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US-12624064-B2 - Linker molecule and use thereof in methods for purifying peptides

US12624064B2US 12624064 B2US12624064 B2US 12624064B2US-12624064-B2

Abstract

The present invention relates to a method for the purification of peptides which are produced by solid phase peptide synthesis (SPPS) and corresponding linker molecules for use in said method. Optionally, the peptide may be modified while bound via said linker molecule on a purification support.

Inventors

  • Robert ZITTERBART
  • Oliver Seitz

Assignees

  • Belyntic GmbH

Dates

Publication Date
20260512
Application Date
20210322
Priority Date
20160129

Claims (16)

  1. 1 . A method for the purification of peptides, comprising the following steps: i. contacting a composition of a full-length peptide to be purified and at least one impurity with a capture compound of the formula X 1 -L-X 2 (1), wherein X 1 is selected from wherein each of R 1 and R 2 is independently from each other selected from H or B, wherein at least R 1 or R 2 is B, wherein R 3 is selected from H or B, wherein B is an acid labile amine protecting group, wherein L is selected from functional linkers, that are cleavable nucleophilically from X 2 under basic conditions, wherein L has the structure -T-U—, wherein T is a spacer between X 1 and U, and T is of the form —CH 2 —C(═O)—NH—(CH 2 ) 2 —, —CH 2 —C(═O)—O—(CH 2 ) 2 —, —C(═O)—O—(CH 2 ) 2 — and U is the cleavage activating part of the functional linker, wherein the activating part is formed to stabilize an anion formed during a basic cleavage from X 2 , wherein U is a moiety of formula (5) or (6) X 2 is of the form —Y—Z, wherein Y is —O—C(═O)—, and Z is an electron-withdrawing leaving group, selected from the consisting of —F, —Cl, —Br, I, —N 3 , —SR 12 , —OCF 3 , —OCH 2 CF 3 , —OSO 2 CF 3 , —SO 2 C 6 H 4 CH 3 , —SO 2 CF 3 , —SO 2 CH 3 , wherein R 12 is a C 1 -C 6 -alkyl, an aryl- or a benzyl residue; and subsequent reaction to form a compound of formula X 1 -L-Y-PEP (12), wherein PEP comprises a full-length peptide bound to Y via its N-terminus; ii. cleaving the acid labile protecting groups by addition of an acid, iii. contacting the composition of ii. with a surface-modified solid support, wherein a covalent hydrazone or oxime bond is formed between the capture compound and the solid support, and a compound of formula D-X 1 ′-L-Y-PEP (13) wherein D is a surface-modified solid support, which is characterized in that the surface is modified by synthetic or natural polymers, wherein X 1 ′ is of the form —NH—O—, —NH—NH— is formed, and iv. cleaving the full-length peptide from the solid support.
  2. 2 . The method according to claim 1 , wherein the solid support comprises on its surface a functional group selected from the group consisting of: aldehyde, ketone, hydroxylamine, and hydrazine.
  3. 3 . The method according to claim 1 , wherein after or during cleaving of the full-length peptide from the solid support, the solid support D is cleaved from the residue X 1 -L of the capture compound and the solid support is regenerated.
  4. 4 . The method according to claim 1 , further comprising step iiia after step iii and before step iv, comprising cyclization of the moiety PEP of the compound of formula (13) by oxidation of two or more residues bearing a nucleophilic thiol to form a disulfide bridge using an oxidizing agent.
  5. 5 . The method according to claim 4 wherein the number of nucleophilic thiols in the peptide is even.
  6. 6 . The method according to claim 5 wherein the peptide comprises 2 to 10 amino acids comprising a nucleophilic thiol.
  7. 7 . The method according to claim 4 wherein the peptide comprises at least two amino acids independently selected from the group consisting of: cysteine, homocysteine and penicillamine.
  8. 8 . The method according to claim 4 wherein cyclization is performed in the presence of oxygen.
  9. 9 . The method according to claim 4 wherein cyclization is performed by basic aqueous solution with a pH >7 to pH 8.5.
  10. 10 . The method according to claim 4 wherein cyclization is performed in the presence of an oxidative additive.
  11. 11 . The method according to claim 10 wherein the oxidative additive is selected from the group consisting of DMSO, iodine, N-chlorosuccinimide, Tl(OAc) 3 , Tl(CF 3 COO) 3 , CH 3 SiCI 3 -Ph(SO)Ph, [Pt(ethylenediamine) 2 Cl 2 ]Cl 2 , 2,2′-Dithiobis(5-nitropyridine), 5,5′-dithiobis-(2-nitrobenzoic acid), trans-[Pt—(CN) 4 Cl 2 ] 2− , glutathione-glutathione disulfide, and K 3 Fe(CN) 6 .
  12. 12 . The method according to claim 1 wherein the impurity comprises an acylated truncated sequence.
  13. 13 . The method according to claim 2 wherein the solid support comprises on its surface —O—CH 2 —CHO.
  14. 14 . The method according to claim 2 wherein the solid support comprises on its surface —O—NH 2 .
  15. 15 . The method according to claim 2 wherein the solid support comprises on its surface N 2 H 3 .
  16. 16 . The method according to claim 1 wherein Z is selected from the group consisting of: —Cl,

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This is a Continuation-in-Part of U.S. patent application Ser. No. 16/073,794, filed Dec. 26, 2018, which is the US National Stage of International Patent Application No. PCT/EP2017/051932, filed on Jan. 30, 2017, and which claims priority to German Patent Application No. 10 2016 101 606.3, filed Jan. 29, 2016. The foregoing patent applications are incorporated by reference herein in their entirety. FIELD OF THE INVENTION The present invention relates to a method for the purification of peptides produced by solid phase peptide synthesis (SPPS) and corresponding linker molecules for use in this method. BACKGROUND Solid phase peptide synthesis is a well-known method for the production of peptides. Apart from the synthesis of the peptides, their purification is also an essential process step. A widely used method for the purification of peptides is the preparative high performance liquid chromatography (HPLC). Disadvantageous at this method is the poor scalability with regard to the desired production quantities, so that different quantities cannot be produced with one and the same system; this causes relatively high acquisition costs for the corresponding complex devices. A further disadvantage is that the correct analytical assessment of the individual fractions requires relatively extensive knowledge; additionally, there is the consumption of solvents and column material (solid phase) during operation. Therefore, methods that are cheaper and less prone to faults would be advantageous for reducing the costs of peptide production. EP 0 552 368 A1 describes a method for the purification of peptides in which a so-called linker is on the one hand covalently bound to the N-terminal end of the synthesized full-length peptide and on the other hand covalently bound by reaction to thioether of a thiol group with functionalized diatomaceous earth. The full-length peptide is thus immobilized and can be purified. The full-length peptide is then released under basic conditions. However, the method is not suitable for thiol-containing peptides such as those comprising the amino acid cysteine or penicillamine. Furthermore, there is the disadvantage that the solid phase used for purification (in this case diatomaceous earth) is not intended or suitable for reuse. EP 2 501 711 B1 proposes an analogous method in which the linker is bound to a solid phase (synthetic hydrophilic polymer, e.g. PEGA) via the N-terminal end of the synthesized full-length peptide and via a 1,3-dipolar cycloaddition between an azide (—N3) and an alkyne (Huisgen reaction). A disadvantage of this method is here the necessity of adding copper or copper-containing compounds to perform the 1,3-dipolar cycloaddition. Many peptides complex copper, particularly those comprising sulphur, i.e. comprising methionine and/or cysteine; arginine and lysine can also bind to copper. The copper is therefore difficult to remove and due to the toxicity of the remaining copper the method is not applicable in all cases, especially not for the purification of peptide therapeutics. Another disadvantage is that the solid phase used for purification is not intended or suitable for reuse. Linear unmodified peptides show low in vivo stability due to the fast degradation by proteases and peptidases. Additionally, they show no cell-permeability and fast clearance rates towards the kidney in mammal bodies (Witt et al. (2001), Peptides (22), 2329-43). Mainly two modifications are used to improve in vivo peptide stability and potency: Lipidation with fatty acids and cyclisation of linear peptides to form macrocycles. Cyclic peptides have many advantages over linear peptides. They are significantly more resistant to both exo- and endoproteases. Also, they show superior binding affinities to desired protein targets most likely because they display a more rigid and arch-like binding motive towards target receptors. Successful cyclic peptide drugs are the anti-cancer drug octreotide (Sandostatin®), the immunosuppressant ciclosporin (Sandimmun®, Sandimmun®, Neoral®) or the neuropeptide hormone oxytocine (Syntocinon® or others). Methods to form peptidic cycles can be separated in two main categories: a) one-component-system, in which peptides can be cyclized by utilizing their intrinsic functional groups to form cycles connected by e.g. amides or disulfides. By incorporation of unnatural amino acids other covalent connections can be created. b) Two-component-system, in which the functional groups of the peptides are used in reactions with a bridging scaffold. Organic molecules can be used, that form together with the peptides mono-, bi- or multi-cycles. When SPPS is used peptides are either cyclized on the synthetic resin or after detachment of peptide from the synthetic resin in solution. Both methods have their pitfalls. Modification on solid support demands orthogonally protected amino acids, that are expensive and need extra steps to be deprote