Method for modification of peptides immobilized on a solid support by traceless reductively cleavable linker molecules

A method for modifying and purifying peptides comprising the steps of

• providing a crude linker-tagged peptide L-P, wherein the crude peptide P is covalently bound to a linker molecule L;
• immobilizing L-P by coupling L-P to a solid support S yielding an immobilized S-L-P;
• modifying S-L-P by introducing a modification into S-L-P yielding an immobilized S-L-mP;
• wherein mP is a modified peptide and the modification is selected from the group consisting of lipidation and cyclization;
• wherein lipidation comprises a compound selected from the group consisting of formula FAx-Spn-A (5a), FAx-Spn-OH (5b), FA-A (5c), and FA-OH (5d);
• wherein
• FA is a —C(═O)—C1-24-alkyl or a —C(═O)—C2-24-alkenyl, wherein the alkyl or the alkenyl moiety may be unsubstituted or substituted by one or more substituents independently selected from —F and —COOH;
• Sp is a spacer 2 to 50 atoms in length and x linking moieties independently selected from the group consisting of —NH—, —O—, —S—, wherein the spacer is connected to FA via the linking moiety;
• x is an integer between 1 and 5;
• n is an integer between 1 and 10;
• A is selected from the group consisting of —F, —Cl, —Br, —I, —N3, —O(C═O)CH2(C═O)OH, —SR14, —OCF3, —OCH2CF3, —OSO2CF3, —SO2C6H4CH3, —SO2CF3, and —SO2CH3,
• wherein R14 is selected from the group consisting of C1-C6-alkyl-, an arylic- and benzylic substituent;
• wherein cyclization comprises disulfide formation;
• and wherein intramolecular or intermolecular cyclization by using an organic scaffold molecule, wherein the peptide comprises at least two amino acids comprising a nucleophilic thiol for intramolecular cyclization by disulfide formation or for intermolecular cyclization using an organic scaffold molecule having at least two electrophilic centers;
• wherein the electrophilic center is formed by halogen substituents and/or Michael acceptors, and is formed by at least two benzylic halogen substituents, at least two acrylamides or at least two acryl esters;
• wherein the organic scaffold molecule is selected from the group consisting of 1,4-Bis(bromomethyl)benzene, m-Xylylene dibromide (mDBX), 1,3,5-Tris(bromomethyl) benzene (TBMB), N,N′-bis(chloroacetyl)-1,2-ethylenediamine, N,N′-bis(chloroacetyl)-1,3-propylenediamine, N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-chloroacetamide), N,N′-bis(bromoacetyl)-1,2-ethylenediamine, N,N′-bis(bromoacetyl)-1,3-propylenediamine, N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))bis(2-bromoacetamide), 1,3,5-Triacryloyl-1,3,5-triazinan (TATA), 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TATB), N,N′,N″-(benzene-1,3,5-triyl)-tris(2-bromoacetamide) (TBAB), N,N′,N″-benzene-1,3,5-triyltrisprop-2-enamide (TAAB), 2,4,6-tris(bromomethyl)-s-triazine (TBMT), N,N′,N″-(Nitrilotris(ethane-2,1-diyl))triacrylamide, N,N′,N″-(Nitrilotris(ethane-2,1-diyl))tris(2-chloroacetamide), N,N′,N″-(Nitrilotris(ethane-2,1-diyl))tris(2-bromoacetamide), N,N′,N″-(Nitrilotris(ethane-2,1-diyl))triethenesulfonamide, N,N′,N″-((1,3,5-Triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))triacrylamide, N,N′,N″-((1,3,5-Triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))tris(2-chloroacetamide), N,N′,N″-((1,3,5-Triazinane-1,3,5-triyl)tris(2-oxoethane-2,1-diyl))triethenesulfonamide, N,N′,N″-(Benzene-1,3,5-triyltris(methylene))triacrylamide, N,N′,N″-(Benzene-1,3,5-triyltris(methylene))tris(2-chloroacetamide), N,N′,N″-(Benzene-1,3,5-triyltris(methylene))triethenesulfonamid, Tri(2-hydroxyethyl)isocyanurate triacrylate, 1,3,5-Tris(oxiran-2-ylmethyl)-1,3,5-triazinane-2,4,6-trione;
• reducing RI by adding a reducing agent yielding a reduced intermediate RI to yield mP;
• wherein L is a compound of formula 1 comprising X-Tb-Va-U—Y;
• wherein
• a. X is selected from the group consisting of 2, 2a, 3, 3a and 4,
• wherein
• i. each R1 and R2 is independently from each other selected from the group consisting of H and B;
• wherein at least one of R1 or R2 is B;
• ii. R3 is selected from the group consisting of H and B;
• iii. R4 is H, C1-C12-alkyl or aryl, wherein the aldehyde or keto group is selected from the group consisting of an unprotected aldehyde or keto group and a protected aldehyde or keto group;
• wherein the protecting group is an acid labile protecting group;
• iv. B is an acid labile amine protecting group;
• b. Tb is a linear or branched spacer with b being 0 or 1;
• c. Va is an electron-withdrawing moiety with a being 0 or 1, wherein the sum of a and b is 1 or 2;
• d. U is a phenyl or a five- or six-membered heteroaryl moiety, that is bound to at least one moiety selected from the group consisting of V, Wq and Ej;
• wherein
• i. Wq is selected from the group consisting of —N3, —NO2, —S(═O)—R8, —S—S—R8, —O—CH2—N3, —O—C(═O)—O—CH2—N3, —N═N-phenyl, —N═N— and R8,
• wherein
• R8 is selected from the group consisting of pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —C1-C6-alkyl and —(CH2)p—NMe2, with q being 1, 2, 3 or 4;
• wherein Wq is in ortho or para position in relation to Y;
• ii. Ej is an electron withdrawing group and j is selected from the group consisting of 0, 1, 2, 3 and 4;
• wherein when U is a phenyl moiety and Y is —(CH2)m—O—C(═O)—, the sum of Hammett constants of V, Wq, Ej under acidic conditions is larger than 0.45;
• and
• e. Y is —(CH2)m—C(═O)— or —(CH2)m—O—C(═O)— with m being 1, 2 or 3.