total cysteine concentration) of a 30 mg / ml solution of Hg (acetate) 2 in 3% aqueous acetic acid was added and the solution was stirred for one hour. The solution was then prepared at 20% in β-mercaptoethanol, loaded on a C4 reverse phase semi-preparation HPLC column and purified with a step gradient. Fractions containing the desired product SEP-3: 3 + SEP-3: 4 were identified by ES-MS and lyophilized overnight.

Stage 3: Ligation # 2 Equal amounts of SEP-3: 3 + SEP-3: 4 and SEP-3: 2 were dissolved together in pure TFE at a concentration of 15 mM. 250 mM phosphate buffer (pH 7.5) containing 6 M guanidinium chloride and 1% thiophenol was added, resulting in a clear solution of the peptide segments. After one day of ligation, the ligation mixture (defined as 1 volume) was added to 2 volumes of a solution of 10 ml of TFE, 10 ml of β-mercaptoethanol, 10 ml of piperidine and 20 ml of guanidinium chloride 6 M, pH 4, and incubated for 20 minutes to remove any remaining protecting group. The solution was acidified with a solution of 15 mg / ml of TCEP in 20% aqueous acetic acid, loaded onto a C4 reverse phase HPLC column of preparation and purified with a linear gradient. Fractions containing the desired bound product SEP3: Acm + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 were identified by ES-MS and lyophilized overnight.

Step 4: Acm Removal For Acm removal, the aqueous acetonitrile solution containing the mixed fractions of SEP-3: Acm + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 was diluted 1 time with HPLC quality water, and solid urea was added for a final concentration of 2 molar. A triple molar excess (relative to the total expected cysteine concentration) of 30 mg / ml of Hg (acetate) 2 solution in 3% aqueous acetic acid was added and the solution was stirred for one hour. The solution was then prepared at 20% in β-mercaptoethanol, loaded on a C4 reverse phase HPLC column of semi-preparation and purified with a gradual gradient. Fractions containing the desired bound product SEP-3: 2 + SEP-3: 3 + SEP-3: 4 were identified by ES-MS and lyophilized overnight.

Stage 5: Ligation # 3 Equal amounts of SEP-3: 2 + SEP-3: 3 + SEP-3: 4 and SEP-3: 1 were dissolved together in pure TFE at a concentration of 15 mM. 250 mM phosphate buffer (pH 7.5) containing 6 M guanidinium chloride and 1% thiophenol was added, resulting in a clear solution of the peptide segments. After one day of ligation, the ligation mixture (defined as 1 volume) was added to 2 volumes of a solution of 10 ml of TFE, 10 ml of β-mercaptoethanol, 10 ml of piperidine and 20 ml of guanidinium chloride 6 M, pH 4, and incubated for 20 minutes to remove some remaining protecting groups. The solution was acidified with a 15 mg / ml solution of TCEP in 20% aqueous acetic acid, loaded onto a C4 reverse phase HPLC column of preparation and purified with a linear gradient. Fractions containing the product SEP3: 1 + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 were identified by ES-MS and lyophilized overnight.

Step 6: Coupling of the GRFNP32 polymer A linear polymer (EDA-Succ) 18 functionalized with maleimide called GRFNP32-maleimide was prepared by functionalizing GRFNP32 with BMPS (NHS ester of propionic 3maleinido acid Pierce, USA) following the manufacturers' protocols to form a polymer (EDA-Succ) 18 functionalized with maleimide [ie, maleimide- (EDA-Succ) 18]. SEP-3: 1 + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 was dissolved in the minimum amount of TFE required. An excess of three times GRFNP32-maleimide was dissolved in 6 M guanidinium chloride, 100 mM phosphate, pH 7.5 and added to the TFE solution. The evolution of the Michael addition reaction was followed by reverse phase analytical HPLC. Once the reaction was over, the solution was loaded on a C4 reverse phase HPLC column of preparation and purified with a linear gradient. Fractions containing product SEP-3: Acm + SEP-3: 1 + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 + pPEG modified with desired polymer [ie 166 residue polypeptide chain complete with four copies of GRFNP32 coupled to the thiols of the side chain of Cys24, Cys38, Cys83 and Cys126 and thus also called SEP-3: Acm + SEP3: 1 + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 + GP32], were identified by ES-MS and lyophilized overnight.

Stage 7: Elimination of Pbo. For the reduction of Pbo, the lyophilized powder of SEP3: Acm + SEP-3: 1 + SEP-3: 2 + SEP3: 3 + SEP-3: 4 + GP32 was dissolved in pure TFA containing 5% ethanedithiol. The Pbo group was then cleaved by the addition of 10% thioanisole and 15% bromotrimethylsilane for 30 minutes. The solution was dried in a rotary evaporator and extracted in aqueous acetonitrile containing 0.1% TFA. The resulting solution was loaded on a semi-preparation reverse phase HPLC column and purified with a gradual gradient. Fractions containing the desired product SEP3: Acm + SEP-3: 1 + SEP-3: 2 + SEP-3: 3 + SEP-3: 4 + GP32-Pbo unprotected by Cys161 were identified by ES-MS and lyophilized overnight.

Step 8: Acm removal For the final removal of Acm from the side chains of Cys7, Cys29 and Cys33, the aqueous acrylonitrile solution containing the mixed fractions of SEP3: Acm + SEP-3: 1 + SEP-3: 2+ SEP3: 3 + SEP-3: 4 + GP32-Pbo was diluted 1 time with HPLC quality water, and solid urea was added for a final concentration of 2 molar. A triple molar excess (relative to the total expected cysteine concentration) of 30 mg / ml of Hg (acetate) 2 solution in 3% aqueous acetic acid was added and the solution was stirred for one hour. The solution was then prepared at 20% in β-mercaptoethanol, loaded on a C4 reverse phase HPLC column of semi-preparation and purified with a gradual gradient. Fractions containing the desired product SEP-3 (1-166) and lyophilized overnight.

Stage 9: Folding: The SEP-3 peptide (1-166) bound whole was dissolved in 200 mM Tris buffer (pH 8.7) containing 6 M guanidinium chloride and 20% TFE and a tenfold molar excess ( with respect to the remains with Cys in SEP-3) of cysteine. This solution was dialyzed overnight against a solution of 200 mM Tris buffer (pH 8.7) containing 3M guanidinium chloride at room temperature. The solution was then dialyzed against a solution of 200 mM Tris buffer (pH 8.7) containing 1 M guanidinium chloride at room temperature for 4 hours at 4 ° C and finally against 10 mM phosphate buffer (pH 7.0 ) for 4 hours at 4 ° C to give the final folded product. Folding was verified by ES-MS for electroatomization and DC spectrometry.

Step 10 Purification: The folded polypeptide was concentrated 5 times in vials with centricon concentrator and loaded onto a 10 mM phosphate equilibrated Resource S cation exchange column, pH 7.0. The folded protein was eluted in a linear saline gradient to 500 mM NaCl in 10 minutes. Fractions containing the desired folded SEP-3L42 product were identified by SDS-PAGE and frozen and stored at -80 ° C.

Example 6

5

Bioactivity assay of erythropoiesis stimulating synthetic proteins

The bioactivity of the erythropoiesis-stimulating synthetic proteins, SEP-0, SEP-1-L26, SEP-1-L30, SEP-1-B50 and SEP-3-L42 was determined using UT / 7 and 32D cell lines 103197, in factor-dependent cell line proliferation assays using commercial recombinant erythropoietin as a reference standard. UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on one of interleukins-3, macrophage and granulocyte colony stimulating factor (GM-CSF) or erythropoietin (EPO) for growth and survival (Miura , Y. et al., Acta Haematol. (1998) 99: 180-184; Komatsu, M. et al., Cancer Res. (1991) 51: 341-8). 32D 103197 is a murine hemopoietic cell line (Metcalf, D., Int. J. Cell

15 Cloning (1992) 10: 116-25).

SEP assembly stock solutions were prepared in a Dulbecco medium modified by Iscove (IMDM), 10% FBS (fetal bovine serum), glutamine and Penstrep, and 2 x serial dilutions of these stock solutions were added to multiwell plates. which were added UT / 7 EPO cells at a concentration of 5,000 cells / 50 µl. Plates were incubated at 37 ° C in the presence of 5% CO2 and growth was monitored daily. After four days, 20 µl of 2.5 mg / ml MTT (methyl thiazole tetrazolium) in PBS (phosphate buffered saline) was added and the plates were incubated for four hours. 150 µl of IPA was added and the absorbance of each well was read at 562 nm. ED50 values (dose effective to reach 50% maximum effect) for SEP compounds were determined and compared with those of rhEPO (recombinant human erythropoietin)

25 produced by CHO cells (Chinese hamster ovary). The results of these experiments showed that all synthetic erythropoiesis stimulating proteins had bioactivity. The results of the ED50 for SEP0, SEP-1-L26, SEP-1-L30 and SEP-1-B50 are shown in Table VI.

Table VI 30

Stimulating protein of

In vitro ED50 values (μM)

erythropoiesis

UT-7 (human) cells 32D 103197 cells (mouse)

SEP-0

1,570 863

SEP-1-L26

46.5 100.8

SEP-1-L30

71.5 182.5

SEP-1-B50

182 6200

rh EPO

32.5 136.3

Sequence listing

35

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<130> 03504.268

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Claims (2)

1. Procedure for synthesizing a desired polypeptide of formula: aaNH2-Q-aax-aay-W-aaCOOH wherein Q and W each indicate the optional presence of one or more additional amino acid residues, aaNH2 indicates the N-terminal amino acid residue of the polypeptide; aax and aay indicate the internal adjacent amino acid residues, which have the x and y side chains, respectively, and in which aay is a ribosomically unspecified amino acid residue and aaCOOH indicates the C-terminal amino acid residue of the polypeptide; Understanding the procedure:

(TO)

bind a first peptide having the formula: aaNH2-Q-aax-COSR, in which R is any group compatible with the thioester group, to a second peptide having the formula: Cys-W-aaCOOH, to thereby form the polypeptide : aaNH2-Q-aaX-Cys-W-aaCOOH; Y

(B)

incubating said polypeptide in the presence of a Raa-X reagent, in which Raa-X is selected from the group consisting of X-CH2COOH, X- (CH2) 2COOH, X- (CH2) -C (O) NH2, X -CH2OH, X-CHOHCH3, X-CH2CHOHCH3, X-CH2CH2OH, X- (CH2) 2NH-GP, X-CH2NH-C (NH2) 2, X- (CH2) 2NH-C (NH2) 2, X-CH2CH3 , X-CH2φ, X-φ-OH (for), X-CH2φ-OH (for), X-CH2φ-OPO3 (for), X-CH2-φ- (OH) 2 (target for), X-CH (COOH) 2, X-CH2-IM-GP, X-CH- (CH3) 2, X-CH2CH- (CH3) 2, X-CH (CH3) -CH2-CH3, X-CH2-CH (CH3) -CH2-CH3, in which X is I or Br, and X-φ, in which X is F, and X-CH2-IN, in which X is F, I or Br, in which φ indicates a benzyl group, IN indicates an indole group, IM indicates an imidazole group and GP indicates a protective group;

said incubation being under conditions sufficient to form said desired polypeptide. 2. A process according to claim 1, wherein R is an aryl, benzyl or alkyl group.