HK1118295A - Method of peptide synthesis - Google Patents

Description

Peptide synthesis method

The present invention relates to a solid phase peptide synthesis method for peptides that are generally difficult to synthesize, and to a representative peptide-solid phase conjugate.

One challenge with solid phase peptide synthesis is that, despite the existence of well established routine methods, successful and efficient synthesis is highly dependent on the sequence of the peptide to be synthesized.

Certain peptide sequences, taken from natural peptide or protein segments, perform much worse than others on the same resin material that is used with high success for many 'normal' peptides. In the literature, it has been proposed to fine-tune the solvent system with frequent care in view of preventing intrachain aggregation on the resin due to beta sheet formation. Furthermore, the use of dichloromethane, N-methylpyrrolidone or dimethylformamide, eventually supplemented with a specific polar solvent such as trifluoroethanol, which stabilizes the spirochete structure, does not provide a universal solution at all for difficult peptides to synthesize.

Thymosin alpha₁(International non-proprietary name: thymalfasin) is an N-acetylated 28-mer peptide hormone produced in the thymus of human beings and is used as a drug for the treatment of chronic hepatitis B.

The linear full-length solid-phase synthesis has been described for a long time, but has been suffered from poor yields to date. Unfortunately, thymosin alpha₁The amino acid sequences of (a) do not provide glycine or proline as spacer residues that are useful in the traditional step-by-step coupling methods of the stepwise synthesis.

Echner et al (Liebigs Ann. chem., 1988, 1095-1097) describe linear solid phase synthesis on Wang-polystyrene resin. Although it is stated that the coupling efficiency was checked with the Kaiser test after each coupling with BOP reagents dissolved in DMF, it only caps the uncoupled peptide with acetylation after every fifth coupling. It cleaves the protected and end-acetylated peptide from the resin using concentrated trifluoroacetic acid; the resin and liquid phases were then separated using filtration and precipitation with diethyl ether was carried out to collect the precipitate. The precipitate collected in this way was quantified and labeled as 'crude product', and the yield was calculated to be 76%. Only thereafter is the crude product subjected to a first chromatographic purification, followed by reverse phase HPLC. The yield of pure product thus obtained is not indicated.

The problem with the Echner method is that incomplete chain coupling of undesirable sequence variants resulting in shortening upon synthesis is not taken into account in the calculation of the yield. 'crude product' is equal to every peptide obtained during synthesis, including incompletely deprotected/alkylated peptides. However, when peptides that are difficult to synthesize are involved, the purity of peptides derived from linear synthesis becomes an issue; interestingly, Echner did not indicate any yield of the purified product thus obtained, nor did it give any HPLC chromatogram as routinely done in the art in its publication to prove the quality of the synthesis. In our practice of the procedure, the synthetic scheme of Echner only produces very low yields of the correct product, with various incorrect peptide products constituting the majority of the crude product.

A further problem associated with the Echner approach (page 1095, left column, paragraph 3) addressed by the authors was the incorporation of thymosin alpha₁The C-terminal amino acid of (1), asparagine, is loaded onto Wang resin; the loading achieved by Echner is 0.15 mmol/g, which is a very low loading. Fmoc-Asn (4, 4' -dimethoxydiphenylmethyl-) -OH amino acid must be used to load the resin without risk of degradation side reactions.

It is an object of the present invention to devise an improved thymosin alpha₁The solid phase synthesis method of (1). This object is solved by the method of claim 1.

According to the invention, a method for synthesizing thymosin alpha on a solid phase is provided₁Or comprises mature thymosin alpha₁C-terminally truncated form of residues 1-27, comprising at least mature thymosin alpha₁N-terminally truncated forms of residues 19-28, or at least comprising mature thymosin alpha₁A method of truncating residues 19-27, said method comprising the steps of:

a. coupling appropriately protected FMOC-Glu or FMOC-Asn residues to a PEG resin, wherein the PEG resin is preferably selected from the group consisting of: polystyrene-PEG mixed resin, PEG polyether-polyester mixed resin, or PEG polyether-polyamide mixed resin and substantially pure PEG resin,

b. peptide chains are extended by FMOC synthesis, where the side chains of individual amino acids can be derivatized with appropriate protecting groups,

c. optionally acetylating the last N-terminal residue, and

d. the peptide was cleaved from the resin.

The present invention enables synthesis of thymalfasin in unprecedented purity and consequently high yields in a single solid phase linear synthesis. According to the invention it is also possible to synthesize only the C-terminal fragment on a solid phase and combine any full-length peptide or at least longer forms of this peptide by means of segment coupling, in a preferred embodiment by means of coupling an N-terminal fragment having a C-terminal Ser or Thr which is protected against racemization by the pseudoproline dipeptide method (W _ hr et al, J.Am.chem.Soc., 118, 9218).

Preferably, the peptide to be synthesized is thymosin alpha having the sequence₁(to distinguish from the precursor peptide also mature active thymosin alpha₁)：

1-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-28

Wherein the individual amino acid chains are unprotected or, as is common knowledge in the art, suitably protected when required for a given individual amino acid residue (see, e.g., Bodansky, below). More preferably, such peptides are N-terminally acetylated in a step immediately preceding the linear synthesis but prior to cleavage from the resin. Natural thymalfasin (tymalfasin), which is also used as a drug, is N-terminally acetylated.

It should be noted that the inventors of the present invention have found that it is primarily the C-terminal portion of the thymalfasin peptide (approximately residues 19-28) that results in most of the impurities due to chain deletion and thus loss of yield of the final product produced during linear phase synthesis. This part of the peptide has been found to be extremely difficult to synthesize.

Vice versa, during linear synthesis it is also possible to use such oxazolidine dipeptides as described in Wohr et al (supra). The benefits of using this dipeptide instead of using individual amino acids protected (preferably Fmoc) are deduced from the sequence of thymalfasin, especially for the Asp-Thr dipeptide; the use of oxazolidine dipeptide derivatives can help to avoid the formation of aspartimides during synthesis in the present invention.

Similarly, the prevention of glutarimide formation may require the use of special protecting groups.

While the present invention strongly prefers Fmoc chemistry for coupling reactions, Boc chemistry can similarly be used in another embodiment to carry out the invention.

Coupling agents for Peptide Synthesis are well known in the art (see Bodansky, M., "Principles of Peptide Synthesis", second edition, Springer Verlag Berlin/Heidelberg, Germany, 1993; see also the discussion therein regarding coupling additives or adjuvants). The coupling agent may be a mixed anhydride (e.g. T3P: propanephosphonic anhydride) or other acylating agent such as an activated ester or acid halide (e.g. ICBF, isobutyl chloroformate), or it may be a carbodiimide (e.g. 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide), an activated benzotriazine derivative (DEPBT: 3- (diethoxyphosphoryloxy) -1, 2, 3-benzotriazin-4 (3H) -one) or a uronium or phosphonium salt derivative of benzotriazole.

More preferably, the coupling agent is selected from the uronium or phosphonium salts of benzotriazoles capable of activating the free carboxylic acid function, and the reaction is carried out in the presence of a base, in view of optimum yield, short reaction time, and prevention of racemization during chain extension. Suitable and similarly preferred examples of such uronium or phosphonium coupling salts are for example: HBTU (O-1H-benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate), BOP (benzotriazol-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate), PyBOP (benzotriazol-1-yloxy-trispyrrolidinophosphonium hexafluorophosphate), PyAOP, HCTU (O- (1H-6-chloro-benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate), TCTU (O-1H-6-chlorobenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate), HATU (O- (7-azobenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium hexafluorophosphate), TATU (O- (7-azobenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate), TOTU (O- [ cyano (ethoxycarbonyl) methyleneamino ] -N, N' -tetramethyluronium tetrafluoroborate), HAPyU (O- (benzotriazol-1-yl) oxybis- (pyrrolidinyl) urea hexafluorophosphate).

Preferably, when DEPBT or a similar uronium or phosphonium salt reagent is used, a further or second weak base reagent is required to carry out the coupling step. This is complexed by a base whose conjugate acid has a pKa value of 7.5 to 15, more preferably 7.5 to 10, with the exception of the alpha amino function of the peptide or amino acid derivative, and which is preferably a sterically hindered tertiary amine. Such and more preferred examples are Hunig-base (N, N-diisopropylethylamine), N' -dialkylanilines with alkyl groups of straight or branched C1-C4 alkyl, 2, 4, 6-trialkylpyridines, or N-alkylmorpholines, more preferably N-methylmorpholine or collidine (2, 4, 6-trimethylpyridine), most preferably collidine.

It is also known to use coupling additives, especially coupling additives of the benzotriazole type (see Bodansky, supra). It is particularly preferred to use highly activated, previously described uronium or phosphonium salt coupling agents. It is therefore preferred that the coupling agent additive is a nucleophilic hydroxy compound capable of forming an activated ester, more preferably one having an acidic, nucleophilic N-hydroxy function, where N is an imide or is an N-acyl or N-aryl substituted triazene, most preferably that the coupling additive is an N-hydroxy-benzotriazole derivative (or 1-hydroxy-benzotriazole derivative) or an N-hydroxy-benzotriazine derivative. Such coupling additives N-hydroxy compounds have been described extensively and extensively in WO94/07910 and EP-410182, and the respective disclosures thereof are incorporated herein by reference. Examples are, for example, N-hydroxy-succinimide, N-hydroxy-3, 4-dihydro-4-oxo-1, 2, 3-benzotriazole (HOOBT), 1-hydroxy-7-azabenzotriazole (HOAt) and N-hydroxy-benzotriazole (HOBt). Particular preference is given to N-hydroxybenzotriazine derivatives, above all and especially HOOBt.

Ammonium salt compounds of coupling additives are known and their use in coupling chemistry has been described, for example, in US 4806641.

In a more particularly preferred embodiment, the uronium or phosphonium salt coupling agent is a uronium salt reagent, preferably which is HCTU, TCTU or HBTU, more preferably which is HCTU or TCTU, and most preferably is used in the reaction with N-hydroxy-3, 4-dihydro-4-oxo-1, 2, 3-benzotriazine or a salt thereof.

In the context of the present invention, it should be noted that HCTU and TCTU are defined to be encompassed by the term 'uronium salt reagent', although analysis of these compounds and possible analogues using crystal structure has shown that they contain an isobitroso moiety rather than a uronium moiety (O.Marder, Y.Shvo and F.Albericio, HCTU and TCTU: novel Coupling agents: Development and Industrial Applications (HCTUand TCTU: New Coupling Reagents: Development and Industrial Applications), Presentation Gordon Conference 2002-02 and Chimica Oggi 2002, 20: 37-41), which is an N-amidino substituent formed on a heterocyclic core due to substitution of the guanidine structure. In the context of the present specification, such compounds are referred to as the 'guanidine-type subclass' of uronium agents according to the present invention.

Base labile na can be deprotected by methods conventional in the art, for example using 20% piperidine in N-methylmorpholine in the case of Fmoc chemistry. The last amino acid to be added, e.g. the N-terminal serine, which is typically mature thymalfasin, may of course carry an N-terminal protecting group other than Fmoc, e.g. it may carry Boc or conveniently an acetyl protecting group, although the latter may conveniently be introduced by acetylation of the N-terminal amino acid, which is typically deprotected from the peptide after coupling. Preferably, orthogonal protecting groups other than Fmoc can be used to protect the last N-terminal residue. Examples are Alloc protecting groups (e.g.for protecting primary amines), all of which can be selectively removed from the peptide by Pd (0) -catalysed transacylation (Gomez-Martinez, N in solid phase peptide synthesis^αTemporary protection of AllocUse of amine-borane complexes as propenyl scavengers (N)^α-Alloc temporal protection in solid-phase peptide synthesis, use of amine-borane complexes as alkyl peptides, J.chem.Soc.Perkin Trans, 1, 1999, 2871-2874), Dde group (Bycroft et al, a novel approach for protecting against lysine in SPPS for branched peptides (A novel lysine protected procedure for SPPS of branched peptides), J.chem.Soc.Commun, 1993, 778-779), dimedone (dimedon) derivatives removable by hydrazinolysis, and functional homologues thereof such as Nde group (N-1- (4-nitro-1, 3-dioxoindan-2-yl) -ethyl, Kethiolam et al, Tehelan et al, Tetrahedron 54, 683 2-yl) -ethyl, Tetrahedron et al, 1998, 6817, 681-681; n-1- (4, 4-dimethyl-2, 6-dioxacyclohexylidene) -3-methylbutyl, Chan, 1995). Dde, its derivatives and homologues are Dde-type protecting groups which in the untreated free state share a dioxyalkylene functional moiety of formula III:

III (-CO)₂C＝C(-R)(-OH)

wherein R is substituted or substituted alkyl, and preferably two carbonyl functions form a single bond consisting of-CH₂-CR’CR”-CH₂-NR '-CO-NR "or-CR' ═ CR" -backbone linked cyclic structures. Preferably, R', R "are alkyl groups or are linked together to form an aryl group.

The use of modified or labelled orthogonal protecting groups is also readily feasible, with the additional benefit of enabling easy purification by subsequent affinity chromatography or affinity modified ` standard ` RP HPLC using such reversible affinity labels. In this case, the synthesized peptide is suitably cleaved from the resin under mild conditions that avoid global deprotection, purified, selectively removed from the N-terminal probe to allow immediate N-terminal acetylation, and finally the peptide is globally deprotected. One such example is described by Ball et al in int.j.peptide Protein res, 1992, 40: 370-379 and Ball et al, J.chromatography, 1994, 686: 73-88.

For the purposes of the present invention, particular preference is given to the removal of the Fmoc group with 20% piperidine-DMF (v/v), for example 2X 1 min, 2X 10 min, 1X 5 min. Similarly, it is preferred that the coupling of Fmoc-aa-OH (5 equivalents) is carried out with the above-mentioned coupling reagent in DMF or a similar solvent for 60 to 120 minutes. In order to optimize the synthesis and in particular the coupling time for the individual sequence positions, the ninhydrin test is carried out after the coupling, and if the test result is positive, the coupling is repeated under the same conditions, otherwise the process is continued. If the ninhydrin test is still positive after the second coupling, Ac is used₂O and DIEA or similar methods are subjected to an acetylation step to cap the deleted peptide chain. This improved coupling and/or capping method is then applied to the repeated synthesis of a particular given sequence position under the same conditions. Notably, we found that the choice of resin strongly affected the sequence position specific coupling problem as exemplified in the experimental section.

According to the invention, the solid phase comprises a PEG resin. According to the present invention, it enables thymalfasin or a core peptide portion of thymalfasin, which is difficult to synthesize, to be synthesized in yields and purities without the previous examples. In the context of the present specification, the definition 'solid phase' includes (in addition to the inert resin matrix) the presence of an integral linker on the inert matrix material to link the peptide or to a further graft linker or handle.

Such resins have been described, for example, in U.S. patent No. 2003078372a1, which achieve amphiphilic properties that make them different from conventional resin materials by virtue of PEG incorporated into the solid phase. They are known to have a 'gel-like' behaviour after swelling rather than the traditional solid-like behaviour of, for example, Merrifield resins. This particular property is believed to provide the benefit of reduced cycle time due to diffusion/liquid exchange during, for example, washing, coupling, deprotection steps, etc. Notably, such PEG resins according to the invention have also been found to significantly enhance the efficiency of difficult and peptide-specific peptide coupling steps, and to enhance purity and yield in a synergistically enhanced manner not expected. The term 'PEG resin' is generally understood in the context of the present specification to comprise at least one polyether copolymeric or block copolymer moiety (polymer share). Different embodiments exist for this type of resin, and the polymerization of carbon monomers (including monomers other than ethylene oxide) is not limited to polyether chemistry. Thus, the solid phase resin matrix may be composed of, for example, amphoteric polystyrene-PEG hybrid resins (e.g., Tartar (Tentagel), please refer to U.S. Pat. No. 4908405) or PEG-polyamide or PEG-polyester hybrid resins, such as Kempe et al, J.Am.Chem.Soc., 1996, 118, 7083; also in U.S. patent No. 5,910,554 a. In general, loading of resins is less efficient and/or chemically stable (especially in acidic matrices) often not comparable to conventional resins, such as conventional Merrifield polystyrene-DVB. polystyrene-PEG hybrid resins can achieve higher loadings, but lower amphoteric properties are obtained because of the reduced PEG content.

PEG resins obtained by grafting a non-PEG resin matrix with a layer of a suitable PEG resin material may also be used, for example, High-load polyethylene glycol-polystyrene (PEG-PS) grafting supports for solid phase synthesis (PEG-PS) Biopolymers, 1998, 47: 365-380.

In a particularly preferred embodiment according to the present invention, the PEG resin is a substantially pure PEG-polyether resin, which preferably does not contain any (eventually further substituted) polystyrene copolymerising or block copolymerising moieties, and more preferably which also does not contain any internal ester or amide functional groups but only polyether functional groups. Most preferably it is a pure PEG resin or PEG-polyether resin. Preferably, 'substantially pure' means that the structure of the above pure PEG resin is defined to be at least 70% of the dry weight of the solid phase material, leaving a minor portion of the other types of polymer additions. This ideal pure PEG resin material provides the most preferred chemical stability and good compatibility and is easily processed with standard solvents in typical Fmoc synthesis. It is noted that in the latter definition, the terminal ester or amide bonds bridging the resin and the peptide, either directly or via an integrated and/or grafted linker, are not taken into account, since they are not internal structural, cross-linking properties and therefore do not influence the definition of 'pure PEG' in this respect. Such 'pure', highly amphiphilic PEG resins are provided by different companies, such as Matrix innovations inc. ('ChemMatrix' brand) of quebec, canada or versamatrix xa.s., denmark, and are described in WO 02/40559. They may provide a loading capacity of greater than 0.5 mmol/g, see for example ChemMatrix brand resin as described in WO' 559 referred to earlier. For the attachment of peptides, such resins may have terminally integrated linkers, such as hydroxymethyl or derived therefrom, e.g. 'quasi-natural' aminomethyl, carboxyl or bromomethyl or iodomethyl, or they may be derived with known solid phase attached integrated or grafted linkers or handles (handles), e.g. Wang, PAL, 4-alkoxy-benzaldehyde or Rink or Sieber amide linkers.

It is noted that the resin according to the invention has a surprising property element that the coupling of Glu at the second amino acid number 27 was suddenly found to be a severely difficult step when compared to conventional resins (e.g. PS-DVB or CTC-PS-DVB), especially when using the side chain anchoring terminal Asp/Asn residue, whereas the coupling efficiency at other important residues of sequences 19-28 was found to be improved but not to be worthless. Thus when using PEG resins, and especially pure or substantially pure PEG-polyether resins, extended and/or repeated coupling of residue No. 27 to the Asn moiety immobilized on the resin is desirable. 'extension' relates to the use of more equivalents of Fmoc amino acids or longer coupling times than the average residue.

A further object of the invention is various peptide-solid phase conjugates obtained in this way. The definitions and explanations set forth above apply equally to these objects.

Such targets are peptide conjugates of formula I:

R1-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn_R2__xR3

I

wherein each amino acid residue is individually protected or unprotected, R3 is a PEG resin solid phase comprising an integral linker, R2 is a grafted linker, x is 0 or 1, R1 is hydrogen, a protecting group or a peptide group, preferably peptide groups in a number of less than 50 and more preferably less than 25 amino acid residues, and wherein if R1 is a peptide group, each amino acid residue on said group is individually protected or unprotected, and the N-terminus of said group is free, acetylated or protected by a protecting group Y.

Thus, preferably R1 is the following peptide residue: Y-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-, Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu or H-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Lys-Asp-Leu-Lys-Glu-Leu-Lys-Glu, Y having the meaning mentioned above.

The linkers defined by Guillier, Orian and Bradley, chem.rev., 100, 2091-: (a) an integrated linker (Integrallinker) in which the solid phase core material part forms or is an inseparable part of the linker, both together forming R3, and (b) a non-integrated or grafted linker (non-integrated or grafted linker) R2, wherein the linker is additionally attached to a solid support on the integrated linker/solid phase R3. Examples of integral linkers are p-Methylbenzhydrylamine (MBHA), 2-chlorotrityl (CTC), amino-methyl, and the like. Integral linkers are necessary and grafted linkers R2 are optional. Typical examples of graft linkers are 4-hydroxymethylphenoxyacetyl-or 4-hydroxymethylbenzoic acid.

Typically, the peptide is attached via its C-terminal residue to the solid phase or graft linker, respectively, through its 1-carboxylic acid functionality.

Preferably, synthesis of thymalfasin, or the previously mentioned fragments and variants thereof, according to the invention is achieved by side chain anchoring of the penultimate Glu or C-terminal Asp or Asn residue. 'penultimate Glu' in the context of this specification can mean a Glu-Asn protected dipeptide immobilized on a solid phase, or it can mean that synthesis of a thymalfasin variant free of the native thymalfasin C-terminal Asn is initiated from the Glu residue. Unexpectedly, anchoring of the side chains was found to result in more efficient synthesis. After cleavage from the resin, the terminal Asp residue, which is immobilized with its beta-carboxy function to the solid phase or to the graft linker, may be subjected to post-synthetic amidation. More preferably, however, Fmoc-Asp with the C carboxyl function suitably protected (preferably by a t-butyl protecting group) is side chain anchored to an amide-generating linker, preferably an amide-generating graft linker; such conjugates are contemplated for the final product after cleavage from the resin and are referred to as 'side-chain anchored Asn' due to the use of an amide-generating structure. Examples and particularly preferred embodiments of such amide-generating structures are, for example, ` Rink amide ` 4- (2 ', 4' -dimethoxybenzyl-aminomethyl) phenoxy-resins, Sieber resins (Tetrahedron Lett., 1987, 28, 2107-2110) or similar 9-amino-xanthenyl-type resins, PAL resins (Albericio et al, 1987, int.J.Pept.protein Research, 30, 206-216). A less preferred example of an amide-generating linker is the specifically substituted tritylamine derivative as described by Meisenbach et al (1997, chem. letters, 1265 onwards). These are examples of (Fmoc chemical compatibility) linker groups from which C.alpha.carboxamides are generated or released upon cleavage of the peptide from the resin. It goes without saying that the use of such an amide linker is of course dependent on the type of solid phase synthesis carried out, i.e. whether it is a conventional Boc or an orthogonal Fmoc protection chemistry currently used for coupling, i.e. whether it is a conventional Boc or a currently customary, orthogonal Fmoc protection chemistry used for coupling, for example; one amide resin linker specific to Boc is PAM. Thus, a solid phase comprising such a linker group is referred to as 'amide producing solid phase' in the context of the present specification.

Incorporation of Fmoc-Asp-OtBu onto a solid phase containing one or two linkers using a coupling reagent has been described (Albericio, van Abel, Barany, int.J.peptide Protein Res., 35, 284-286, 1990) and may be applied in the context of the present specification. Generally, a uronium-type coupling agent is preferred for achieving this.

The advantages of this side chain anchoring strategy are:

(i) incorporation of the first amino acid into the solid support is carried out in relatively quantitative yields via an amide bond, and thus the same reagents used for peptide bond formation can be used. Furthermore, the incorporation of asparagine derivatives into Wang-type resin systems occurs in low yields with the risk of racemization and formation of β -cyanoalanine if the side chain amide is unprotected (Katsoyannis et al, 1958, am. chem. soc., 80: 2558). Moreover, asparagine, unprotected in the side chain, may be incorporated into the halogen resin via both 1-carboxy and carboxamide functional groups, which requires the use of, for example, appropriately protected Fmoc-Asn (Mbh) -OH amino acids, which may cause further problems in the final deprotection stage of the peptide.

(ii) Only side chain anchoring strategies involving the use of acid-labile t-Bu protecting groups for the Lys side chain (tBu ester of the 1-carboxylic acid of the first residue and the omega-carboxylic acids of Asp and Glu, tBu ester of the Ser and Thr side chains) and acid-labile Boc contribute to the final overall deprotection due to simple scavengers such as H₂O may be used. In contrast, if trityl, xanthyl, 2, 4, 6-trimethoxybenzyl protecting groups are used, the presence of an additional scavenger is required.

(iii) The strategy of anchoring the backbone peptide chain into the side chain will be more flexible than in the 1-carboxy immobilization strategy, since the immobilization is via the side chain.

Scheme I outlines the most preferred mode of synthesis of thymalfasin (zadaxin) according to the invention on PEG resin, most preferably on pure or substantially pure PEG resin, where linker I is an amide-generating linker and linker 2 is an integrating linker:

scheme I

(i) Fmoc-Asp-OtBu is incorporated onto a Solid Support (SS) containing one or two linkers using a coupling reagent (Albericio, van Abel, Barany, int. J. peptide Protein Res., 35, 284-286, 1990).

(ii) The peptide chain was extended with the remaining Fmoc-protected amino acids (tBu for Asp, Glu, Ser and Thr; Boc for Lys).

(iii) And (4) acetylation.

(iv) The peptide is cleaved from the solid support and the protecting group is removed, which can be done in one (high acid concentration solution) or two steps (first with low acid concentration solution followed by high acid concentration solution).

Examples

General methods. Fmoc-Sieber-PS-resin was purchased from NovaBiochem (L _ ufelfingen, Sweden), ChemMatrix resin from Matrix Innovation (Quebec, Canada), 2-Cl-TrtCl-resin from CBL (Greece, Patras), HCTU from Luxembourg Industries Ltd. (Israel, Tel Aviv), protected Fmoc-amino acid derivatives from IRIS Biotech (Marktredwitz, Germany).

Solid phase synthesis was performed in a glass funnel equipped with a filter or a polypropylene syringe (50 ml) equipped with a polyethylene porous disc. The solvent and soluble reagents are removed by aspiration. Removal of the Fmoc group was performed with piperidine-DMF (2: 8, v/v) (2X 1 min, 2X 10 min, 1X 5 min). Washing between deprotection and coupling with DMF (5X 0.5 min) and CH₂Cl₂(5X 0.5 min) and deprotection step was performed using 10 ml of solvent per g of resin each time. Transformation and washing of peptide synthesis was performed at 25 ℃. The synthesis on the solid phase is carried out byPeptide-resin aliquots (approximately 2 mg) were washed with TFA-H₂The intermediate obtained after one hour of O (95: 5) cleavage was subjected to HPLC for control. HPLC reversed phase column Symmetry^TM C₁₈4, 6X 150mm, 5 μm (column A) was purchased from Waters corporation (Ireland). Analytical HPLC was performed on a Waters instrument containing two solvent delivery pumps (Waters 1525), an autosampler (Waters 717 autosampler), a dual wavelength detector (Waters 2487). UV detection is at 215nm or 220nm, with 30% to 100% CH₃CN (+ 0.036% TFA) to H₂A linear gradient of O (+ 0.045% TFA) was performed over 15 min.

MALDI-TOF and ES-MS analysis of peptide samples were performed in a PerSeptive Biosystems Voyager DE RP using an ACH matrix (ACH matrix).

Example 1

Ac-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asp (Rink-ChemMatrix-resin) -OtBu

Step 1

Side chain anchoring of Fmoc-Asp (Rink-ChemMatrix-resin) -OtBu

Fmoc-Rink-ChemMatrix-resin (0.45 mmol/g) (5 g, 2.25 mmol) was placed in a glass funnel equipped with a porous disc. The resin was washed/treated with CH₂Cl₂(3X 0.5 min), DMF (3X 0.5 min), and piperidine as specified in the general procedure, and DMF (5X 0.5 min). Fmoc-Asp-OtBu (4.11 g, 5 equiv.) and DIEA (3.4 ml, 10 equiv.) in DMF (9 ml) were then added followed by HCTU (3.96 g, 4.8 equiv.) in DMF (5 ml). The mixture was left under mechanical stirring for 2 hours and the resin was washed with DMF (3X 0.5 min) and the ninhydrin test was negative. Ac in DMF (14 ml) was added₂O (0.5 mL, 5 mmol) and DIEA (1.7 mL, 5 mmol), maintaining mechanical stirring for 15 minutes, and the resin was removedFiltered and washed with DMF (3 × 0.5 min).

Successful loading was determined to reach 0.4 mmol/g.

Step 2

H-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBr) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asp (Rink-ChemMatrix-resin) -OtBu

The Fmoc group was removed and immediately afterwards Fmoc-aa-OH (5-10 equivalents) was added to the above peptidyl-resin together with DIEA (3.4 ml, 10 equivalents) dissolved in DMF (9 ml) (step 1), followed by HCTU (3.96 g, 4.8 equivalents) dissolved in DMF (5 ml). The mixture was kept under mechanical stirring for 1-2 hours and the resin was washed with DMF (3X 0.5 min) for ninhydrin test. If the test is positive (blue) the coupling reaction is repeated, whereas if the test is negative (brown) Ac in DMF (14 ml) is used₂O (0.5 ml, 5 mmol) and DIEA (1.7 ml, 5 mmol) were subjected to an acetylation step and mechanically stirred for 15 minutes. A small portion of the resin was sampled after piperidine treatment. At positions E27, K19, the double coupling reaction was performed for each position using 10 equivalents of Fmoc amino acid, the reaction time per single coupling being at least 35 minutes.

Step 3

Ac-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asp (Rink-ChemMatrix-resin) -OtBu

Ac in DMF (14 ml) was used₂The final acetylation was performed by mechanically stirring a solution of O (0.5 ml, 5 mmol) and DIEA (1.7 ml, 5 mmol) for 15 minutes, with negative results in the ninhydrin test. The resin was dried to yield 16.5 grams.

Step 4

Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (Zadaxin, thymalfasin)

Cut from the resin in a single step and perform global deprotection. The 5 g resin fraction from step 3 was placed in a glass funnel equipped with a multi-well plate and washed with cold TFA-H₂O (95: 5) (35 ml) for 2H, then the solution was filtered and the resin was treated with TFA-H₂O (95: 5) (10 mL) was further washed. The combined TFA solution was poured onto cold tert-butyl methyl ether (350 ml) and the mixture was centrifuged. The solid was isolated by pouring. MALDI-TOF-MS, calculated 3106.50. Measured value: m/z 3107.36[ M + H ]]+。

RP-HPLC analysis of the crude peptide showed the product to be predominantly pure, reaching a 90% yield (FIG. 1). The two smaller contaminating peaks can be easily removed by RP-HPLC on C18 Hypersil with 10-30% acetonitrile/water over 45 minutes, yielding a pure product with the correct product peak almost completely recovered. (FIG. 2)

Example 2: comparative examples

Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (Zadaxin, thymalfasin)

The experimental procedure as described in examples 1 and 2 was used, but Fmoc-Rink-ChemMatrix-resin was replaced by Fmoc-Sieber-PS resin (0.59 mmol/g) (3.33 g, 2 mmol).

MALDI-TOF-MS, calculated 3106.50. Measured value: m/z 3107.24[ M + H ] +. RP-HPLC analysis of the mixture of crude products gave a purity and yield of only 40% as determined by peak area (FIG. 3), with a wide range of impurities.

Example 3: comparative examples

Ac-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn-Trt-O- (2-Cl-Trt) -resin

Step 1

Anchoring the C alpha-terminus to the CTC resin, resulting in Fmoc-Asn (Trt) -O-TrtCl-resin

2-Cl-TrtCl resin (0.2 g, 1.64 mol/g; Ambersynth)^TMCTC, Rohm from Paris, France&Haas corporation, made with polystyrene-1% divinylbenzene) into a 10 ml polypropylene syringe equipped with a polyethylene filter disc. Then using CH₂Cl₂The resin was washed (5X 0.5 min) and Fmoc-Asn (Trt) -OH (0.7 eq.) and DIEA (241. mu.l) in CH was added₂Cl₂(2.5 ml) the mixture was stirred for 15 minutes and the mixture was stirred for 45 minutes while more DIEA (121 μ l, 7 equivalents total) was added. After stirring for 10 min, the reaction was quenched by addition of MeOH (160. mu.l). Fmoc-Asn (Trt) -O-TrtCl-resin was subjected to the following reaction with CH₂Cl₂(3X 0.5 min.) and DMF (3X 0.5 min). The loading was calculated to be 0.8 mmol/g by Fmoc assay.

Step 2

H-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn-Trt-ClTrt-PS-resin

The Fmoc group was removed and Fmoc-aa-OH's (10 equivalents) and DIEA (20 equivalents) dissolved in DMF (9 ml) were added sequentially to the above peptide-resin (step 1); HCTU (9.6 equivalents) in DMF was then added using an ABI autosynthesizer 433A at a coupling time of 60 minutes.

Step 3

Ac-Ser (tBu) -Asp (OtBu) -Ala-Ala-Val-Asp (OtBu) -Thr (tBu) -Ser (tBu) -Glu (OtBu) -Ile-Thr (tBu) -Lys (Boc) -Asp (OtBu) -Leu-Lys (Boc) -Glu (OtBu) -Lys (Boc) -Glu (OtBu) -Val-Glu (OtBu) -Ala-Glu (OtBu) -Asn-Trt-ClTrt-PS-resin

Final acetylation with Ac in DMF (2 mL)₂O (0.5 mmol) and DIEA (5 mmol) were carried out for 15 minutes with intermittent manual stirring, wherein the ninhydrin test was negative.

Step 4

Ac-Ser(tBu)-Asp(OtBu)-Ala-Ala-Val-Asp(OtBu)-Thr(tBu)-Ser(tBu)-Ser(tBu)-Glu(OtBu)-Ile-Thr(tBu)-Thr(tBu)-Lys(Boc)-Asp(OtBu)-Leu-Lys(Boc)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Glu(OtBu)-Val-Val-Glu(OtBu)-Glu(OtBu)-Ala-Glu(OtBu)-Asn(Trt)-OH

With TFA-Et₃SiH-CH₂Cl₂(1: 98) (5X 30 seconds) the protected peptide from step 3 above was cleaved off. At H₂The filtrate was collected on O (4 mL) and H was added₂Part O was removed under reduced pressure. Acetonitrile (ACN) was then added to dissolve in the removal of H₂Solids appeared during O and the solution was lyophilized.

Step 5

Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (Zadaxin, thymalfasin)

Dissolving the protected peptide from step 4 in TFA-H₂O (95: 5, 5 ml) and the mixture was stirred for 1 hour. The solution was then filtered off and the resin was further treated with TFA-H₂O (95: 5) (1 ml) wash. The combined TFA solutions were poured into cold tert-butyl methyl ether (25 ml) and the mixture was centrifuged. The solid was isolated by pouring. Then, H is added₂O (5 ml) and lyophilized.

MALDI-TOF-MS, calculated 3106.50. Measured value: m/z [ M + H]⁺3107.98。

Crude purity and yield, as determined by RP-HPLC peak area, achieved disappointing 20% correct product with strong background smear bands for many incorrect chain variants (fig. 4). Solid phase synthesis of the shortened fragment (Lys19 to Asn28) was carefully repeated with manual coupling and ninhydrin testing after each step, showing that chain synthesis often effectively terminates after addition of V22; three sequential couplings, E21, K20 and K19, proved to be highly inefficient, requiring capping of the unreacted chains. The double coupling, extended coupling time and use of more equivalents of Fmoc amino acids did not satisfactorily overcome this problem and thus a significantly more efficient synthesis could not be established in this way (figure 5).

Example 4: comparative examples

Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (Zadaxin, thymalfasin)

Experimental procedure was essentially as in example 2, except that Fmoc-Rink handle was added to MBHA resin (4-methylbenzhydrylamine resin LL on a polystyrene-1% divinylbenzene substrate, NovaBiochem^TMMerck/Germany). Fmoc-Asp-Otbu side chains were anchored to the Rink aminomethyl grafted linker after removal of the Fmoc group from the Rink moiety as in example 1, step 1, with a loading of 0.66 mmole/g. Coupling was carried out with HCTU (10 equivalents) for 35 min, while double coupling was carried out at the positions D28 (i.e. N28), E27, E21, K20, K19. After acetylation as described above with TFA-H₂O (95: 5) was cleaved in a single step and deprotected. The crude purity and yield obtained were 27% as determined by RP-HPLC (FIG. 6).

Example 5

NH2-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (fragment 19-28 of thymalfasin)

Side chain anchoring and synthesis were performed on Rink-Chemmatrix resin essentially as described in example 1, except that no double coupling was performed at the sequence positions indicated in example 1 but a consistent single coupling over 35 minutes was performed. Deletion of Glu residue No. 27 is the only but major chain termination reaction byproduct observed in the HPLC profile (fig. 7). This by-product can be successfully inhibited by extension, repeated coupling at E27, as shown in example 1.

Example 6

NH2-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH (fragment 19-28 of thymalfasin)

Side chain anchoring and synthesis were performed essentially as described in example 1, except that PEG-MBHA resin from Millipore-Waters (MeO [ PEG ] 2000-CO-Orn-MBHA-PS-resin, which is similar to Kates et al in "High-load PEG-polystyrene grafting support for solid phase synthesis" was used grafted with Rink aminomethyl linker-Rink-the loading of Rink-PEG-MBHA resin was determined to be 0.55 mmol/g-in particular, double coupling was performed at sequence positions E27, E21, K20, K19-the crude purity and yield obtained was 92% as determined by RP-HPLC (fig. 8).

Claims (9)

1. A peptide conjugate of the formula I,

R1-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn_R2__xR3I

wherein each amino acid residue is individually protected or unprotected, R3 is a PEG resin solid phase comprising an integral linker, R2 is a grafted linker, x is 0 or 1, R1 is hydrogen, a protecting group or a peptide group, preferably peptide groups in a number of less than 50 and more preferably less than 25 amino acid residues, and wherein if R1 is a peptide group, each amino acid residue on said group is individually protected or unprotected, and the N-terminus of said group is free, acetylated or protected by a protecting group Y.

2. Peptide conjugate according to claim 1, wherein the protecting group Y is an Fmoc group, a Dde-type group, a Boc group or a derivative of the protecting group, preferably a derivative of an Fmoc or Dde-type group, comprising a moiety allowing reversible affinity binding of such protected peptide to a suitable chromatographic solid matrix material.

3. Peptide conjugate according to claim 3, characterised in that the protecting group Y is capable of reversible affinity binding to an immobilized metal affinity chromatography solid matrix material.

4. Peptide conjugate according to any of the preceding claims, wherein R1 is Y-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-, Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu or H-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Lys-Asp-Leu-Lys-Glu-Leu-Lys-Glu, y has the meaning mentioned above.

5. Synthesis of thymosin alpha on solid phase₁Comprising mature thymosin alpha₁Thymosin alpha residues 1-27₁C-terminally truncated form of (a), comprising at least mature thymosin alpha₁Thymosin alpha residues 19-28₁Or at least comprises mature thymosin alpha₁A method of truncating residues 19-27, said method comprising the steps of:

a. coupling appropriately protected FMOC-Glu, FMOC-Asp or FMOC-Asn residues to a PEG resin solid phase, wherein the PEG resin is preferably selected from: polystyrene-PEG mixed resin, PEG polyether-polyester mixed resin, or PEG polyether-polyamide mixed resin and substantially pure PEG resin,

b. peptide chains are extended by FMOC synthesis, where the side chains of individual amino acids can be derivatized with appropriate protecting groups,

c. optionally acetylating the last N-terminal residue, and

d. the peptide was cleaved from the resin.

6. The method of claim 5, wherein the synthesized peptide is thymosin alpha having the sequence₁：

1-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-28

Wherein each amino acid chain is unprotected or suitably protected.

7. The method of claim 5, wherein the PEG resin is an amphoteric resin.

8. The method of claim 7, wherein the PEG resin has a loading capacity of at least 0.5 mmol/g, and preferably is substantially free of polystyrene co-moieties.

9. The method according to claim 7, wherein the PEG resin is a substantially pure polyether-PEG resin, preferably a pure PEG-polyether resin, except for the integrating and/or grafting linker.