HK1113577A - Method for solid phase peptide synthesis - Google Patents

Description

Method for solid phase peptide synthesis

The present invention relates to and improves the solid phase peptide synthesis of the anticoagulant peptide bivalirudin, the so-called "hirulog". The invention also relates to the corresponding peptide-solid phase conjugation product containing the still protected peptide bound to the resin.

Thrombin inhibitors are considered to be promising antithrombotic agents: the proteolytic process by thrombin is a key to the control of blood coagulation. Hirudin is an effective therapeutic thrombin peptide inhibitor obtained from the blood-sucking leech, Hirudo medicinalis, which contains 65 amino acids.

Peptide fragment short peptide analogues of the amino acids 45-65 of hirudin, the so-called bivalent hirudins, have proven effective in the treatment of thrombosis, a life-threatening condition.

Okayama et al (1996, chem.Pharm.Bull.44: 1344-1350) and Steinmetzer et al (1999, Eur.J.biochem.265: 598-605) invented solid phase synthesis of different hirudins on Wang resin by ester-forming binding of the C-terminus of the Fmoc amino acid to the resin esterified to the p-benzyloxy-benzyl alcohol group. The use of Wang resin requires cleavage of the peptide from the resin with concentrated trifluoroacetic acid, where resin cleavage is equivalent to concomitant global deprotection of the peptide.

Cleavage by acid hydrolysis (acidic cleavage) from Wang resin was performed under strongly acidic conditions and it is known that this method inevitably causes poor alkylation of Trp residues as a side reaction if no scavenger is used during acid hydrolysis (Giraud et al, 1999, J.peptide Science 5: 457-461). This side reaction is particularly prone to C-terminal Trp (Atherton et al, 1988, Tetrahedron 44: 843-. Alkylation is caused by aromatic carbonium ions generated by the Wang resin linker phenoxy moiety. When the hirudins do not contain a Trp residue, they contain a Tyr residue in a position adjacent to the C-terminus. We have found and reported here for the first time that this Tyr residue also tends to be unstable to alkylation upon cleavage from Wang resin and has a negative impact on product purity.

The object of the present invention is to eliminate the disadvantages of the prior art and to provide a different or improved method for the synthesis of Hirulog peptide (Hirulog peptide).

This object is achieved by a peptide-resin conjugate and a corresponding synthesis method of the invention.

In the present invention, a method is provided for the isolation and deprotection of a peptide-solid phase conjugate, resulting in a peptide, preferably having the formula D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Tyr-Leu. The peptide-solid phase conjugate comprises a 2-chloro-trityl arm (handle) of formula I:

wherein: a ═ Boc-D-Phe-Pro-Arg (R2) -Pro-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-, or a ═ Fmoc-D-Phe-Pro-Arg (R2) -Pro-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-, or a ═ NH₂-D-Phe-Pro-Arg (R2) -Pro-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-, and wherein R2, R3, R4, R5, R7, R8, R9 are amino side chain protecting groups and R1 is an insoluble solid phase.

The above peptide sequence is that of hirudin-8 (Hirulog-8) (described in EP-489070). It is a 20-mer bivalent derivative of hirudin (a 65-mer, a naturally occurring potent thrombin inhibitor). It consists of functionally important, linked structural motifs on hirudin: active site binding motif D-Phe-Pro-Arg-Pro and carboxy-terminal sequence Asn on hirudin⁹To Leu²⁰Bridged by a tetraglycine spacer. '-D-Phe-' as used herein means D-phenylalanine, as opposed to the natural L-enantiomer of a given amino acid, here Phe.

Optionally, in another object of the invention, the group a in formula I may be any of the following groups:

a ═ P-X1-Tyr (R9) -X2-, wherein X1 is a peptidyl moiety, optionally comprising a protecting group on each individual amino acid side chain, having 0-200, preferably 1-100, most preferably 2-50 amino acids, wherein X2 is a single amino acid residue optionally protected on the side chain or the end, linked to the solid phase by-O-or-NH-, X2 is preferably not Trp, Cys or Arg, and wherein P is H (i.e. to give NH —)₂) Or a protecting group, preferably an orthogonal protecting group (orthogenic protection group) or a protecting group removable under strongly acidic conditions as defined below, more preferably selected from: boc, Fmoc, Dde, Nps, Alloc, Z.

It will be appreciated that, as described above, when the side chain of its X2 residue comprises an amino functional group (e.g. lysine, homolysine or a structural isomer such as 3-lysine), the X2 residue may be bound to the resin solely through the aminoether functional group. Conversely, -O-linkage may be an ester or ether linkage to the arm or linker through a pendant or C.alpha.carboxyl or pendant hydroxyl functionality.

A ═ P-X1-Tyr (R9) -Leu-O or P-X1-Tyr (R9) -X2

A ═ P-X1-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O or P-X1-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -X2

A ═ P-X1- [ Gly ]0-3-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O or P-X1- - [ Gly ]0-3-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -X2

A ═ P-X1-Arg (R2) -Pro-Gly-Gly-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-or P-X1-Arg (R2) -Pro-Gly-Gly-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -X2

P, X1, X2 are commonly used in the various possible embodiments of A and the resulting peptide-solid phase conjugates.

We have found and reported here for the first time: the Tyr residue is also prone to irregular alkylation when cut from Wang resin, which can adversely affect the purity of the product. For bivalent hirudin, this modification appears to be promoted by an ortho effect, similar to the results of Atherton et al on Trp; however, alkylation of Tyr (e.g., in arginine deprotection) has never been reported as a general phenomenon, which is clearly different from that of Trp (Atherton et al, 1989, Solid phase synthesis: A practicallappacach, Solid phase synthesis: progress of practice:. IRLpress, Oxford). Furthermore, Atherton's study was limited to C-terminal Trp, whereas the Tyr residue in the bivalent hirulog peptide synthesized in the C-terminal to N-terminal direction was only ortho-terminal (juxtaproximal), the penultimate residue at the C-terminal end of the growing peptide chain. It was later discovered, without being bound to any theory, that this may be because the phenoxy moiety is more reactive in electrophilic substitution than the general aryl compounds. In fact, phenols are scavengers of acidolytic cleavage from resins (d.s.king et al, 1990, int.j.peptid Protein res., 36, 255). The skilled person has not described or mentioned the side-reactions so far, and so far it is believed that only the terminal Trp is readily randomly alkylated. Therefore, Wang resin has been widely used for the synthesis of hirudins until now.

Peptide-solid phase conjugates of the invention can be synthesized by conventional solid phase methods well known in the art, such methods being described in detail by Principles of Peptide Synthesis by Bodanszky et al, second edition, Springer Verlag Berlin Heidelberg 1989, which is incorporated by reference. Due to the acid lability of the solid phase linkage, coupling reactions during solid phase synthesis must be performed using Fmoc chemistry in such a synthetic strategy. Only the last terminating D-Phe residue may be Boc-or Fmoc-protected. The Fmoc protection can be eliminated while the peptide is still attached to the resin, and can be performed using standard processing, e.g., 20% piperidine or other Fmoc deprotection base reagent, and obtained therefromThe peptide-resin conjugate (conjugate) of the invention is described, but carries a free N-terminal amino group. However, such early Fmoc deprotection, which early exposes the N-terminus, makes or makes it possible to make the free N-terminal D-Phe residue more susceptible to racemization when the global deprotection is carried out while separation from the resin by acidolysis, especially under strongly acidic conditions. It is therefore more preferred that the terminating D-Phe residue is Boc-protected or protected with another protecting group which is easily removable under strongly acidic conditions, thereby eliminating the need for a separate Fmoc deprotection step. For the sake of clarity, this includes, for example, a Z- (benzyloxy-carbonyl-) protecting group which can be cleaved under the (in particular) strongly acidic conditions described in the present invention, although hydrogenolysis or HF-promoted cleavage is known to be more effective. Again, a separate Fmoc deprotection step on the end-capped D-Phe revealed early the exposed N-terminus (end-capped with free alpha-amino group, denoted H-D-Phe-or NH in formula I)₂-D-Phe, synonymous) which, when isolated from the resin by acidolysis with global deprotection, allows racemisation of the deprotected free N-terminal D-Phe easily, although this is not preferred, but is still one of the possible embodiments of the invention. The one-step separation or simultaneous cleavage and global deprotection can be carried out in a solvent mixture (e.g., aqueous TFA and DCM mixture).

In general, according to the invention, the protected peptide of formula I can be cleaved from the resin either simultaneously with deprotection or complete deprotection of the amino acid side chain (preferably the N-terminal protecting group) or in a previous initial step. In the latter embodiment, cleavage from the resin is first performed under weak acid conditions followed by removal of all remaining protecting groups under strong acid conditions (global deprotection).

Either under either condition, especially 2-chloro-trityl-resin (CTC resin for short) and e.g. the commercially available 4-methoxy-or 4-methyl-trityl-resin, or, to the same or a considerable extent, other resins encompassed by the present invention are well suited for avoiding undesired modification of the proximal tyrosine upon cleavage and/or deprotection. This prevents undesired alkylation of the proximal tyrosine (i.e. the penultimate tyrosine at the C-terminus) when deprotected simultaneously with cleavage of the peptide from the resin. Due to the halogenation, the CTC resin allows the protected peptide and tyrosine to be efficiently cleaved from the resin under very mild acidolysis conditions (e.g., in 0.5% trifluoroacetic acid (TFA) in Dichloromethane (DMC)) under which most of the side chain and N-terminal protecting groups are not normally affected, thereby preventing alkylation by timely separation of the different deprotection events. Hereinafter, specific example defaults (tacidly) performed with CTC resins (as the most preferred embodiment of solid phase or resin) may also be performed with other resins described in the present specification and claims.

By strongly acidic conditions as opposed to weakly acidic conditions is meant that a trifluoroacetic acid (TFA) solution is employed at a concentration of at least 50% (v/v), as defined herein. And, conversely, the protecting group that needs to be removed with strongly acidic conditions is one that can be removed with at least 80% TFA. Thus, the above definitions are not included throughout the present invention for protecting groups that require a stronger acid (e.g., HF). Weak acidic conditions are defined as having 0.01% (v/v) to < 50% TFA, preferably 0.1% to 30% TFA.

In either mode, it is noteworthy that the peptidyl moieties of the invention exhibit unexpectedly no undesired alkylation of the proximal tyrosine and no diketopiperazine side reactions at all (another side reaction that may occur when the peptide is cleaved from the resin and is known to be particularly sensitive to the nature of the last two C-terminal amino acids). Without wishing to be bound by theory, it is speculated that the tyrosine in the 2 nd position of the arm of the CTC resin on the peptide chain is just at an optimal distance and spacing, thus exhibiting a degree of stabilization and hydrophobic packing of the aromatic phenyl moiety, avoiding for example the cyclisation phenomenon, which is a prelude to diketopiperazine formation.

Loading (loading) of CTC resins is generally performed by nucleophilic substitution of a biphenyl-2' -chlorophenyl-chloromethane derivative (hence called CTC, short for chloro-trityl chloride), which is known to be effective. Alternatively, there are also commercially available Fmoc-amino-acid-CTC pre-loaded resins.

Protecting groups and their chemistry are well known in the art and widely documented (see Bodanszky, supra). Needless to say, different protecting groups R2-R9 are suitable for protecting the unique individual amino acid side chains, respectively, since different protecting groups need to be used for different chemical moieties. For example, histidine is typically protected with a trityl or Boc, lysine with Boc or allyloxycarbonyl, and aspartic acid with tert-butyl ether or allyl ester. Threonine, serine and tyrosine are also commonly protected as tertiary butyl ethers. Protection of arginine is also discussed below. Different deprotection modes can be applied, for example the laborious (laborously) removal of allylated protecting groups by Pd-catalysed reductive transacylation. The Z (benzyloxycarbonyl) group is inconvenient to use because it requires hydrogenolysis to remove it efficiently. Preferably, the protecting groups R2-R9 are acid labile, 'labile' meaning that the cleavage rate of each protecting group is at least 20% upon incubation in DCM solution for 5 hours under weakly or strongly acidic conditions. According to the present invention, it is more preferable that the protecting groups R2 to R9 are removed under and only under the strongly acidic conditions described above (i.e., by acidolysis under strongly acidic conditions).

R1 is an insoluble solid phase, typically a polymeric solid phase, such as a crosslinked polystyrene/1% divinylphenol copolymer. Typically (but not strictly required), the solid phase R1 may of course also exhibit multiple 2-chloro-trityl-arm moieties functionalized with peptidyl-a in the practice of the invention, rather than being limited to the one explicitly shown in formula I. More importantly, for use in the first proposed solid phase synthesis by Merrifiled, the minimum particle size of the polymeric solid phase needs to be capable of forming a true suspension of easily filterable or easily precipitable particles of sufficient size rather than colloidal character. Except that CTC arms or linkers are used directly (e.g., Bayer's 4-carboxytrityl liner (liner), Bayer et al, 13^thAmerican Peptide Symposium, codes of Hodges et al, ESCOM,leiden, 1994, page 156) derivatized polystyrene host polymer (base polymer) the individual benzene moieties in the host polymer have been derivatized to participate in the formation of the 2-chloro-trityl function), but also other host polymers, such as pure PEG or mixed PEG resins (e.g., Tentagel) or optionally mixed or grafted resins (hybrid or grafted resins), for example, by grafting a 2-CTC linker (e.g., Bayer linker) onto the polystyrene-based polymer through a PEG spacer rather than directly reacting the linker with the polystyrene-based polymer. The addition of PEG to the resin provides a more amphiphilic resin and thus better handling, e.g. in one step separation and deprotection in a DCM/TFA mixture, although loading capacity may be problematic. It should be noted that there are, of course, insoluble PEG resins in the strict sense. One such PEG polymer-based technique that mimics the principles of solid phase separation but works strictly in solution is described in Bayer et al (Nature 1972, Vol. 237, p. 512 f), where the peptide-resin conjugate remains soluble and provides a homogeneous single phase system. Such resin properties are included in the definition of "insoluble" according to the present invention in its preferred meaning in the context of the present invention, as they essentially allow a rapid and simple size-based separation on a microscopic level by microfiltration or ultrafiltration techniques. In a more preferred meaning, "insoluble" means that one phase of the two-phase system is a true solid suspended phase in a given solvent system used for peptide synthesis.

Preferably, the solid phase has a particle size of less than 700 mesh (particle size as defined by the US Bureau of standards, and is retrievable, for example, in R ö mpps Chemie-Lexikon, 7.Auflage, 1973, Franck' sche Verlagshandlung, W.Keller & Co. Stuttgart/Germany).

Preferably, the particle size of the 2-chloro-trityl-functionalized solid phase of the present invention is in the range of 50 to 600 mesh (as defined by the US Bureau of standards), more preferably in the range of 60 to 400 mesh, most preferably in the range of 100-300 mesh.

The tyrosine of the present invention may be protected with different protecting groups, such as tertiary butyl ether, or a Z-, or more preferably a 2-bromo-Z-ester. Likewise, trityl alcohol protecting groups such as 2-chloro-trityl or 4-methoxy or 4, 4' methoxy-trityl may also be employed. Preferably, R9 is a trityl or tert-butyl protecting group. More preferably, R9 is a tert-butyl (tBu) protecting group, which means that the tyrosyl side chain is modified to a tert-butyl ether. the tBu group can only be removed effectively under strongly acidic conditions.

Preferably, alone and especially in combination with other preferred embodiments, the arginine protecting group R2 is selected from: pentamethyldihydrobenzofuranyl (Pbf), adamantyloxy-and isobornyl-oxy-carbonyl, pentamethylchromosulfonyl (Pmc), 4-methoxy-2, 3, 6-trimethylbenzenesulfonyl (Mtr) and its 4-tert-butyl-2, 3, 5, 6-tetramethyl homologue (Tart) or Boc, which are cleaved only under the strongly acidic conditions described above. R2 is more preferably Pbf, Pmc, Mtr, most preferably Pbf; after complete deprotection of the side chains, which is carried out under strongly acidic conditions and usually in aqueous medium, no bystander-alkylation of the deprotected tyrosine is observed with Pmc, Mtr and especially Pbf protection. The cleavage rate of Pbf was highest.

It is well known that carboxy-protecting groups can be used for Glu, Asp, e.g., Mpe, O-1-adamantyl, O-benzyl, and even simpler alkyl esters, although not so commonly used. For the sake of simplicity, tert-butyl is generally and preferably used independently as protecting groups R4, R5, R6, R7, R8.

The protecting group R3 is of critical importance because it is present in the abovementioned sequence Gly-Asp in the divalent hirudin-8, which dipeptide sequence is particularly prone to side reactions with formation of aspartimide. The formation of aspartimide occurs in the protected peptide during each subsequent coupling reaction in the linear synthesis, to a slight extent (0.1-0.5%), but may eventually exhibit an accumulating effect. Also, while it is preferred to use a trityl protecting group or 2-chloro and 4-methyl or 4-methoxy derivatives thereof, adamantyl protecting groups may also be used. Most preferably, a trityl protecting group is used.

It should also be noted that N α -alkyl protected dipeptide modules can be used in linear synthesis instead of side chain and N α protected amino acid coupling; the dipeptide has the function of interfering with secondary structure, so that the synthesis yield and purity are better. For example, Fmoc-Gly- (N-Hmb) Gly-OH and Fmoc-Gly- (N-Dmb) Gly-OH are commercially available from EMD Biosciences (Novabiochem). It is to be understood that the N-alkyl groups are not considered protecting groups in the present invention and, therefore, their use or presence is optional and is not excluded from the structure of formula I.

In a preferred method of isolation and deprotection of a peptide-conjugate of formula I substantially as described in the claims, a two-step sequential scheme is used, first acid hydrolysis under weakly acidic conditions to cleave the protected peptide from the CTC resin, and then removal of the remaining protecting groups under strongly acidic conditions.

The reason for this approach is that the complete deprotection product and the hydrophobic ligation educt in a one-step global deprotection of the peptide-solid phase conjugate of formula I contradict the solvent requirements, and therefore compromising treatment negatively affects both product purity and yield. This continuous multi-step process eliminates the above-mentioned inherent drawbacks, enabling a better control of the different reactions and thus an optimization of the yields. Its surprising effect according to the invention also includes a complete suppression of diketopiperazine formation as a side reaction.

Accordingly, a method of isolating and deprotecting a peptide-solid phase conjugate of formula I as hereinbefore described to obtain a peptide of formula D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu has been devised, the method being characterized in that: in the first step, the protected peptide is cleaved from the 2-chlorotrityl arm by treatment under weakly acidic conditions, preferably using a 0.1-10% solution of TFA in a polar aprotic solvent, and in the second step, the protecting group is removed under strongly acidic conditions as described above.

Preferably, the first step is carried out in a polar aprotic solvent, which is dichloromethane. This is the best solvent for carrying out the reaction compared to other solvents (e.g. NMP, N-methylpyrrolidone). It is also possible, but not necessary, to include a scavenger in the solvent, especially the solvent system used in the second deprotection step, which scavenger is present in an amount of 0.1-10% (w/w) of the reaction solution (reaction broth) for preventing undesired alkylation of the aromatic nucleus of tyrosine from occurring again. The scavenger blocks the reactive alkyl-carbonium ion intermediate generated when the protecting group is removed (which may have occurred slightly during the shearing reaction in the first step).

An example of a scavenger is thiobenzol (which also has a second acidolysis promoting effect-this second effect and a replacement for the thiobenzol are discussed in Bodanszky M et al, int.j.peptide Protein res.23: 287). Examples of other scavengers not having this acidolysis effect are phenol and/or trialkylsilanes (Stierandova et al, int.J. peptide Protein Res.43, 1994, 31-38).

Preferably, after the first step of cleavage or isolation from the resin, the reaction is quenched directly by mixing with pyridine and the product of step 1 is recovered by mixing with water. The product can be recovered most simply and efficiently by this process.

In another embodiment of the invention, the peptide involved is essentially required to be attached to a peptide-solid phase conjugate of formula I, the only difference being that the thrombin cleavage site-Arg (R2) -Pro-is not a standard peptide bond but a chemically modified pseudo-cleavable bond (pseudo-cleavage) or a 'psi' bond (replacement of the amide bond is indicated by an atom with a 'psi' prefix in added parentheses, see Rudin et al, Drug Desgin Vol.II, editors, Aries, E., academic Press, New York, p.319(1971) more preferably, the psi substitution is-Arg [ psiCH2NH ] Pro- (Kline, T et al, 1991, high role peptides with cleavage site residues resistant to thrombin cleavage, antithrombin cleaved bivalent hirulog peptide with cleavable bond substitution, biochem. Biophys. Res. Commun.177, 1049-substituted 1055) the simplest is, for example, the psi bond was introduced during solid phase synthesis immediately by normal coupling of the growing conjugated peptide with a preformed Fmoc-protected psi dipeptide.

It is a further object of the present invention to extend the above embodiments and methods to peptide-solid phase conjugates comprising a resin moiety other than the CTC resin described above, such that after extension the method can still cleave the peptide moiety from the resin under mildly acidic or mildly acidic conditions as described above.

As another object, peptide-resin conjugates of the formula A-W are contemplated, wherein A may be any of the previously described embodiments of A, optionally comprising amino acid side chain protecting groups, wherein R2-R9 are present as described above, and wherein W is a preferably insoluble solid phase or solid phase complex which can be cleaved at weakly acidic conditions at the peptide moiety and has a resin arm or linker as shown below

a. Formula II

Provided that A comprising residue X2 is always attached to the indicated arm or linker via-O-,

wherein R * is a solid phase, R '1, R' 2, R '3 are the same or different and are each independently hydrogen, 4-or 4' - (C)₁-C₄Alkyl) or 4-or 4' - (C)₁-C₄Alkoxy) but R '1 and R' 2 may not both be hydrogen, R 'is optionally 2-Cl when R' 1 is H, and wherein more preferably or most preferably the arm or linker of formula II is selected from 2-chloro-trityl, 4-methoxy-trityl, 4-methyltrityl,

b. or formula III

(which may be derived from acylated amino-or hydroxy-functionalized resins with Bayer's 4-carboxytrityl linker) provided that A, which also contains the residue X2, is attached to the arm or linker via-O-, R * is as defined above,

c. or formula IV

Wherein R * is a solid phase or a polymer resin and R "1, R" 2, R "3 are the same or different and are independently hydrogen, C₁-C₄Alkyl or C₁-C₄Alkoxy, but R '1 and R' 2 cannot both be hydrogen, wherein L is A (L ═ A) or wherein L is of the formula V

In another preferred embodiment, the resin arm has the formula VI, R *, R '1 and R' 2 are as defined above,

as previously mentioned, it is more preferred that the resin and resin arms have the formula VII, R *, R '1 and R' 2 are as defined above,

in another more preferred embodiment, A, optionally including residue X2, is attached via-O-to an arm or linker of formula VII, R "1 and R" 2 are independently hydrogen, methyl or methoxy when R "1 and R" 2 are not both hydrogen; when A comprising residue X2 is attached via-N-to an arm or linker of formula VII, R "1 and R" 2 are independently methyl or methoxy, preferably methoxy. More preferably, A, also containing X2, is bound to the arm through an-O-functional group, R "1 is hydrogen, R" 2 is methyl or methoxy, and preferably A is a resin or resin arm. Most preferably, R' 2 is methyl.

The resin or resin arm combination part may in principle be any resin used in synthesis, for example polystyrene-stilbene resins used by Merrifield together with hydroxybenzyl-phenyl linker parts (as part of the resin) or by Wang together with hydroxy-benzyl-p-benzyloxy parts, including for example those to which more acid-labile linkers may be further grafted, or the latter linkers may be made integral or directly attached to the resin. In principle, the solid phase resin used for synthesis must contain at least one linker or arm as part of it, which is part of the solid phase core material; the linker or arm can be considered an immobilized protecting group (Guillier et al, chem. Rev.100, 2091-2157, 2000). Examples are for instance: sieber resins, related xanthenyl (related xanthenyl) PAL-arm resins, Rink amide resins, Rink acid resins, more complex PEG grafted polystyrene resins (e.g. tentagel based Novasyn TG (Novabiochem, Merck Biosciences, germany), ready-made such resins having different grafting arms (e.g. 2' -chloro-trityl)), or resins constructed by grafting functional arms onto a matrix material (e.g. silica gel). Preferably, if the resin is a trityl resin or resin arm, it is selected from 4-methoxy or 4, 4' -dimethoxy-trityl resin. The resin used in the present invention has a standard particle size of about 50 to 500 mesh, more preferably 100-400 mesh. Resin or solid phase R * as shown in formula IV is understood to comprise a cross-linked polymer matrix material which may be attached to the arm parts as shown in formulas IV-VII via any kind of chemically inert alkyl, alkoxy, aryloxy or alkyl ester spacer or linker, which is considered to be a constituent of R *. However, it should be noted that in addition to having an effect on the conditions of cleavage from the resin, the properties of the resin material, particularly the chemistry of the arm groups, will greatly affect the effectiveness of the coupling and lactamisation reactions (lactamisation), but the mechanism of the effect is not well understood. The yield of mature peptide in the on-resin stage (on-resin stage) varies depending on the type of resin or resin arm used. For this reason, in a preferred embodiment of the invention, the resin or resin arm is of formula IV (as claimed), more preferably of formula VI (as claimed), most preferably of formula VII (as claimed). Examples of such resins or resin arms are (4-methoxyphenyl) -methyl-and (4-methylphenyl) -methyl-polystyrene (Atkinson et al, 2000, j.org.chem.65, 5048), resins linked to peptide moieties with O-or N-linkages, and PEG-resin derivatives thereof. Other examples are for example: acid-labile HMPB-MBHA or HMPB-BHA resins (Sieber et al, 1987, Tetrahedron Lett.28, 6147), acid-labile Rink amide resins or Rink acid resins (Rink et al, 1987, Tetrahedron Lett.28, 3787). The term "acid labile" means that substantially quantitative cleavage occurs at ambient temperature in 2-10% TFA in dichloromethane for at least 1 hour. Surprisingly, the use of such preferred resins having a biphenyl-methyl structural core motif increases the efficiency of the coupling and lactamization reactions during linear synthesis; it is noted that the resin can be used at lower reaction temperatures, e.g. 15-25 ℃, whereas the standard temperature required to achieve efficient coupling on e.g. triphenylmethane resins is 40 ℃.

Experiment of

Synthesis of Boc-D-Phe-Pro-Arg (Pbf) -Pro-Gly-Gly-Gly-Asn (Trt) -Gly-Asp (tBu) -Phe-Glu (tBu) -Ile-Pro-Glu (tBu) -Tyr (tBu) -Leu-O-2-CTC (protected bivalent hirudin-8, see for details EP-489070, ester bond to 2-CTC resin via carboxyl terminus)

All reagents were obtained from EMD Biosciences (Madison, Wis.; Novabiochem-brand). Polystyrene-based 2-ClTrt (CTC) resin preloaded with Fmoc-Leu-OH (Cbl Patras, Greece) was 100-200 mesh based on its base polymer and 60-200 mesh based on the final CTC resin product after preloading. The loading density was about 0.60 mmol/g. Each amino acid is provided as an Fmoc amino acid or, when it is D-Phe, as Boc-protected Boc-D-Phe. Coupling was carried out with TCTU in dichloromethane/N-methylpyrrolidone (NMP) solution in the presence of Hunig-Base (diisopropyl-ethyl-amine, DIEA). Typically, 1.5 equivalents of Fmoc amino acid or Boc protected amino acid are used, but 2.5 equivalents are used for Fmoc-Arg (Pbf) coupling. Similarly, the coupling reaction time for Fmoc-Arg (Pbf) was extended from the standard 60 min (30 ℃) to 90 min. In the process, Kaiser test or Chloranil test method is adopted to control the coupling efficiency.

Deprotection of Fmoc was performed at 30 ℃ with 3-4 cycles of 20% piperidine in NMP during which time NMP was used for appropriate washes.

Synthesis of Boc-D-Phe-Pro-Arg (Pbf) -Pro-Gly-Gly-Gly-Asn (Trt) -Gly-Asp (tBu) -Phe-Glu (tBu) -Ile-Pro-Glu (tBu) -Tyr (tBu) -Leu-OH

Cleavage from 48.3g of resin (prepared as described in example 1) was accomplished by 3 cycles of 15 min, 15 deg.C, 2% (w/w) TFA, 1% (w/w) Triethylsilane (TES), and reaction in dichloromethane. Stirring the reactants by nitrogen bubbling; the color of the reaction changed from yellow/orange to brown with the number of reaction rounds. After each reaction, the shear reaction was quenched directly by injecting the entire reaction solution into dilute pyrimidine (pyrimidine/ethanol 1: 9 (v/v)). The resin was then separated by filtration through a frit (frit) for the next round of reaction. All filtrates were pooled, concentrated under vacuum (RotaVap) to an orange semi-fluid, washed with DCM, resuspended in 400mk double distilled water, stirred at room temperature, filtered, washed with water and dried. 28,8g of a pale yellow powder of analytical grade (. about.90% purity) were obtained. The product was analyzed by HPLC and LC-MS.

3. Global deprotection of NH₂Synthesis of-D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH

Global deprotection was performed in DCM dilutions (DCM: ' CC ═ 1: 10(v/v)) of the sheared mixture (cleavage cocktail, ' CC '). 'CC' consists of TFA/thioanisole/phenol/water/TES in a mixing ratio (% w/w) 89: 2.5: 5.0: 1. 0.1g of the dried product of experiment 2 was dissolved in 10ml of DCM diluted as ` CC ` as described above and stirred at room temperature for 5 hours. The product is then recovered by adding 50ml of methyl-tert-butyl ether (MTBE, Fluka Chemie, Buchs/Switzerland), cooling the reaction to 0 ℃ in a water bath for 30 minutes with stirring, and filtering off the salt precipitate formed during this. The filter cake is washed several times with MTBE and dried at room temperature to give 0.8g of crude product, about 55% pure by HPLC. The combined overall yield of steps 2 and 3 was about 55%.

Comparative cleavage experiments and LC-MS analysis of the Synthesis of hirudin-8 or its C-terminal tetrapeptide fragment on Wang resin or CTC resin

HPLC LC-MS analysis showed that the peptide product using Wang resin was 1-10% alkylated after cleavage from the resin and global deprotection under strong acidic conditions, and no such modification was observed after cleavage from the CTC resin. The modification on the tyrosyl residue was localized by MS analysis. The synthesis procedure is as described above.

Claims (7)

1.A peptide-resin conjugate of formula I comprising a 2-chloro-trityl arm,

wherein A ═ Boc-D-Phe-Pro-Arg (R2) -Pro-Gly-Gly-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-or A ═ Fmoc-D-Phe-Pro-Arg (R2) -Pro-Gly-Gly-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-orA＝NH₂-D-Phe-Pro-Arg (R2) -Pro-Gly-Asn (R3) -Gly-Asp (R4) -Phe-Glu (R5) -Glu (R6) -Ile-Pro-Glu (R7) -Glu (R8) -Tyr (R9) -Leu-O-, and wherein R2, R3, R4, R5, R7, R8, R9 are amino side chain protecting groups and R1 is an insoluble solid phase.

2. The conjugate according to claim 1, wherein the solid phase is polymeric and has a particle size of less than 700 mesh (US bureau of standards), preferably a particle size of 50-600 mesh.

3. The conjugate of claim 1, wherein R2 is pentamethyldihydrobenzofuranyl, adamantyloxy-carbonyl or isobornyloxycarbonyl, R9 is tert-butyl or a derivative thereof, and R3-R8 are acid labile protecting groups.

4. The conjugate of claim 3, wherein R2 is Pbf and R4-R9 are acid labile protecting groups that require at least 50% or more concentrated trifluoroacetic acid.

5. The conjugate of claim 4, wherein R3 is trityl and R4, R5, R6, R7 and R8 are tert-butyl.

6. The conjugate of claim 5, wherein R9 is tert-butyl.

7. The conjugate of claim 1, wherein the N-terminal Boc group is replaced with another protecting group that is cleavable under strongly acidic conditions.