WO2004050686A2

WO2004050686A2 - Process and supports for synthesis of peptides comprising a thioester or thioacid

Info

Publication number: WO2004050686A2
Application number: PCT/GB2003/004967
Authority: WO
Inventors: David John Evans
Original assignee: Avecia Limited
Priority date: 2002-11-29
Filing date: 2003-11-17
Publication date: 2004-06-17
Also published as: AU2003283579A8; GB0227876D0; AU2003283579A1; WO2004050686A3

Abstract

A process for the Fmoc synthesis of a peptide comprising a thioester or thioacid which comprises removing the Nα-Fmoc blocking group with a base selected from optionally substituted piperazine or optionally substituted 2,6-C1-4dialkyl-piperidine or a mixture thereof. Also novel solid supports suitable for the solid-state synthesis of peptide thioesters and thioacids and processes for making these supports.

Description

PROCESS AND SUPPORTS FOR PEPTIDE SYNTHESIS

This invention relates to a process for producing peptide thioesters and peptide thioacids using the Fmoc blocking group, also to novel supports suitable for use in solid state peptide thioester synthesis.

Peptides comprising up to about 60 amino acids may be effectively produced using solid phase synthesis (Schnolzer et al., Int. J. Pept. Protein Res. 1992, 40, 180- 193). However with larger synthetic objectives the accumulation of low-level impurities makes purification of the desired peptide extremely demanding. This is especially so when the desired peptide has to be produced on a commercial scale.

The favoured method for overcoming this problem is to synthesise short unprotected peptide fragments and chemically ligate them to form the desired product. A number of different functionalities have been tried. However the method most favoured uses a ligation reaction between a peptide fragment bearing a C-terminal thioester (Dawson et al., Science, 1994, 266, 776) or a C-terminal thioacid (Liu et al., Tet. Lett., 1996, 37, 933) and a peptide fragment bearing an N-terminal residue, such as cysteine, with a thiol side chain (for a review see Dawson and Kent in Annu. Rev. Biochem 2000, 69, 923-960).

Production of peptide thioesters and thioacids has in the main relied on tert- butyloxycarbonyl (Boc) solid phase synthetic methods (Blake and Li, Proc. Natl. Acad. U.S.A., 1981 , 78, 4055-4058; Canne et al., Tet. Lett, 1995, 1217-1220; Hojo et al., Bull. Chem. Soc. Jpn., 1993, 66, 2700-2706; Hackend et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 10068-10073).

Synthesis of peptide thioesters and thioacids using the more convenient fluorenylmethyloxycarbonyl (Fmoc) technology has proved to be problematic. This is because the established and most convenient methods employ a thioester linkage to the solid phase and this bond is susceptible to cleavage by piperidine, a base routinely used to remove the Fmoc protecting group. Despite this, some methods have been developed using Fmoc technology. These include introducing the labile thioester at the end of the synthesis (Aisina et al., J. Org. Chem., 1994, 64, 8761-8769); utilising the sulphonamide "safety catch" linker (Biancalana et al., Lett, in Pep. Science, 2001 , 7, 291-297); activating the sulphonamide linked to an assembled peptide by alkylation and then cleaving with a thiol nucleophile to yield a thioester (Ingenito et. al., J. Am. Chem. Soc, 1999, 121 , 11369- 11374; Shin et al., J. Am. Chem. Soc.,1999, 121 , 11684-11689); and generating a thioester peptide utilising a para-hydroxymethylphenoxyacetic acid linker and cleaving with a thiol in the presence of (CH₃)₂AICI (Swinnen and Hilvert, Organic Lett., 2000, 2, 2439-2442). These methods all have drawbacks and their usefulness for producing large amounts of peptide thioesters and peptide thioacids is limited. In a recent paper (Tet. Lett., 2002, 43, 2419-2422) Bu et al. have noted that by using 1,8-diazabicydo[5.4.0]undec-7-ene (DBU) rather than piperidine to remove Fmoc the thioester peptide is left attached to the solid support.

However, DBU, although known for use in peptide synthesis, suffers from serious drawbacks which limit its use in large scale production. Thus, DBU is known to cause considerable racemisation of cysteine and other susceptible residues leading to downstream purification problems and yield loss. In addition DBU promotes aspartimide formation and so the use of DBU usually requires the use of the expensive back bone protection amino acid Gly(Hmb). Furthermore, during Fmoc removal by DBU the unassociated dibenzofulvene afforded has the potential to react with the peptide. This is not the case when piperidine is used to remove Fmoc since the piperidine is able to scavenge the dibenzofulvene as it is formed.

Alternative methods for the manufacture of peptide thioesters and peptide thioacids are therefore required. The present invention provides a process for the Fmoc synthesis of a peptide comprising a thioester or thioacid which comprises removing the Nα-Fmoc blocking group with a base selected from optionally substituted piperazine or optionally substituted 2,6-C_Ϊ. ₄dialkyl-piperidine or a mixture thereof.

Preferably the peptide comprises a C-terminal thioester or C-terminal thioacid. When the base is optionally substituted 2,6-C^dialkyl-piperidine the C^alkyl groups in the 2 and 6 positions may be varied independently. Preferably when the base is optionally substituted 2,6-C₁-₄dialkyl-piperidine it is optionally substituted 2,6-dimethyl- piperidine.

Most preferably the base is unsubstituted piperazine or unsubstituted 2,6-dimethyl- piperidine.

When the piperazine or 2,6-C^dialkyl-piperidine bases are optionally substituted then the optional substituent must be selected so that the substituted base is still able to remove the Nα-Fmoc blocking group yet leave thioester linkage intact.

The optional substituents present on either piperazine or 2,6-C₁_₄dialkyl-piperidine may be any unreactive group that under the reaction employed does not interfere with the synthesis of the target peptide. When piperazine or 2,6-C^dialkyl-piperidine does carry a substituent it is preferably selected from optionally substituted alkyl (preferably C^-alkyl), optionally substituted alkoxy (preferably C^-alkoxy), aryl optionally carrying an electron donating substituent (preferably phenyl), optionally substituted aryloxy (preferably phenoxy), optionally substituted heterocycle; polyalkylene oxide (preferably polyethylene oxide or polypropylene oxide), carboxy, phosphato, sulpho, nitro, cyano, halo, ureido, - SO₂F, hydroxy, ester, -NR^aR^b, -COR^a, -CONR^aR^b, -NHCOR³, carboxyester, sulphone, and -SO₂NR^aR^b wherein R^a and R^b are each independently H or optionally substituted alkyl (especially C^-alkyl). The base used in the process of the present invention may be either free in solution or immobilised on a macroporous or microporous solid support. The solid support resin is preferably as described below.

Fmoc synthetic protocols are well known and documented in the art and are reviewed in Chan and White "Fmoc Solid-phase Peptide Synthesis" Oxford University Press, 2000, which is incorporated herein, in its entirety, by reference.

Preferably the process of the present invention is a solid phase peptide synthesis.

More preferably, the present invention provides a process for the solid phase synthesis of a peptide comprising a C-terminal thioester or C-terminal thioacid which process comprises the steps of:

(a) attaching a first amino acid with an α-nitrogen Fmoc protecting group to a solid support by means of a linkage comprising a thioester;

(b) (i) removing the α-nitrogen Fmoc protecting group using optionally substituted piperazine or optionally substituted 2,6-C^dialkyl-piperidine or a mixture thereof under conditions such that the attached amino acid remains connected to the solid support; and (ii) coupling an additional amino acid with an α-nitrogen Fmoc protecting group to the unprotected α-nitrogen of the first attached amino acid;

(c) (i) removing the α-nitrogen Fmoc protecting group using optionally substituted piperazine or optionally substituted 2,6-C^dialkyl-piperidine or a mixture thereof under conditions such that the attached amino acids remain connected to the solid support; and

(ii) coupling an additional amino acid with an α-nitrogen Fmoc protecting group to the unprotected α-nitrogen of the attached amino acids and repeating until the desired peptide minus the desired N-terminal residue is assembled on the solid support;

(d) (i) removing the α-nitrogen Fmoc protecting group using optionally substituted piperazine or optionally substituted 2,6-C^dialkyl-piperidine or a mixture thereof under conditions such that the attached amino acids remain connected to the solid support; and (ii) coupling the N-terminal residue of the desired peptide with an α-nitrogen protecting group to the unprotected α-nitrogen of the attached amino acids to yield the desired peptide;

(e) removing the protecting group from the terminal α-nitrogen of the peptide; and

(f) cleaving the link between the first amino acid of the peptide and the solid support and optionally removing any side chain protecting groups to release a peptide with a C- terminal thioester or a C-terminal thioacid. Any solid support suitable for use in solid-state peptide synthesis with a linker which before or after reaction with the Cα-carboxyl group, or activated form thereof, of the first amino acid comprises a thioester group may be used in step (a). Examples of such supports may be found in Tet.Lett., 2002, 43, 2419-2422 and Tet.Lett., 1995, 36, 1217- 1220 the supports of which references are incorporated herein by reference.

A preferred solid support comprises an optionally substituted thiol-trityl linker.

Favored thiol-trityl linkers comprise thiol-2-chlorotrityl or thiol-4-methyltrityl and particularly thioI-4-methoxytrityl. These linkers react with the Cα-carboxyl group, or activated form thereof, of the first amino acid to form a thioester bond which, after synthesis of the desired peptide, may be cleaved to yield a peptide with a C-terminal thioacid residue.

Another preferred solid support is of Formula (1):

HS — R¹ — ^LL-0 — Resin

Formula (1) wherein:

L represents the residue of a acid labile linker which on cleavage liberates an acid; and

R¹ is optionally substituted C_halky!. Preferably R¹ is optionally substituted C^alkyl and more preferably, optionally substituted C^alkyl. It is especially preferred that R¹ is -C₂H -.

The optional substituent on R¹ may be any unreactive group that under the reaction employed does not interfere with the synthesis of the target peptide. When R¹ does carry an optional substituent it is preferably selected from the group consisting of: optionally substituted alkoxy (preferably C^-alkoxy), aryl optionally carrying an electron donating substituent (preferably phenyl), optionally substituted aryloxy (preferably phenoxy), polyalkylene oxide (preferably polyethylene oxide or polypropylene oxide), carboxy, phosphato, sulfo, nitro, cyano, halo, ureido, -SO₂F, hydroxy, ester, -NR^aR^b, - COR^a, -CONR^aR^b, -NHCOR³, carboxyester, sulfone, and -SO₂NR^aR^b, wherein R^a and R^b are each independently H or optionally substituted alkyl (especially C^-alkyl). Optional substituents for any of the substituents described for R¹ may be selected from the same list of substituents.

A preferred solid support of Formula (1 ) is of Formula (2):

Formula (2) wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker.

Solid supports of Formula (2) may be prepared by reacting a solid support comprising 4-hydroxymethylphenoxyacetyl (HMPA) linker with 2,2-dithiodipropionic acid, dithiodiglycolic acid or, more preferably, 3-mercaptopropionic disulfide, preferably in the presence of a catalyst such as 4-(N,N-dimethylamino)pyridine. This adduct is then reduced, for example by tris(carboxyethyl)phosphine (TCEP) in DMF, to yield a support of Formula (2):

An alternate process by which a solid support of Formula (2) may be made comprises reacting a substituted trityl chloride, preferably 2-chlorotrityl chloride, 4- methyltrityl chloride and especially 4-methoxytrityl chloride, with 3-mercaptopropionic acid, or analogue or derivative thereof, to give a compound of Formula (4):

Formula (4)

wherein each X is independently a substituent which may be present on 1 or more of the phenyl rings and n is 1 to 3.

Preferably each X is independently chloro, methyl or methoxy. Preferably n is 1.

Preferably X is 2-chloro, 4-methyl or 4 methoxy.

The compound of Formula (4) may then be esterified with a solid support comprising a 4-hydroxymethylphenoxyacetyl and the substituted trityl group selectively removed, for example, by treating the resin with 1 to 5% trifluoroacetic acid in dichloromethane, to yield a solid support of Formula (2).

The Cα-carboxyl group of the first amino acid may then be reacted with the free thiol of the support of Formula (1) or (2) and the desired peptide synthesised. At the end of the synthesis a peptide thioester of Formula (3):

o

Peptide -s — R¹ — COOH

Formula (3) may be released by treating the peptide bound to the solid support resin with an acid, preferably trifluoroacetic acid under standard conditions. A third preferred solid support is of Formula (5):

Formula (5) wherein: each Y is independently an optional substituent; and R² is optionally substituted d.₁₀alkyl. A preferred solid support of Formula (5) is of Formula (6):

Formula (6)

wherein each Y independently is an optional substituent.

A solid support of Formula (6) may be prepared by reacting a solid support comprising an optionally substituted trityl chloride linker with 3-mercaptopropionic disulfide. This adduct is then reduced, for example by tris(carboxyethyl)phosphine in DMF, to yield a support of Formula (6). Other supports of Formula (5) may be prepared by an analogous procedure except that 3-mercaptopropionic disulfide should be replaced by which ever disulfide is required to give the desired R¹.

Preferably the solid support of Formula (6) comprises 4-methoxytrityl, 4-methyltrityl and especially 2-chlorotrityI.

A further preferred solid support may be prepared by reacting a solid support comprising an acid labile linker, such as 4-hydroxymethylphenoxyacetyl, with a thiol containing amino acid, preferably cysteine or cysteine, wherein the α-nitrogen is blocked, preferably with Fmoc, and the thiol side chain is also blocked, preferably with 4- methoxytrityl. The blocking group is removed from the α-nitrogen which is then acylated. The thiol side chain blocker is then removed to yield the support of Formula (7): Resin

Formula (7)

wherein:

-R³-SH is the side chain of a thiol containing amino acid; and Linker is the residue of an acid labile linker. The acyl group is preferably acetyl and acylation may be achieved by any means known in the art. For example, by treating with acetic anhydride (10 equivalents with respect to the solid support loading) and N.N-diisopropylethylamine (DIPEA) in DMF.

When acid labile linker is 4-hydroxymethylphenoxyacetyl and L-cysteine is used as the thiol containing amino acid a preferred solid support, of Formula (8), is obtained:

Resin

Formula (8)

wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker. The solid support may be either a microporous or a macroporous support. Preferably the solid support resin is based on a polystyrene or polydimethylacrylamide polymer. More preferably the support is a copolymer of styrene with about 0.5 to 2% divinyl benzene as a cross-linking agent or a polydimethylacrylamide polymer comprising N,N-dimethylacrylamide, N,N-bisacryloylethylenediamine and acryloylsarcosine methyl ester monomers. Details of these preferred supports and other suitable supports may be found in Chan and White "Fmoc Solid Phase Peptide Synthesis" Oxford University Press, 2000 pages 14 to 15 the solid supports of which are incorporated herein by reference.

Particularly preferred supports are supports with a thiol trityl linker and 4- hydroxymethylphenoxyacetyl (HMPA) linker supports incorporating 3-mercaptopropionic acid as shown in Formula (1) and (2).

Step (a) may be carried out under conditions and in solvents such as those commonly used in linking amino acids to solid supports in solid-phase peptide synthesis, these conditions and solvents are well known in the art. Methods suitable for carrying out step (a) are described in many standard texts on the subject such as Atherton and Sheppard, "Solid-Phase Peptide Synthesis a Practical Approach", IRL Press at Oxford University Press, 1989 and Chan and White "Fmoc Solid Phase Peptide Synthesis" Oxford University Press, 2000 which are incorporated herein by reference.

Step (a) typically comprises dissolving the appropriate Fmoc amino acid in a suitable solvent, activating the carboxyl group, preferably with a diimide such as an alkyl carbodiimide particularly diisopropylcarbodiimide and reacting with the linker attached to the solid support in the presence of a catalyst such as 4-(N,N'-dimethylamino) pyridine. After the reaction, the resin is collected by filtration and washed with a suitable solvent, typically N,N-dimethylformamide.

The coupling cycles of steps (b), (c) and (d) may be carried out using standard conditions for peptide solid-phase synthesis that would be well known to one skilled in the art. For further detail reference is made, for example, to Atherton and Sheppard, " Solid- Phase Peptide Synthesis a Practical Approach ", IRL Press at Oxford University Press, 1989 and Chan and White "Fmoc Solid Phase Peptide Synthesis" Oxford University Press, 2000 the coupling conditions and procedures of which are incorporated herein by reference.

The conditions used to remove the Fmoc protecting group are selected to avoid substantial premature release of the amino acid or peptide via cleavage of the base labile thioester bond with the linker. Thus, it is particularly favoured that Fmoc removal is effected with from 0.1% to 5% piperazine, particularly 1% to 5% piperazine, especially 5% piperazine preferably in solution in N,N-dimethylformamide or with from 10% to 30% 2,6- dimethyl piperidine, particularly 20% 2,6-dimethyl piperidine, preferably in solution in N,N- dimethylformamide or with mixtures of piperazine and 2,6-dimethyl piperidine.

Amino acid activation is carried out using conditions known to the art of peptide synthesis. Those agents and conditions most commonly used are summarised in Chan and White "Fmoc Solid Phase Peptide Synthesis" Oxford University Press, 2000 on page 28 and on pages 52 to 60 which are incorporated herein by reference.

Preferably amino acid activation is carried out in N,N-dimethylformamide in the presence of 1-hydroxybenzotriazole and diisopropylcarbodiimide.

In steps (b), (c), (d) and (e) side chain protecting groups may be used to protect the side chains of susceptible amino acids such as Cys, Arg, Ser, Tyr, Thr, Lys, Orn, Asp, Glu, Trp, His, Asn, Gin, Har, Dpr, Dab, Dht, Gla, Hser, Hyp and Pen. When side chain protecting groups are used during the solid phase synthesis then preferably the peptide is fully deprotected while still attached to the support or the protecting groups are removed simultaneously with release of the peptide from the support. Alternatively a chemical modification of the peptide may be carried out post solid-state synthesis to yield the desired side chain.

In steps (d) and (e) the α-nitrogen protecting group on the N-terminal residue of the desired peptide may be any group known in the art but preferably is Fmoc or Boc and more preferably Boc. In step (e) the α-nitrogen protecting group on the N-terminal residue may be removed by any method known in the art for that particular protecting group.

In step (f) any agent may be used which is able to cleave the link between the first amino acid of the support bound peptide and yield a thioester or thioacid. Preferably any side chain protecting groups are removed at the same time the peptide is released from the solid support.

It is particularly preferred that step (e) and (f) are carried out simultaneously. For example when the α-nitrogen protecting group in steps (e) and (f) is Boc then the Boc protecting group and tertiary butyl based side chain protecting groups may be removed and the peptide released from the support by treatment with a suitable concentration of trifluoroacetic acid.

Isolation and purification of the peptide thioester from step (f) may be achieved using standard procedures and techniques that would be well known to one skilled in the art. Typically, this would involve precipitation of the peptide thioester in a solvent that does not effect the integrity of the peptide thioester such as diisopropylether followed by preparative HPLC.

According to a second aspect of the invention there is provided a process for the Fmoc synthesis of a peptide comprising a thioester or thioacid which comprises removing the Nα-Fmoc blocking group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C^dialkyl-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from: N,N-dimethylformamide (DMF); a haloalkane, especially dichloromethane (DCM); a haloalcohol, especially trifluoroethanol (TFE); and dimethylacetamide (DMA).

Preferably the peptide comprises a C-terminal thioester or C-terminal thioacid. When the base is optionally substituted 2,6-C^dialkyl-piperidine it is preferably optionally substituted 2,6-dimethyl-piperidine.

When the piperazine, 2,6-d^dialkyl-piperidine or 1-methylpyrrolidine bases are optionally substituted then the optional substituent is selected so that the substituted base is still able to remove the Nα-Fmoc blocking group while leaving the thioester linkage intact.

Optional substituents may be selected from those described above in the first aspect of the invention.

Most preferably the base is unsubstituted piperazine, unsubstituted 2,6-dimethyl piperidine or unsubstituted 1-methylpyrrolidine. The base used in the process of the second aspect of the present invention may be either free in solution or immobilised on a support.

Preferably the solvent is selected from N,N-dimethyIformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) and mixtures thereof. More preferably the solvent comprises a mixture of DMF and DCM. In a preferred embodiment the solvent is DMF in the substantial absence of other solvents.

More preferably, the second aspect of the present invention provides a process for the solid phase synthesis of a peptide comprising a C-terminal thioester or C-terminal thioacid which process comprises the steps of:

(b) (i) removing the α-nitrogen Fmoc protecting group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C^dialkyI-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) under conditions such that the attached amino acid remains connected to the solid support; and

(ii) coupling an additional amino acid with a α-nitrogen Fmoc protecting group to the unprotected α-nitrogen of the first attached amino acid;

(c) (i) removing the α-nitrogen Fmoc protecting group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C^dialkyl-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) under conditions such that the attached amino acids remain connected to the solid support; and

(ii) coupling an additional amino acid with a α-nitrogen Fmoc protecting group to the unprotected α-nitrogen of the attached amino acids and repeating until the desired peptide minus the desired N-terminal residue is assembled on the solid support;

(d) (i) removing the α-nitrogen Fmoc protecting group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-d^dialkyI-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) under conditions such that the attached amino acids remain connected to the solid support; and

(ii) coupling the N-terminal residue of the desired peptide with an α-nitrogen protecting group to the unprotected α-nitrogen of the attached amino acids to yield the desired peptide; (e) removing the protecting group from the terminal α-nitrogen of the peptide; and

(f) cleaving the link between the first amino acid of the peptide and the solid support and optionally removing any side chain protecting groups to release a peptide with a C- terminal thioester or a C-terminal thioacid.

The conditions and preferences for the second aspect of the invention are as described and preferred in the first aspect of the invention.

The bases used in the processes of the present invention may exist in tautomeric forms. These tautomers are included within the scope of the present invention.

The processes of the present invention may use manual synthesis techniques or any automated synthesizer, following the instructions provided in the instruction manual supplied by the manufacturer.

A third aspect of the invention provides a solid support of Formula (1)

HS— ¹- -O- -Resin

Formula (1) wherein:

R¹ is optionally substituted Cι_ι₀alkyl.

L and R¹ are as described and preferred in the first aspect of the invention. A preferred solid support of Formula (1), according to the third aspect of the invention, is of Formula (2):

HMPA -Resin

HS J^~ -O—

Formula (2)

wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl linker. A fourth aspect of the invention provides a solid support of Formula (5):

Formula (5) wherein: each Y is independently an optional substituent; and R² is optionally substituted Cι.ι₀alkyl.

Y and R¹ are as described and preferred in the first aspect of the invention. A preferred solid support of Formula (5) is of Formula (6):

Formula (6)

wherein each Y independently is an optional substituent.

A fourth aspect of the invention provides a solid support of Formula (7):

Resin

Formula (7)

wherein: -R³-SH is the side chain of a thiol containing amino acid; and

Linker is the residue of an acid labile linker. The acyl group is preferably acetyl and acylation may be achieved by any means known in the art. For example, by treating with acetic anhydride (10 equivalents with respect to the solid support loading) and DIPEA in DMF. When the acid labile linker is 4-hydroxymethylphenoxyacetyl and L-cysteine is used as the thiol containing amino acid a preferred solid support, of Formula (8), is obtained:

Resin

Formula (8) wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker. A fifth aspect of the invention provides a process for preparing a solid support of Formula (2):

Formula (2)

which comprises reacting a solid support comprising 4- hydroxymethylphenoxyacetyl (HMPA) linker with 2,2-dithiodipropionic acid, dithiodiglycolic acid or, more preferably, 3-mercaptopropionic disulfide, preferably in the presence of a catalyst such as 4-(N,N-dimethylamino)pyridine, this adduct is then reduced, for example by tris(carboxyethyl)phosphine (TCEP) in DMF, to yield a solid support of Formula (2). The solid support resin component is as described and preferred in the first aspect of the invention.

A sixth aspect of the invention provides a process for preparing a solid support of Formula (2):

Formula (2)

which comprises the following steps:

(i) reacting a substituted trityl chloride with 3-mercaptopropionic acid, or analogue or derivative thereof, to give a compound of Formula (4):

Formula (4) wherein each X is independently a substituent which may be present on 1 or more of the phenyl rings and n is 1 to 3;

(ii) esterifying the compound of Formula (4) with a solid support comprising a 4- hydroxymethylphenoxyacetyl linker; and

(iii) selectively removing the substituted trityl group to yield a solid support of Formula (2).

Preferably X is 2-chloro, 4-methyl or 4 methoxy.

Thus, the substituted trityl chloride is preferably 2-chlorotrityl chloride, 4-methyltrityl chloride and especially 4-methoxytrityl chloride.

In step (ii) the conditions for esterifying the compound of Formula (4) with the 4- hydroxymethylphenoxyacetyl would be well known to one skilled in the art.

The substituted trityl group may be selectively removed in step (iii), for example, by treating the solid support with 1 to 5% trifluoroacetic acid in dichloromethane, to yield a solid support of Formula (2).

The invention is illustrated but not limited by the following Example.

Example 1

Analytical Method

The Analysis method used was:

Buffer A 0.1% TFA (thfluroacetic acid) in water Buffer B 0.1 % TFA (trifluoroacetic acid) in Acetonitrile

Column Vydac C18 RP 5μm 300A 250 x 4.6mm

Gradient 1 to 90% buffer B in buffer A over 30 minutes

Flow rate 1.0 cm³, min¹ Detection 214nm Temp Ambient

Stability of a Model Thioester Nα-Benzyloxycarbonyl-L-phenylalanine-benzyl thioester (Z- L-Phe-S-CH₂-CgHg) to Various Bases in DMF

Samples (0.050g, 0.123mmole) of Z-L-Phe-S-CH₂-C₆H₅ were accurately weighed into snap top vials. To each sample was charged 1 ml of the following base reagents:

(A) 20% v/v piperidine in DMF;

(B) 10% v/v piperidine in DMF;

(C) 50% v/v N-methylmorpholine in DMF;

(D) 5% w/v piperazine in DMF; (E) 20% v/v 2,6-dimethylpiperidine in DMF; (F) 50% v/v morpholine in DMF;

(G) 50% v/v 1-methylpyrrolidine in DMF; (H) 50% v/v 1-methylpyrrole in DMF; and (I) 20% v/v 1 ,4-dimethylpiperazine in DMF. The Z-L-Phe-S-CH₂-C₆H₅ derivative dissolved immediately in all the various media.

The solutions were gently agitated over a 56 or 70 hour period. Representative samples (100μl) were removed periodically, diluted with DMF (200μl) then quenched by the addition of 50% w/v HOBt in DMF (300μl). These samples were analysed by HPLC analysis against standards of Z-L-Phe-S-CH₂-C₆H₅, benzyl disulphide, benzyl mercaptan, Z-L-Phe-OH and HOBt. Degradation of the Z-L-Phe-S-CH₂-C₆H₅ standard was followed by monitoring the HPLC peak area. The thioester bond was stable in the following samples:

(C) 50% v/v N-methylmorpholine in DMF;

(D) 5% w/v piperazine in DMF; (E) 20%) v/v 2,6-dimethylpiperidine in DMF;

(G) 50% v/v 1-methylpyrrolidine in DMF; (H) 50% v/v 1-methylpyrrole in DMF; and (I) 20% v/v 1 ,4-dimethylpiperazine.

Lability of the Nα-Fmoc protecting Group to Various Bases in DMF

Samples (1.00g, 2.58mmole) of Fmoc-Phe-OH were accurately weighed into snap top vials. To each sample were charged 20ml of the individual base reagents:

(A) 20%) v/v piperidine in DMF;

(B) 10% v/v piperidine in DMF; (C) 50% v/v N-methylmorpholine in DMF;

(D) 5% w/v piperazine in DMF;

(E) 20% v/v 2,6-dimethylpiperidine in DMF; (G) 50% v/v 1-methylpyrrolidine in DMF; (H) 50% v/v 1-methylpyrrole in DMF; and (I) 20%) v/v 1 ,4-dimethylpiperazine in DMF.

The Fmoc-Phe-OH derivative dissolved immediately in all the samples. The solutions were then gently agitated over a 63 minute period. Representative samples (200μl) were removed at 5, 15, 25, 35, 45, 55 and 63 minutes respectively. These were then quenched by the addition of 50% w/v HOBt in DMF (700μl) followed by dilution with DMF (1000μl). These solutions were analysed by HPLC against standards of Fmoc-Phe- OH, H-Phe-OH and HOBt. The reaction was followed by monitoring the Fmoc-Phe-OH HPLC elution peak area. The Fmoc protecting group was removed in the following samples: (A) 20% v/v piperidine in DMF;

(B) 10% v/v piperidine in DMF;

(D) 5% w/v piperazine in DMF;

(E) 20% v/v 2,6-dimethylpiperidine in DMF; (G) 50% v/v 1-methylpyrrolidine in DMF;

(I) 20% v/v 1 ,4-dimethylpiperazine in DMF.

In particular, the reagents (A), (B), (D), (E) and (G) were able to remove the majority of Nα-Fmoc protecting group in less than 15 mins. The Nα-Fmoc protecting group was removed by (I) at a very slow rate, less than 50% over 63 minutes. Evaluation of both experiments indicated that the reagents (D) 5% w/v piperazine in DMF, (E) 20% v/v 2,6-dimethylpiperidine in DMF and (G) 50% v/v 1-methylpyrrolidine in DMF were able to remove Fmoc sufficiently rapidly to be of use in a commercial process without cleaving the thioester bond.

Claims

1. A process for the Fmoc synthesis of a peptide comprising a thioester or thioacid which comprises removing the Nα-Fmoc blocking group with a base selected from optionally substituted piperazine or optionally substituted 2,6-C^dialkyl-piperidine or a mixture thereof.

2. A process according to claim 1 wherein the peptide comprises a C-terminal thioester or C-terminal thioacid.

3. A process according to either claim 1 or claim 2 wherein the base is unsubstituted piperazine or unsubstituted 2,6-dimethyl piperidine.

4. A process according to any one of the preceding claims which is a solid phase peptide synthesis.

5. A process according to claim 4 for the solid phase synthesis of a peptide comprising a C-terminal thioester or C-terminal thioacid which process comprises the steps of:

(d) (i) removing the α-nitrogen Fmoc protecting group using optionally substituted piperazine or optionally substituted 2,6-C^dialkyI-piperidine or a mixture thereof under conditions such that the attached amino acids remain connected to the solid support; and (ii) coupling the N-terminal residue of the desired peptide with an α-nitrogen protecting group to the unprotected α-nitrogen of the attached amino acids to yield the desired peptide;

6. A process according to claim 5 wherein the solid support comprises an optionally substituted thiol-trityl linker.

7. A process according to claim 5 wherein the solid support comprises a solid support of Formula (1):

HS — R¹ — ^L0 — L Resin

Formula (1) wherein:

R¹ is optionally substituted C_halky!.

8. A process according to claim 7 wherein the solid support comprises a solid support of Formula (2):

HS s -o— HMPA -Resin

Formula (2)

wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker.

9. A process according to claim 5 wherein the solid support comprises a solid support of Formula (5):

Formula (5)

wherein: each Y is independently an optional substituent; and R² is optionally substituted Cι-ι₀alkyl.

10. A process according to claim 9 wherein the solid support comprises a solid support of Formula (6):

Formula (6)

wherein each Y independently is an optional substituent.

11. A process according to claim 5 wherein the solid support comprises a solid support of Formula (7):

Resin

Formula (7)

wherein: -R³-SH is the side chain of a thiol containing amino acid; and

Linker is the residue of an acid labile linker.

12. A process according to claim 5 wherein the solid support comprises a solid support of Formula (8): Resin

Formula (8)

wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker.

13. A process according to any one of the preceding claims wherein Fmoc removal is effected with from 0.1% to 5% piperazine.

^' 14, A process according to claim 13 wherein Fmoc removal is effected with from 1% to 5% piperazine.

15. A process according to any one claims 1 to 12 wherein Fmoc removal is effected with from 10% to 30% 2,6-dimethyl piperidine.

16. A process according to any one of claims 13 to 15 wherein the piperazine and/or 2,6-dimethyl piperidine is in solution in N,N-dimethylformamide.

17. A process according to any one of claims 5 to 16 wherein in steps (d) and (e) the α-nitrogen protecting group on the N-terminal residue of the desired peptide is Boc.

18. A process for the Fmoc synthesis of a peptide comprising a thioester or thioacid which comprises removing the Nα-Fmoc blocking group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C^dialkyl-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from: N.N-dimethylformamide; a haloalkane; a haloalcohol; and dimethylacetamide.

19. A process according to claim 18 wherein the peptide comprises a C-terminal thioester or C-terminal thioacid.

20. A process according to either claim 18 or claim 19 wherein the base is unsubstituted piperazine, unsubstituted 2,6-dimethyl piperidine or 1-methylpyrrolidine.

21. A process according to any one of claims 18 to 20 wherein the solvent is DMF in the substantial absence of other solvents.

22. A process according to any one of claims 18 to 21 which is a solid phase peptide synthesis.

23. A process according to claim 22 for the solid phase synthesis of a peptide comprising a C-terminal thioester or C-terminal thioacid which process comprises the steps of:

(b) (i) removing the α-nitrogen Fmoc protecting group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C^dialkyl-pipehdine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) under conditions such that the attached amino acid remains connected to the solid support; and

(c) (i) removing the α-nitrogen Fmoc protecting group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C₁.₄dialkyl-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) under conditions such that the attached amino acids remain connected to the solid support; and

(d) (i) removing the α-nitrogen Fmoc protecting group using a composition which comprises a base selected from optionally substituted piperazine, optionally substituted 2,6-C₁-₄dialkyl-piperidine and optionally substituted 1-methylpyrrolidine and also comprises a solvent selected from N,N-dimethylformamide (DMF), dichloromethane (DCM), trifluoroethanol (TFE) and dimethylacetamide (DMA) under conditions such that the attached amino acids remain connected to the solid support; and

24. A solid support of Formula (1 ): o

HS — R¹ — "—O— —Resin

Formula (1) wherein:

L represents the residue of a acid labile linker which on cleavage liberates an acid; and R¹ is optionally substituted C^oalkyl.

25. A solid support according to claim 24 of Formula (2):

Formula (2)

wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker.

26. A solid support of Formula (5):

Formula (5)

wherein: each Y is independently an optional substituent; and R² is optionally substituted C_halky!.

27. A solid support according to claim 26 of Formula (6):

Formula (6)

wherein each Y independently is an optional substituent.

28. A solid support of Formula (7):

Resin

Formula (7) wherein:

-R³-SH is the side chain of a thiol containing amino acid; and Linker is the residue of an acid labile linker.

29. A solid support according to claim 28 of Formula (8):

Formula (8)

wherein HMPA is the residue of a 4-hydroxymethylphenoxyacetyl (HMPA) linker.

30. A process for preparing a solid support of Formula (2):

Formula (2) which comprises reacting a solid support comprising 4-hydroxymethylphenoxyacetyl (HMPA) linker with 2,2-dithiodipropionic acid, dithiodiglycolic acid or 3-mercaptopropionic disulfide and reducing the adduct so formed to yield a support of Formula (2).

31. A process for preparing a solid support of Formula (2):

HS _f -O— HMPA -Resin

Formula (2)

which comprises the following steps:

Formula (4)

wherein each X is independently a substituent which may be present on 1 or more of the phenyl rings and n is 1 to 3;