A METHOD FOR MODIFICATION OF PEPTIDES IMMOBILIZED ON A SOLID SUPPORT BY TRACELESS REDUCTIVELY CLEAVABLE LINKER MOLECULES
20220363713 · 2022-11-17
Assignee
Inventors
Cpc classification
C07K1/1133
CHEMISTRY; METALLURGY
C07K1/1075
CHEMISTRY; METALLURGY
A61K47/542
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a method for modifying and purifying peptides comprising an immobilizing step, a modification step and a releasing step. In the immobilizing step, a crude linker-tagged peptide L-P is coupled to a solid support yielding an immobilized linker-tagged peptide S-L-P. Subsequently, the immobilized linker-tagged peptide S-L-P is modified with one or more organic molecules yielding an immobilized linker-tagged peptide S-L-mP. Finally, the modified peptide is released via a reduced intermediate RI. The linker molecule is a compound of formula 1, X—Tb—Va-U—Y—Z (1), which can be coupled to a purification resin via the moiety X and to a peptide via the moiety Y under the release of the leaving group Z. T is an optional spacer moiety and V is an optional electron withdrawing moiety. U is an aryl or 5- or 6-membered heteroaryl moiety bound to at least one electron withdrawing moiety V, W or E. The linker is stable under acidic conditions and releases the peptide upon addition of a reducing agent.
Claims
1. A method for modifying and purifying peptides comprising the steps of providing a crude linker-tagged peptide L-P, wherein the crude peptide is covalently bound to a linker molecule L, in an immobilizing step, coupling the linker-tagged peptide L-P to a solid support yielding an immobilized linker-tagged peptide S-L-P, in a modification step, modifying the immobilized linker-tagged peptide S-L-P with one or more organic molecules yielding an immobilized linker-tagged peptide S-L-mP, in a releasing step, adding a reducing agent yielding a reduced intermediate RI and subsequent decomposition of the reduced intermediate RI yielding the modified peptide mP, wherein the Linker L is a compound of formula 1, X—Tb—V.sub.a—U—Y— (1), wherein a. X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more particularly of formula 2 or 2a, ##STR00046## wherein i. each R.sup.1 and R.sup.2 is independently from each other selected from H or B, wherein at least R.sup.1 or R.sup.2 is B, ii. R.sup.3 is selected from H or B, iii. R.sup.4 is selected from H, C.sub.1-C.sub.12-alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group, iv. B is an acid labile amine protecting group, b. T is a linear or branched spacer with b being 0 or 1, c. V 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 of the moieties V, W.sub.q and E.sub.n and that may optionally be substituted by C.sub.1-6-alkyl, wherein i. W is selected from —N.sub.3, —NO.sub.2, —S(═O)—R.sup.8, —S—S—R.sup.8, —O—CH.sub.2—N.sub.3, —O—C(═O)—O—CH.sub.2—N.sub.3, —N═N-phenyl, —N═N—R.sup.8, ##STR00047## wherein R.sup.8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —C.sub.1-C.sub.6-alkyl or —(CH.sub.2).sub.p—NMe.sub.2, with q being 0, 1, 2, 3 or 4, particularly with q being 1, 2, 3 or 4, ii. E is an electron withdrawing group under acidic conditions, with n being 0, 1, 2, 3 or 4, in case of U being a phenyl moiety and Y being —(CH.sub.2).sub.m—O—C(═O)—, the sum of Hammett constants of V, W, E under acidic conditions is larger than 0.45, and wherein e. W is in ortho or para position in relation to Y,Y is —(CH.sub.2).sub.m—C(═O)— or —(CH.sub.2).sub.m—O—C(═O)— with m being 1, 2 or 3.
2. The method according to claim 1, wherein the linker-tagged peptide L-P is provided by connecting a crude peptide P, that has been synthesized on a synthesizing support SR, with a free linker Lf yielding a linker-tagged, supported crude peptide L-P-SR by releasing an electron withdrawing leaving group Z, wherein Lf is a linker X-T.sub.b-V.sub.a—U—Y—Z with X, T, b, V, a, U and Y as defined according to claim 1 and Z being an electron withdrawing leaving group, cleavage of the linker-tagged peptide L-P from the synthesizing support SR yielding the linker-tagged peptide L-P.
3. The method according to claim 2, wherein the cleavage of the linker-tagged peptide L-P- is achieved under acidic conditions, particular by TFA, HBr, HCl or acetic acid.
4. The method according to claim 1, wherein the solid support is aldehyde-modified, particularly the solid support is aldehyde-modified and selected from agarose, hydroxylated poly(methyl acrylate), poly(glycidyl acrylate), poly(glycidyl methacrylate), polylysine, polyethylene glycol, polyamide, polyacrylamide, polystyrene and copolymers of those, more particularly, the solid support is aldehyde-modified and selected from agarose and hydroxylated poly(methyl acrylate) beads.
5. The method according to claim 1, wherein the immobilizing step is performed at a pH<5, particularly at pH 3-4.
6. The method according to claim 1, wherein the reducing agent in the releasing step is selected from triphenylphosphine, dithiothreitol (DTT), dithioerythritol (DTE), trimethylphosphine, triethylphosphine or tris(2-carboxyethyl)phosphine, particularly dithiothreitol, dithioerythritol or triphenylphosphine.
7. The method according to claim 1, wherein the reduced intermediate RI is decomposed by a pH change, particularly by changing the pH to a pH that is lower than the pH used during reduction of the linker.
8. The method according to claim 1, wherein the peptide comprises an amino acid comprising at least one nucleophilic amine in an amino acid side chain, wherein in particular the amino acid is selected from lysine, ornithine, α-aminoglycine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, 2,6-diamino-4hexynoic acid, β-(1-piperazinyl)alanine, 4,5-dehydrolysine and d-hydroxylysine, in particular from lysine and ornithine, or the nucleophilic amine is the N-terminus of the peptide.
9. The method according to claim 8, wherein the organic molecule is a compound of formula 5a, 5b, 5c or 5d, particularly 5a or 5c,
FA.sub.x-Sp.sub.n-A (5a),
FA.sub.x-Sp.sub.n-OH (5b),
FA-A (5c),
or FA-OH (5d), wherein FA is a —C(═O)—C.sub.1-24-alkyl or a —C(═O)—C.sub.2-24-alkenyl, particularly a —C(═O)—C.sub.5-17-alkyl or a —C(═O)—C.sub.5-17-alkenyl, more particularly a —C(═O)—C.sub.5-17-alkyl, 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 comprising 2 to 50 atoms in length, optional a terminal —C(═O)-moiety, and x linking moieties independently selected from —NH—, —O—, —S—, wherein the spacer is connected to FA via the linking moiety, x is an integer between 1 and 5, particularly between 1 and 3, n is an integer between 1 and 10, particularly 1 and 4, more particularly 1 and 2, A is selected from: —F, —Cl, —Br, —I, —N.sub.3, —O(C═O)CH.sub.2(C═O)OH, —SR.sup.14, —OCF.sub.3, —OCH.sub.2CF.sub.3, —OSO.sub.2CF.sub.3, —SO.sub.2C.sub.6H.sub.4CH.sub.3, —SO.sub.2CF.sub.3, —SO.sub.2CH.sub.3, ##STR00048## in particular, —Cl, —Br, —I, ##STR00049## more particularly ##STR00050## —Cl, —Br, —I, wherein R.sup.14 is an C.sub.1-C.sub.6-alkyl-, an arylic- or benzylic substituent.
10. The method according to claim 9, wherein Sp is independently selected from a linear or branched alkyl substituted by 1 to 5, particularly 1 to 3, linking moieties, —NH—[C.sub.r-alkyl-O].sub.s—C.sub.t-alkyl-C(═O)— with r being independently selected from 1, 2 and 3, s being an integer between 1 and 10, particularly 1, 2, 3 or 4, and t being 1, 2 or 3, and an amino acid which may be protected or unprotected, wherein the N-terminus and/or an amine in the side chain is replaced by —NH— and the C-terminus and/or a carboxylic acid moiety in the side chain is replaced by —C(═O)—, particularly Sp is independently selected from a linear alkyl substituted by 1 to x linking moieties, 2-(2-(2-aminoethoxy)ethoxy)acetic acid ##STR00051## alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, Glu-tBu, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, more particularly Sp is independently selected from —O-ethyl-, ##STR00052## 2-(2-(2-Aminoethoxy)ethoxy)acetic acid, cysteine, glutamic acid or Glu-tBu.
11. The method according to claim 8, wherein the organic molecule of formula 5b or 5d is coupled to the at least one nucleophilic amine in the modification step in the presence of an activating reactant, particularly in the presence of an activating reactant selected from tetramethylaminium tetra uoroborate (TBTU), (3-(Diethoxy-phosphoryloxy)-1,2,3-benzo[d] triazin-4(3H)-one) (DEPBT), (2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylaminium hexa uoro-phosphate) (HCTU), tetramethylaminium hexa uorophosphate (HATU), (N-(3-Dimethylaminopropyl)-N′-ethylcar-bodiimide (EDC), Dicyclohexylcarbodiimide (DCC), Ethyl 2-cyano-2-(hydroximino)acetate (Oxyma), 1-Hydroxybenzotriazole (HOBt), 1-Hydroxy-7-aza-1H-benzotriazole (HOAt), NHS and diisopropylcarbodiimide (DIC).
12. The method according to claim 1, wherein the peptide comprises at least two amino acids comprising a nucleophilic thiol, in particular at least two amino acids independently selected from cysteine, homocysteine or penicillamine.
13. The method according to claim 12, wherein the organic molecule is an organic scaffold molecule comprising a scaffold having at least two electrophilic centers, wherein in particular the electrophilic center is formed by halogen substituents and/or Michael acceptors, particularly by at least two benzylic halogen substituents, at least two acrylamides or at least two acryl ester, more particularly at least two acrylamides or at least two benzylic halogen substituents, in particular the organic scaffold molecule is selected from 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-ethylenedi amine, 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.
14. The method according to claim 12, wherein the modification step comprises a reducing step followed by a cyclisation step, wherein the immobilized linker-tagged peptide S-L-P is treated with a reducing agent, particularly DTT or TCEP, in the reducing step, and the organic scaffold molecule is coupled to the at least two nucleophilic thiols under basic conditions in the cyclisation step.
15. The method according to claim 12, wherein the number of nucleophilic thiols is even, and the nucleophilic thiols are oxidized by basic aqueous solutions with a pH>7 in the modification step to yield macrocyclic bridged peptides.
16. The method according to claim 12, wherein the number of nucleophilic thiols is even, and the nucleophilic thiols are oxidized in the modification step to yield macrocyclic bridged peptides in the presence of air, particularly in the presence of oxygen, and/or in the presence of an oxidative additive, in particular an oxidative additive selected from DMSO, iodine, N-chlorosuccinimide, Tl(OAc).sub.3, Tl(CF.sub.3COO).sub.3, CH.sub.3SiCI.sub.3-Ph(SO)Ph, [Pt(ethylenediamine).sub.2Cl.sub.2]Cl.sub.2, 2,2′-Dithiobis(5-nitropyridine), 5, 5′-dithiobis-(2-nitrobenzoic acid), trans-[Pt—(CN).sub.4Cl.sub.2].sup.2−, glutathione-glutathione disulfide, K.sub.3Fe(CN).sub.6.
17. The method according to claim 1, wherein X is selected from a moiety of formula 2, 2a, 3, 3a or 4, in particular of formula 2, 2a, 3 or 3a, more particularly of formula 2 or 2a, ##STR00053## wherein each R.sup.1 and R.sup.2 is independently from each other selected from H or B, wherein at least R.sup.1 or R.sup.2 is B, R.sup.3 is selected from H or B, R.sup.4 is selected from H, C.sub.1-C.sub.12-alkyl or aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group, B is an acid labile amine protecting group, T is a linear or branched spacer comprising at least one, particularly 1 to 5, of the moieties —C.sub.1-12-alkyl-, (—C.sub.2H.sub.4O—).sub.1-12, —C(═O)—, —C(═O)—, -JR.sup.9—, -JR.sup.9—C(═O)—, -JR.sup.9—, phenyl, 5- or 6-membered heteroaryl, wherein J is C or N, in particular N, in particular T is a spacer selected from —C.sub.1-C.sub.12-alkyl-, in particular C.sub.1-6-alkyl, more particularly C.sub.1-3-alkyl, —R.sup.5—C(═O)—, —R.sup.5—C(═O)—NR.sup.9—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—, —C(═O)—NR.sup.9—R.sup.6—, —R.sup.5—NR.sup.9—C(═O)—R.sup.6—, —R.sup.5—NR.sup.9—R.sup.5′—NR.sup.9′C(═O)—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—R.sup.5′—NR.sup.9′—C(═O)—R.sup.6—, —R.sup.5—NR.sup.9—, —R.sup.5—NR.sup.9—R.sup.6—, —R.sup.5—NR.sup.9—R.sup.5′—NR.sup.9′—R.sup.6—, —R.sup.5—C(═O)—NR.sup.9—R.sup.5′—NR.sup.9′—R.sup.6—, —R.sup.5—C(═O)—O—R.sup.6—, —C(═O)—O—R.sup.6—, —R.sup.5-phenyl-R.sup.6—, —R.sup.5-phenyl-, -phenyl-R.sup.6—, -phenyl-, —R.sup.5-pyrroyl, —R.sup.5-pyrazoyl, —R.sup.5-imidazoyl, R.sup.5-piperazinyl-, —R.sup.5-pyridinyl, —R.sup.5-pyrimidinyl, —R.sup.5-pyrazinyl, —R.sup.5-pyridazinyl, —R.sup.5-pyrroyl-R.sup.6—, —R.sup.5-pyrazoyl-R.sup.6—, —R.sup.5-imidazoyl-R.sup.6—, —R.sup.5-piperazinyl-R.sup.6—, —R.sup.5-pyridinyl-R.sup.6—, —R.sup.5-pyrimidinyl-R.sup.6—, —R.sup.5-pyrazinyl-R.sup.6—, —R.sup.5-pyridazinyl-R.sup.6—, pyrroyl-R.sup.6—, pyrazoyl-R.sup.6—, imidazoyl-R.sup.6-piparazinyl-R.sup.6—, pyridinyl-R.sup.6—, pyrimidinyl-R.sup.6—, pyrazinyl-R.sup.6—, pyridazinyl-R.sup.6—, pyrroyl, pyrazoyl, imidazoyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl and pyridazinyl, wherein R.sup.5, R.sup.5′ and R.sup.6 are independently from each other selected from C.sub.1-C.sub.12-alkyl or (—C.sub.2H.sub.4O—).sub.1-12, in particular C.sub.1-C.sub.6 alkyl, particularly C.sub.1-C.sub.3 alkyl, and wherein R.sup.9 and R.sup.9′ are independently from each other selected from H, C.sub.1-4-alkyl, —C.sub.1-6-alkyl-NH.sub.2, —C.sub.1-6-alkyl-NHB, —C.sub.1-6-alkyl-NB.sub.2, —R.sup.15, —C.sub.1-6-alkyl-R.sup.15, —C.sub.1-6-alkyl-NH—R.sup.15, in particular from H and C.sub.1-2-alkyl, more particularly R.sup.9 is H, wherein B is an independently selected acid labile amine protecting group, R.sup.15 is a blocking agent that is able to react with an aldehyde moiety, in particular R.sup.15 is selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethandithiole, hydroxylamine, O-methylhydroxylamine, N-methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more particularly cysteinyl, wherein amine and/or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group B, particularly Boc, and/or an acid labile thiol protecting group, particularly trityl, b is 0 or 1, in particular 1, V is an electron-withdrawing moiety selected from —NR.sup.11—C(═O)—, —C(═O)—NR.sup.11—, —S(═O)—, —NR.sup.12—(CH.sub.2).sub.p—, -piperazinyl-(CH.sub.2).sub.p—,-pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, ##STR00054## —C(═O)—, —C(═O)—O—, in particular —NR.sup.11—C(═O)—, —C(═O)—NR.sup.11—, —S(═O)—, —NR.sup.12—(CH.sub.2).sub.p—, -piperazinyl-(CH.sub.2).sub.p—, -pyridinyl-, pyrimidinyl, more particularly from —NH—C(═O)—, —C(═O)—NH—, —N—(CH.sub.3)—, -piperazinyl-(CH.sub.2).sub.p—, -pyridinyl-, pyrimidinyl, wherein R.sup.11 is selected from H and C.sub.1-4-alkyl, in particular from H and C.sub.1-2-alkyl, more particularly R.sup.11 is H, R.sup.12 is selected from H and C.sub.1-4-alkyl, in particular from H and C.sub.1-2-alkyl, more particularly R.sup.12 is methyl, p is 0, 1 or 2, particularly 0 or 1, a is 0 or 1, wherein the sum of a and b is 1 or 2, U is a phenyl or a five- or six-membered heteroaryl moiety, in particular a phenyl or a six-membered heteroaryl moiety, more particularly a phenyl, that is bound to at least one of the moieties V, W.sub.q and E.sub.n and that may optionally be substituted by C.sub.1-6-alkyl, in particular C.sub.1-3-alkyl, wherein V is defined as described above, W is selected from —N.sub.3, —NO.sub.2, —S(═O)—R.sup.8, —S—S—R.sup.8, —O—CH.sub.2—N.sub.3, —O—C(═O)—O—CH.sub.2—N.sub.3, —N═N-phenyl, —N═N—R.sup.8, ##STR00055## in particular —N.sub.3, —N═N—R.sup.8, —O—CH.sub.2—N.sub.3, —S—S—R.sup.8, wherein R.sup.8 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —C.sub.1-C.sub.6-alkyl or —(CH.sub.2).sub.p—NMe.sub.2, in particular pyridyl or —C.sub.1-C.sub.6-alkyl, with p being 1, 2, 3 or 4, E is an electron withdrawing group under acidic conditions, n is an integer between 0 and 4, in particular 0 and 2, more particularly 0 or 1, and q is an integer between 0 and 4, in particular 0 and 2, more particularly 0 and 1, wherein the sum of n and q is equal or lower than 4, and wherein in case of U being a phenyl moiety and Y being —(CH.sub.2).sub.m—O—C(═O)—, the sum of Hammett constants of V, W, E under acidic conditions is larger than 0.45, and wherein W is in ortho or para position in relation to Y, Y is —(CH.sub.2).sub.m—C(═O)— or —(CH.sub.2).sub.m—O—C(═O)— with m being 1, 2 or 3, in particular 1 or 2, more particularly 1, Z is an electron-withdrawing leaving group.
18. The method according to claim 1, wherein E is selected from piperidinyl, piperazinyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —N(C.sub.2H.sub.4).sub.2NH.sub.2, —N(C.sub.2H.sub.4).sub.2N—B, —N═N-phenyl, —N═N—R.sup.8, —(CH.sub.2).sub.r—NH—C.sub.1-6-alkyl, —(CH.sub.2).sub.r—N(C.sub.1-6-alkyl).sub.2-, —F, —Cl, —Br, —I, —CN, —NO.sub.2, —N.sub.3, —CF.sub.3, —SO.sub.3H, —CO.sub.2H, —C(═O)NH.sub.2, —SO.sub.2Me, —SOMe, —SO.sub.2Et, —SOEt, in particular pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, —N═N-phenyl, —N═N—R.sup.8, —F, —Cl, —Br, —I, —CN, —NO.sub.2, —N.sub.3, —CF.sub.3, —SO.sub.3H, —CO.sub.2H, more particularly pyridyl, pyrimidinyl, pyridazinyl or —Br with R.sup.8 being pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, —C.sub.1-C.sub.6-alkyl or —(CH.sub.2).sub.p—NMe.sub.2, in particular pyridyl or —C.sub.1-C.sub.6-alkyl, with p being 1, 2, 3 or 4 B being an acid labile amine protecting group, and r being 0, 1, 2, 3 or 4, particularly 0, 1 or 2.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0407]
[0408]
TABLE-US-00001 (H-AK(Palm-γE-OH)EFIAWLVRGRG-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of trail cleavage of crude peptide sample before linker coupling to P1. 2.) Chromatogram of P1 after linker (X1) coupling and final TFA-cleavage. 3.) Chromatogram of P1 after modification by Palm-γGlu(OH)-OtBu coupling and purification as described in claims 1-9.
[0409]
TABLE-US-00002 (H-HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of trail cleavage of crude peptide sample before linker (X1) coupling to P1. 2.) Chromatogram of P2 after modification by Palm-γGlu(OH)-OtBu coupling and purification as described in claims 1-9.
[0410]
[0411]
[0412]
[0413]
[0414]
[0415]
[0416]
[0417]
[0418]
[0419]
[0420]
[0421]
[0422]
[0423]
[0424]
[0425]
[0426]
[0427]
[0428]
[0429]
[0430]
[0431]
[0432]
TABLE-US-00003 P6 (H-ACSWPARCLHQDLCA-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of trial cleavage of crude peptide sample before linker coupling to P6. 2.) Chromatogram of P6 after linker (LuS1) coupling and TFA cleavage from SPPS resin. 3.) Chromatogram of P6 after intramolecular bicyclization with TATB and purification as described in Example 12 using aldehyde-modified Agarose as solid phase and DMSO+10 vol. % Immobilization Buffer 2 as peptide solvent for immobilisation. 4.) Chromatogram of P6 after intramolecular bicyclization with TATB and purification as described in Example 13 using aldehyde-modified poly (methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilisation. 5.) Chromatogram of P6 after intramolecular bicyclization with TATB and purification as described in claims Example 14 using aldehyde-modified Agarose as solid phase and 1 M SDS in DMSO+10 vol. % Immobilization Buffer 2 as peptide solvent for immobilisation. 6.) Chromatogram of P6 after intramolecular bicyclization with TATB at 1° C. and purification as described in Example 16 using aldehyde-modified poly (methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilisation.
[0433]
TABLE-US-00004 P8 (H-ACYNEFGCEDFYDICA-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of trial cleavage of crude peptide sample before linker coupling to P8. 2.) Chromatogram of P8 after linker (LuS1) coupling and TFA cleavage from SPPS resin. 3.) Chromatogram of P8 after intramolecular bicyclization with TATB and purification as described in Example 12 using aldehyde-modified Agarose as solid phase and DMSO+10 vol. % Immobilization Buffer 2 as peptide solvent for immobilisation. 4.) Chromatogram of P8 after intramolecular bicyclization with TATB and purification as described in Example 13 using aldehyde-modified poly (methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilisation. 5.) Chromatogram of P8 after intramolecular bicyclization with TATB and purification as described in Example 14 using aldehyde-modified Agarose as solid phase and 1 M SDS in DMSO+10 vol. % Immobilization Buffer 2 as peptide solvent for immobilisation. 6.) Chromatogram of P8 after intramolecular bicyclization with TATB at 1° C. and purification as described in Example 16 using aldehyde-modified poly (methyl acrylate) beads as solid phase and pure HFIP as peptide solvent for immobilisation.
[0434]
TABLE-US-00005 P3 (H-CRVPGDAHHADSLC-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of P3 after linker (X1) coupling and final TFA-cleavage. 2.) Chromatogram of P3 after disulfide formation and purification as described in claims 1-9.
[0435]
TABLE-US-00006 P4 (H-VRCPGAAHHADSLC-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of trail cleavage of crude peptide sample before linker coupling to P3. 2.) Chromatogram of P4 after linker (X1) coupling and final TFA-cleavage. 3.) Chromatogram of P4 after disulfide formation and purification as described in claims 1-9.
[0436]
TABLE-US-00007 P9 (H-CYFQNCPRG-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of P9 after linker (X1) coupling and final TFA-cleavage. 2.) Chromatogram of P9 after disulfide formation and purification as described in claims 1-9.
[0437]
TABLE-US-00008 PY (H-KCNTATCATQRLANFLVHSSNFGPILPPTNVGSNTY-NH.sub.2)
by usage of inventive linker molecule X1. A=absorption (210 nm), B=time/min; 1.) Chromatogram of PY after linker (X1) coupling and final TFA-cleavage. 2.) Chromatogram of PY after disulfide formation and purification as described in claims 1-9.
[0438]
[0439]
EXAMPLES
[0440] General Methods
[0441] Solid Phase Peptide Synthesis (SPPS) Linker Coupling and TFA-Cleavage:
[0442] The peptide sequences P1-P8 were synthesized in 100 μmol scale under standard solid phase peptide synthesis conditions on a Intavis MultiPep RSi, whereby the synthetic resin was treated with acetic anhydride and pyridine after each amino-acid coupling to block unreacted amino groups. The resin was preswollen for 15 min prior to linker coupling. Linker X1 was coupled to P1-P17 on resin by usage of 4 eq linker X1 (301 mg), 6 eq oxyma (86 mg) and 6 eq diisopropylamine (DIEA, 105 μL) for 2 h in 1.3 mL DMF. The amounts used were based on the loading of the SPPS resin (Rinkamide RAM). The reaction mixture was shaken for 120 min, filtered off, washed (3×DMF, 3×DCM) and dried.
[0443] The dried resin was incubated with TFA-Cleavage cocktail (TFA/H.sub.2O/PhOH/PHSH/Ethandithiol, 82.5:5:5:52.5, v/v/w/v/v, 10 mL per 100 μmol synthetic scale) for 120 min. The cleavage solution was separated from the synthesis resin by filtration and collected in a Falcon tube 10 times the volume of the solution. By adding 9-fold the volume of refrigerated diethyl ether, the peptide was precipitated. The vessel was centrifuged at 5000 rpm for 3.5 min, the supernatant solution decanted off and the peptide pellet washed again with diethyl ether followed by decantation. The peptides were then dissolved in H.sub.2O/MeCN/TFA, (99.95:99.95:0.1, v/v/v), frozen with liquid nitrogen and lyophilized under high vacuum.
[0444] General Procedure to Modify and Purify Peptides by the Inventive Method
[0445] Dissolution and Immobilization of Crude Linker-Tagged Peptides
[0446] The crude linker-tagged peptide mixture was dissolved in pure DMSO (4.5 mL for 100 μmol synthetic scale). After complete dissolution 10 vol. % Immobilisation Buffer 2 (0.1 M citric Acid/Na.sub.2CO.sub.3 pH 3.5 with 8 M guanidinium chloride, 0.45 mL for 100 μmol synthetic scale) was added.
[0447] The linker-tagged peptide is immobilized on 1.5 times the amount of aldehyde-modified agarose loaded with 100 μmol/1 mL settled beads (100 μmol/2 mL of slurried beads) or on hydroxylated poly(methyl acrylate) beads. After aliquoting, the beads material is washed with 3× milli-Q water and 3× immobilization buffer 1 (0.1 M citric Acid/Na.sub.2CO.sub.3 pH 4.5). The dissolved crude linker-tagged peptide mixture was then added to the agarose and shaken. Of note, the beads should have a free and good fluctuation in the immobilization solution. After a reaction time of 90 minutes, the immobilization solution is filtered off with suction.
[0448] Washing
[0449] After immobilization the supernatant was removed and beads linker-connected to the peptides were washed each, three-times 5 mL (for 100 μmol scale) with DMSO. Thereafter a mixture of L-Cysteine in 0.1 M citric Acid/Na.sub.2CO.sub.3 pH 4.5 is added, and reactors are shaken for 15 min or longer. This mixture was removed and beads were washed three-times with 5 mL (for 100 μmol scale) with the following solvents and solutions: [0450] 1) DMSO with 0.9 M guanidinium chloride [0451] 3) 70% ethanol in with 0.1 M NaCl Milli-Q water
[0452] Modification of Immobilized Peptide on Solid Support
[0453] Generally, modification of target peptides was preformed after immobilization of the linker-tagged peptide on the modification/purification support and washing of said peptide-support conjugate.
[0454] In case of fatty acid modification of peptide, after immobilization and washing of target peptide, possibly build imines of lysine side chains with excess aldehydes and said aldehydes were transformed into oximes by addition of O-methylhydroxylamine hydrochloride or L-Cysteine. Thereafter the fatty acid is added together with a coupling reagent, with organic base after a preactivation time to the peptide on support. Thereafter excess of reagents are washed out and the peptide is released as described below.
[0455] In case of macrocycle formation via electrophilic scaffolds, after immobilization and washing of target peptide possibly build disulfide were reduced by addition of a small amount of DTT. After washing DTT out, the electrophilic scaffold was added to the support. If necessary, this step can be repeated to increase conversion to the macrocyclic product. Thereafter excess of reagents are washed out and the peptide is released as described below.
[0456] In case of disulfide formation, after immobilization and washing of target peptide, possibly build imines of lysine side chains and thioacetals of cysteine side chains with excess aldehydes and said aldehydes were transformed into thiazolidines by addition guanidinium chloride. Disulfide were formed in a 1 to 1 mixture of DMSO and 0.4 M (NH.sub.4).sub.2CO.sub.3, 6M GdmCl on the support. Thereafter the oxidation buffer was washed out and the peptide is released as described below.
[0457] Release of Modified Peptides
[0458] 500 mg of dithiothreitol (DTT) are dissolved in 5 ml of 5 w. % NaHCO.sub.3 pH 8, added to the agarose or poly(methyl acrylate) material were 5 mL MeCN was already on the beads and shaken for 15 min. The supernatant is discarded, and the agarose material is washed 3× with water and 2× with MeCN to remove excess DTT and oxidized DTT. Subsequently, 2 mL TFA/H.sub.2O (2:3) are added and shaken for a further 60 min. Then another 2 mL of pure TFA are added to the solution, the mixture is eluted and collected in a Falcon tube. It is eluted two more times with 2 mL each of TFA/H.sub.2O (95:5). The linker peptide is precipitated by addition of 9 times the volume of deep-frozen diethyl ether and then centrifuged 5000 rpm for 3.5 min and the ether phase is decanted off. The peptide is now present as a pellet and can be lyophilized if necessary.
Experimental Description for Organic Synthesis of FA-A and FA-Sp-A
[0459] Synthesis of 1-bromopropyl-2,3-dipalmitate (Pam.sub.2-Br) 1,2-Dipalmitoyl-sn-glycerol (0.4 g, 0.7 mmol, 1 eq) was dissolved in 9 mL DCM and tetrabromomethane (0.5 g 1.5 mmol, 2.1 eq) was added. The solution was cooled to 0° C. and triphenylphosphine (0.4 g 1.5 mmol, 2.1 eq) was added in portions within 5 min. After stirring for 30 min at 0° C., the ice bath was removed and stirring was continued at rt for 3 h. The solvent was removed under reduced pressure. Cyclohexane was added and the mixture was sonicated for about 10 min. After centrifugation, the supernatant was transferred into a clean tube and the solvent was removed under reduced pressure to obtain the crude product. To remove residual O═PPh.sub.3, the product was treated with MeOH in a sonication bath. After centrifugation, the solvent was decanted. The wash with methanol was repeated twice to afford 1-bromopropyl-2,3-dipalmitate (Pam.sub.2-Br) as an off-white solid (0.17 g, 0.27 mmol, 39%).
[0460] R.sub.f=0.68 (chloroform/ethyl acetate 96:4); UPLC-MS: t.sub.R=5.69 min (gradient 0-3 min 40-100% D (0.1% TFA in 9:1 MeCN/iPrOH) in C (0.1% TFA in 8:1:1 H2O/MeCN/iPrOH), 3-6 min 100% D); UPLC-purity (210 nm) 98%; ESI-MS: (calculated [M+Na].sup.+: 653.41, 655.41, found: 653.21, 655.68; calculated [M-OCOC.sub.15H.sub.31].sup.−+: 375.19, 377.19, found: 375.27, 377.26).
[0461] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.21 (qd, J=5.5, 4.4 Hz, 1H), 4.29 (ddd, J=17.5, 11.9, 5.0 Hz, 2H), 3.50 (qd, J=10.9, 5.5 Hz, 2H), 2.34 (m, 4H), 1.62 (m, 4H), 1.28 (m, 48H), 0.88 (t, J=6.9 Hz, 6H).
Synthesis of 1-iodopropyl-2,3-dipalmitate (Pam.SUB.2.-1)
[0462] 1,2-Dipalmitoyl-sn-glycerol (0.5 g, 0.88 mmol, 1 eq), imidazole (0.12 g, 1.76 mmol, 2 eq) and triphenylphosphine (0.5 g 1.99 mmol, 2.3 eq) was dissolved in 7.5 mL THF. I.sub.2 (0.32 g 1.67 mmol, 2.0 eq) was slowly added to the solution. After 4 h stirring at rt, 250 mg NaHCO.sub.3 and 1 mL water was added to quench the reaction. The color of the reaction mixture disappeared. The solvent was evaporated and the residue was washed with 4 mL water. The suspension was centrifuged and the supernatant was decanted. The crude product was treated 6 mL MeOH in a sonication bath for several minutes. After centrifugation, the supernatant was decanted. The precipitate was washed another four times with 6 mL MeOH. The product was dried in vacuo to afford 0.47 g (0.69 mmol, 78%) 1-iodopropyl-2,3-dipalmitate as an off-white solid.
[0463] R.sub.f=0.66 (chloroform/ethyl acetate 96:4); UPLC-MS: t.sub.R=5.83 min (gradient 0-3 min 40-100% D in C, 3-6 min 100% D); UPLC-purity (210 nm) 90%; ESI-MS: (calculated [M+Na].sup.+: 701.40, found: 701.50; calculated [M-OCOC.sub.15H.sub.31].sup.−+: 423.18, found: 423.28).
[0464] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 5.00 (m, 1H), 4.26 (ddd, J=17.6, 11.8, 5.0 Hz, 2H), 3.31 (ddd, J=27.4, 10.7, 5.8 Hz, 2H), 2.33 (m, 4H), 1.63 (m, 4H), 1.28 (m, 48H), 0.88 (t, J=6.8 Hz, 6H).
Synthesis of 1-Tosylpropyl-2,3-dipalmitate (Pam.SUB.2.-OTs)
[0465] 4-Toluenesulfonyl chloride (2 g, 10.5 mmol, 10 eq) was dissolved in 50 mL THF. The solution was cooled to 0° C. with an ice bath. 1,2-Dipalmitoyl-sn-glycerol (0.6 g, 1.05 mmol, 1 eq) was dissolved in 14.4 mL THF at rt and 2.84 mL 15 w % aq. NaOH (10.7 mmol, 10 eq) was added. Slowly, the 4-toluenesulfonyl chloride solution was added and the reaction mixture was stirred for 4 h at rt. The solvent was evaporated under reduced pressure and the residue was taken up in chloroform. The organic phase was washed thrice with brine and thrice with water and was then dried over Na.sub.2SO.sub.4. After filtration, the solvent was removed under reduced pressure to obtain the crude product as yellow liquid, which turned into a solid after drying in vacuo. 5 mL chilled MeOH was added and the resulting mixture was sonicated for 10 min. After centrifugation, the supernatant was decanted. The residue was treated another five times with MeOH to remove excess tosyl chloride. The product was dried in vacuo to afford 0.47 g (0.64 mmol, 61%) off-white solid.
[0466] R.sub.f=0.64 (chloroform/ethyl acetate 96:4); UPLC-MS: t.sub.R=4.99 min (gradient 0-3 min 40-100% D in C, 3-6 min 100% D); UPLC-purity (210 nm)=93%; ESI-MS: (calculated [M+Na].sup.+: 745.51, found: 745.68).
[0467] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 7.80 (t, J=8.0 Hz, 2H), 7.35 (dd, J=8.2, 3.1 Hz, 2H), 4.88 (m, 1H), 4.18 (m, 4H), 2.45 (s, 3H), 2.23 (m, 4H), 1.56 (m, 4H), 1.27 (m, 48H), 0.88 (t, J=7.0 Hz, 6H).
Synthetic Steps for the Synthesis of 2-iodoethyl palmitate (Pam-Et-I)
[0468] Palmitoyl Chloride
[0469] Thionyl chloride (7.2 mL, 100 mmol, 10 eq) slowly added dropwise to palmitic acid (2.6 g, 9.9 mmol, 1 eq) in a round-bottom flask. After stirring for 30 min at rt, the reaction mixture was stirred for 2 h at 75° C. The solution was diluted with DCM and washed three times with water. The organic layer was dried over Na.sub.2SO.sub.4. The salt was removed by filtration and the solvent was removed under reduced pressure. The resulting solution was dried under vacuo to give the product as colorless solid (2.47 g, 9 mmol, 90%).
[0470] R.sub.f=0.52 (chloroform/ethyl acetate 96:4).
2-iodoethyl palmitate (Pam-Et-I)
[0471] To a solution of 2-iodoethanol (0.71 mL, 9.1 mmol, 5 eq) in 18 mL DCM under nitrogen atmosphere was added triethylamine (0.5 mL, 3.6 mmol, 3 eq). The solution was stirred for 30 min at 0° C. Palmitoyl chloride (0.5 g, 1.8 mmol, 1 eq) was added at 0° C. Stirring was continued for 4 h at rt. The solution was successively washed three times each with water and saturated aq. NaHCO.sub.3. After drying over Na.sub.2SO.sub.4 and filtration, the solvent was removed under vacuo. The resulting crude product was treated with MeOH in a sonication bath and the suspension was centrifuged. The supernatant was decanted and the residue was washed another two times with MeOH. The MeOH solutions from the second and third wash were combined and kept at room temperature overnight to crystalize the product as colorless needles. The solvent was removed and the crystals were dried in vacuo to afford 0.15 g (0.36 mmol, 20%) product.
[0472] R.sub.f=0.43 (chloroform/ethyl acetate 96:4.
[0473] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 4.33 (t, J=6.8 Hz, 2H), 3.29 (t, J=6.8 Hz, 2H), 2.33 (t, J=7.5 Hz, 2H), 1.64 (p, J=7.4 Hz, 2H), 1.26 (m, 24H), 0.88 (t, J=6.9 Hz, 3H).
Synthesis of Myristic Acid N-hydroxysuccinimide ester (Myr-OSu)
[0474] To the stirred solution of 1 g myristic acid (4.33 mmol, 1 eq) and 554 mg N-hydroxysuccinimide (4.77 mmol, 1.1 eq) in 44 mL acetonitrile was added 994 mg dicyclohexyl carbodiimide (DCC, 4.77 mmol, 1.1 eq) and the mixture was stirred for 1.5 h. The formed precipitate was removed by filtration and the filtrate was washed with 50 mL of acetonitrile to. Then 1.430 g (4.33 mmol, quant. Yield) of the desired white crystalline title compound was gained by evaporation of the solvent and drying under reduced pressure. ESI-MS: (calculated [M+Na].sup.+: 348.22, found: 348.28).
Synthesis of 22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontanoic N-hydroxysuccinimide ester (tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu)
[0475] To the stirred solution of 260 g 22-(tert-butoxycarbonyl)-43,43-dimethyl-10,19,24,41-tetraoxo-3,6,12,15,42-pentaoxa-9,18,23-triazatetratetracontanoic acid (0.3 mmol, 1 eq) and 39 mg N-hydroxysuccinimide (0.33 mmol, 1.1 eq) in 6 mL acetonitrile was added 67 mg DCC (0.33 mmol, 1.1 eq) and the mixture was stirred for 1.5 h. The formed precipitate was removed by filtration and the filtrate was washed with 10 mL of acetonitrile to. Then 0.305 g (0.30 mmol, quant. yield) of the desired title compound was gained as a colorless oil after evaporation of the solvent and drying under reduced pressure. ESI-MS: (calculated [M+H].sup.+: 943.59, found: 943.72).
Example 1: Formation of Lipidated Peptides P1-P2a Bound to Purification Solid Support with Palmitoyl-L-Glu(OH)-tBu-Building Block
[0476] The inventive method for the modification and purification of peptides was applied to two peptides of different length and polarity, these were
TABLE-US-00009 (SEQ ID NO: 1) H-AKEFIAWLVRGRG-NH.sub.2 (P1) a fragment 25-37 (K34R) of the Glucagon-like peptide-1 (GLP-1) and (SEQ ID NO: 2) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-NH.sub.2 (P2a) is a longer sequence 7-37 (K34R) of the Glucagon-like peptide-1 (GLP-1).
[0477] The peptide was immobilized and washed according to the General Methods in a 5 μmol scale, as a deviation from the general method, after immobilization and removal of the immobilization supernatant a O-methylhydroxylamine hydrochloride solution (4 Aq., 0.1 M) in immobilization Buffer 1 (0.1 M citric Acid/Na.sub.2CO.sub.3 pH 4.5) was added and shaken for 20 min. After washing according to the General Methods, it was washed 3× with a 10 vol. % pyridine-solution in DMF and 3×DMF. Followed by the addition of a 0.3 M coupling solution consisting of 2-(6-Chlor-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium-hexafluorophosphat (HCTU) or diisopropylcarbodiimide (DIC), 10 eq Oxyma and 12 eq of the Palmitoyl-L-Glu(OH)-tBu building block in DMF. This solution was pre-activated for 20 min prior to addition to the agarose beads. After addition beads were shaken for 2 hours and afterwards the supernatant the filtered of and the solid support was washed with 3×DMF, 3×H.sub.2O and 3×MeCN and the modified peptide was released under the conditions given at General Methods\Release of Modified Peptides using PPh.sub.3 in MeCN/AcOH/H.sub.2O (9:0.5:0.5, v/v/v) instead of DTT. To remove the tBu protection of the introduced Palmitoyl-L-Glu-tBu group, released peptide pellet was dissolved in TFA/TIS/H.sub.2O (96:2:2, v/v/v) and precipitated after 2 h in a 10-fold volume of Et.sub.2O.
TABLE-US-00010 H-AK(Palm-γE-OH)EFIAWLVRGRG-NH.sub.2:
The peptide precursor P1 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block Palm-γGlu(OH)-OtBu purified according to the above described method. Results are shown in
[0478] H-HAEGTFTSDVSSYLEGQAAK(Palm-γE-OH)EFIAWLVRGRG-NH.sub.2: The peptide precursor P2 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block Palm-γGlu(OH)-OtBu purified according to the above described method. Results are shown in
Example 2: Formation of Macrocycles of Peptides P3-P5 Bound to Purification Solid Support with m-Xylylene Dibromide (Intermolecular Cyclization)
[0479] The inventive method for the modification and purification of peptides was applied to three peptides of cysteine positions and polarity, these were
TABLE-US-00011 (SEQ ID NO: 3) H-CRVPGDAHHADSLC-NH.sub.2 (P3), (SEQ ID NO: 4) H-VRCPGAAHHADSLC-NH.sub.2 (P4) and (SEQ ID NO: 5) H-VRVPGCAHCADSLY-NH.sub.2 (P5).
[0480] The peptide was immobilized and washed according to the general method in a 25 μmol scale. To the immobilized peptide was added DTT in H.sub.2O (4 eq, 0.3 M) and the reactor was shaken for 15 min. After removal of the DTT solution by filtration, the bead material was washed with 3×H.sub.2O and 3×MeCN. Subsequently, m-Xylylene dibromide (mDBX) in acetonitrile (4 eq, 0.6 M) was added and subsequently the same volume of cyclization buffer (200 mM NH.sub.4HCO.sub.3, pH 7.9) was added. The syringe reactor was shaken for 45 min, the reaction mixture was filtered off and the bead material was washed with 3×MeCN and 3×H.sub.2O. The protocol was then repeated twice after the DTT treatment and finally the cyclized peptide was released under the conditions given at General Methods\Release of Modified Peptides using PPh.sub.3 in MeCN/AcOH/H.sub.2O (9:0.5:0.5, v/v/v) instead of DTT.
TABLE-US-00012 H-CRVPGDAHHADSLC-NH.sub.2
(underlined C residues are connected by the scaffold): The peptide precursor P3 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with m-Xylylene dibromide (mDBX) scaffold and purified according to the above described method. Results are shown in
TABLE-US-00013 H-VRCPGAAHHADSLC-NH.sub.2
(underlined C residues are connected by the scaffold): The peptide precursor P4 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with mDBX scaffold and purified according to the above described method. Results are shown in
[0481] H-VRVPGCAHCADSLY-NH.sub.2 (underlined C residues are connected by the scaffold): The peptide precursor P4 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with mDBX scaffold and purified according to the above described method. Results are shown in
Example 3: Formation of Bicyclic Macrocycles of Peptides P6-P7 Bound to Purification Solid Support with 1,3,5-Tris(Bromomethyl)Benzene (Intermolecular Cyclization)
[0482] The inventive method for the modification and purification of peptides was applied to two different peptides these were
TABLE-US-00014 (SEQ ID NO: 6) H-ACSWPARCLHQDLCA-NH.sub.2 (P6) and (SEQ ID NO: 7) H-ACREGFLRCLHRPTVCG-NH.sub.2 (P7).
[0483] The peptide was immobilized and washed according to the General Methods in a 5 μmol scale. To the immobilized peptide was added DTT in H.sub.2O (4 eq, 0.3 M) and the reactor was shaken for 15 min. After removal of the DTT solution by filtration, the bead material was washed with 3×H.sub.2O and 3×DMF. Subsequently, 1,3,5-Tris(bromomethyl)benzene (TBMB) in dimethylformamide (4 eq, 0.45 M) was added and subsequently half of that volume of cyclization buffer (400 mM NH.sub.4HCO.sub.3, 8 M GdmCl, pH 8.0) was added. The syringe reactor was shaken for 45 min, the reaction mixture was filtered off and the bead material was washed with 3×DMF and 3×H.sub.2O. The protocol was then repeated twice after the DTT treatment and finally the cyclized peptide was released under the conditions given at General Methods\Release of Modified Peptides using PPh.sub.3 in MeCN/AcOH/H.sub.2O (9:0.5:0.5, v/v/v) instead of DTT.
TABLE-US-00015 H-ACSWPARCLHQDLCA-NH.sub.2
(underlined C residues are connected by the scaffold): The peptide precursor P6 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with 1,3,5-Tris(bromomethyl) benzene (TBMT) scaffold and purified according to the above described method. Results are shown in
TABLE-US-00016 H-ACREGFLRCLHRPTVCG-NH.sub.2
(underlined C residues are connected by the scaffold): The peptide precursor P7 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with 1,3,5-Tris(bromomethyl) benzene (TBMT) scaffold and purified according to the above described method. Results are shown in
Example 4: Formation of Bicyclic Macrocycles of Peptide P8a Bound to Purification Solid Support with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (intermolecular cyclization)
[0484] The inventive method for the modification and purification of peptides was applied to the peptide H-ACYNEFGCEDFYDICA-NH.sub.2 (SEQ ID NO: 8) (P8a).
[0485] The peptide was immobilized and washed according to the General Methods in a 5 μmol scale. To the immobilized peptide was added DTT in H.sub.2O (4 eq, 0.3 M) and the reactor was shaken for 15 min. After removal of the DTT solution by filtration, the bead material was washed with 3×H.sub.2O and 3×MeCN. Subsequently, the 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TATB) in MeCN/cyclization buffer (400 mM NH.sub.4HCO.sub.3, 8 M GdmCl, pH 7.1; 9:1) was added. The syringe reactor was shaken for 120 min, the reaction mixture was filtered off and the bead material was washed with 3×H.sub.2O and 3×MeCN. The protocol was then repeated twice after the DTT treatment and finally the cyclized peptide was released under the conditions given at General Methods\Release of Modified Peptides using PPh.sub.3 in MeCN/AcOH/H.sub.2O (9:0.5:0.5, v/v/v) instead of DTT.
TABLE-US-00017 H-ACYNEFGCEDFYDICA-NH.sub.2
(underlined C residues are connected by the scaffold): The peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TBMT) scaffold and purified according to the above described method. Results are shown in
Example 5: Formation of Bicyclic Macrocycles of Peptide P8b Bound to Purification Solid Support with 1,3,5-Triacryloyl-1,3,5-triazinan (intermolecular cyclization)
[0486] The inventive method for the modification and purification of peptides was applied to the peptide H-ACYNEFGCEDFYDICA-NH.sub.2 (SEQ ID NO: 8) (P8b).
[0487] The peptide was immobilized and washed according to the General Methods in a 5 μmol scale. To the immobilized peptide was added DTT in H.sub.2O (4 eq, 0.3 M) and the reactor was shaken for 15 min. After removal of the DTT solution by filtration, the bead material was washed with 3×H.sub.2O and 3×MeCN. Subsequently, the 1,3,5-Triacryloyl-1,3,5-triazinan (TATA) in MeCN/cyclization buffer (400 mM NH.sub.4HCO.sub.3, 8 M GdmCl, pH 7.1; 9:1) was added. The syringe reactor was shaken for 120 min, the reaction mixture was filtered off and the bead material was washed with 3×H.sub.2O and 3×MeCN. The protocol was then repeated twice after the DTT treatment and finally the cyclized peptide was released under the conditions given at General Methods\Release of Modified Peptides using PPh.sub.3 in MeCN/AcOH/H.sub.2O (9:0.5:0.5, v/v/v) instead of DTT.
TABLE-US-00018 H-ACYNEFGCEDFYDICA-NH.sub.2
(underlined C residues are connected by the scaffold): The peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TBMT) scaffold and purified according to the above described method. Results are shown in
Example 6: Formation of Macrocycle of Peptide P3b Bound to Purification Solid Support with by Disulphide (Intramolecular Cyclization)
[0488] The inventive method for the modification and purification of peptides was applied to the peptide
TABLE-US-00019 (SEQ ID NO: 3) H-CRVPGDAHHADSLC-NH.sub.2 (P3b).
[0489] The peptide was immobilized and washed according to the general method in a 10 μmol scale. Subsequently a one to one mixture of DMSO and 0.4 M (NH.sub.4).sub.2CO.sub.3, 6 M GdnCl, pH 8.7 was added and the syringe reactor was shaken for 120 min, the reaction mixture was filtered off and the bead material was washed with 3×H.sub.2O. The cyclized peptide was released under the conditions given at General Methods\Release of Modified Peptides yielding 22% of disulfide cyclized peptide using PPh.sub.3 in MeCN/AcOH/H.sub.2O (9:0.5:0.5, v/v/v) instead of DTT.
[0490] H-CRVPGDAHHADSLC-NH.sub.2 (underlined C residues are connected by the scaffold): The peptide precursor P3 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified by air oxidation and purified according to the above described method. Results are shown in
Example 7: Formation of Lipidated Peptides P1-P3 Bound to Purification Solid Support with Building Blocks 1-bromopropyl-2,3-dipalmitate (a), 1-iodopropyl-2,3-dipalmitate (b), 1-tosyl-2,3-dipalmitate (c), or palm-OC.SUB.2.H.SUB.4.I (d)
[0491] The inventive method for the modification and purification of peptides was applied to two peptides of different length and polarity, these were the model peptide
TABLE-US-00020 (SEQ ID NO: 12) H-KKKKSC-NH.sub.2 (P12), (SEQ ID NO: 13) H-KEKFSINSEDNNGC-NH.sub.2 (P13), and (SEQ ID NO: 14) H-CRG-NH.sub.2 (P14).
[0492] The linker-tagged peptide was immobilized according to the General Methods in a 5 μmol scale. After removal of the immobilization solution, the agarose beads were washed 3× with DMSO. 250 μL of a solution of 2 w % L-cysteine (5 mg) in buffer 1 was added and shaken for 15 min. The solution was filtered off with suction and washing of the beads linker-connected to the peptides was carried out as described in General Methods. For the reduction of the linker and removal of the StBu-protecting group of cysteine residues in the peptide, a reducing solution of 25 mg (162 μmol, 32 eq) DTT in 250 μL 5 w % aqueous NaHCO.sub.3 was prepared. 250 μL MeCN was added to the agarose beads followed by the reducing solution. The mixture of peptides with StBu protecting groups on cysteine (P1-P2) was shaken for 60 min, without StBu (P3) for 15 min and the solution was filtered off by suction. The agarose beads with the linker-connected peptide were washed 3× with water, 2× with MeCN and 3× with DMF. A sonicated suspension of 3.2 mg (10 μmol, 2 eq) Cs.sub.2CO.sub.3 in 200 μL DMF was added. A solution of 20 μmol palmitoyl building block (4 eq, a) Palm.sub.2-propyl-Br=12.6 mg, b) Palm.sub.2-propyl-I=13.6 mg, c) Palm.sub.2-propyl-OTs=14.5 mg, d) Palm-OC.sub.2H.sub.4I) in 300 μL DMF was added. The mixture was shaken for 18-76 h. In case some precipitate was formed over time, another 200-400 μL DMF was added. The supernatant was removed, and the beads were washed 5× with DMF, 3× with water, and 3× with MeCN. 200 μL TFA/water (40:60) was added and shaken for 60 min to release the peptide. 200 μL TFA was added to the cartridge and the solution was collected in a 15 mL centrifuge tube. The agarose beads were eluted 2× with 200 μL TFA/water (95:5) into the same tube. 10 mL chilled Et.sub.2O was added to the TFA/water solution with lipidated peptide P1 or P2 to precipitate the peptide. The precipitate was collected and washed once with ether. The peptide was dissolved in water/MeCN (1:1) and lyophilized. For samples with lipidated peptide P3 the TFA/water solution was concentrated under airstream. Water/MeCN (1:1) as added and the sample was lyophilized.
[0493] H-KKKKSC(C.sub.3H.sub.5O.sub.2-diPalm)-NH.sub.2 (P12a): The peptide precursor P12a has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block 1-bromopropyl-2,3-dipalmitate (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1 and
[0494] H-KKKKSC(C.sub.3H.sub.5O.sub.2-diPalm)-NH.sub.2 (P12b): The peptide precursor P12b has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block 1-iodopropyl-2,3-dipalmitate (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1,
[0495] H-KKKKSC(C.sub.2H.sub.4O—Palm)-NH.sub.2 (P12d): The peptide precursor P12d has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block Palm-OC.sub.2H.sub.4I (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1 and
[0496] H-KEKFSINSEDNNGC(C.sub.3H.sub.5O.sub.2-diPalm)-NH.sub.2 (P13a): The peptide precursor P13a has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block 1-bromopropyl-2,3-dipalmitate (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1 and
[0497] H-KEKFSINSEDNNGC(C.sub.3H.sub.5O.sub.2-diPalm)-NH.sub.2 (P13b): The peptide precursor P13b has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block 1-iodopropyl-2,3-dipalmitate (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1 and
[0498] H-KEKFSINSEDNNGC(C.sub.2H.sub.4O—Palm)-NH.sub.2 (P13d): The peptide precursor P13d has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block Palm-OC.sub.2H.sub.4I (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1 and
[0499] H—C(C.sub.3H.sub.5O.sub.2-diPalm)RG-NH.sub.2 (P14b): The peptide precursor P14b has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block 1-iodopropyl-2,3-dipalmitate (time for palmitoylation=76 h) according to the above described method. Results are shown in Table 1 and
[0500] H—C(C.sub.3H.sub.5O.sub.2-diPalm)RG-NH.sub.2 (P14b): The peptide precursor P14c has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block 1-tosylpropyl-2,3-dipalmitate (time for palmitoylation=76 h) according to the above described method. Results are shown in Table 1 and
[0501] H—C(C.sub.2H.sub.4O—Palm)RG-NH.sub.2 (P14d): The peptide precursor P13c has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block Palm-OC.sub.2H.sub.4I (time for palmitoylation=18 h) according to the above described method. Results are shown in Table 1, and
Example 8: Formation of Lipidated Peptide P2b Bound to Purification Solid Support with Palmitoyl-L-Glu(OSu)-tBu-Building Block
[0502] The inventive method for the modification and purification of peptides was applied to
TABLE-US-00021 (SEQ ID NO: 2) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH (P2b)
is a longer sequence 7-37 (K34R) of the Glucagon-like peptide-1 (GLP-1) and known as the peptide drug Liraglutide when Palm-Glu-OH is attached to the Lys side chain.
[0503] The peptide was immobilized and washed according to the General Methods in a 25 μmol scale. After washing according to the General Methods, it was washed 3×DMF. Followed by the addition of 8 eq Palmitoyl-L-Glu(OSu)-tBu building block with 2 eq DIPEA in 600 μL N-methylpyrrolidone. After addition of this solution beads were shaken for 1 hours and afterwards the supernatant the filtered of and the solid support was washed with 3×DMSO, 3×DMF, 3× and the modified peptide was released under the conditions given at General Methods\Release of Modified Peptides. To remove the tBu protection of the introduced Palmitoyl-L-Glu-tBu group, the released peptide pellet was re-dissolved in TFA/DTT/H.sub.2O (94:4:2, v/v/v) and precipitated after 1 h in a 10-fold volume of Et.sub.2O.
[0504] H-HAEGTFTSDVSSYLEGQAAK(Palm-Glu-OH)EFIAWLVRGRG-OH: The peptide precursor P2 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with palmitic acid (Palm) building block Palm-Glu(OSu)-OtBu purified according to the above described method. Results are shown in
Example 9: Formation of Lipidated Peptide P15 Bound to Purification Solid Support with tBuOC(O)-Ste-Glu(Aeea-Aeea-OSu)-OtBu Building Block
[0505] The inventive method for the modification and purification of peptides was applied to
TABLE-US-00022 (SEQ ID NO: 15) H-HAibEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-OH (P15)
is a longer sequence 7-37 (K34R, Aib8A) of the Glucagon-like peptide-1 (GLP-1) and known as the peptide drug Semaglutide when CO.sub.2H-Ste-Glu(aeea-aeea-OH)—OH is attached to the Lys side chain.
[0506] The peptide was immobilized and washed according to the General Methods in a 25 μmol scale. After washing according to the General Methods, it was washed 3×DMF. Followed by the addition of 8 eq tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu building block with 2 eq DIPEA in 600 μL N-methylpyrrolidone. After addition of this solution beads were shaken for 1 hours and afterwards the supernatant the filtered of and the solid support was washed with 3×DMSO, 3×DMF, 3× and the modified peptide was released under the conditions given at General Methods\Release of Modified Peptides. To remove the tBu protections of the introduced tBuOC(0)-Ste-Glu(aeea-aeea-OSu)-OtBu group, the released peptide pellet was re-dissolved in TFA/DTT/H.sub.2O (94:4:2, v/v/v) and precipitated after 1 h in a 10-fold volume of Et.sub.2O.
[0507] H-HAibEGTFTSDVSSYLEGQAAK(CO.sub.2H-Ste-Glu(aeea-aeea-OH)-OH)EFIAWLVRGRG-OH: The peptide precursor of P15 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with tBuOC(O)-Ste-Glu(aeea-aeea-OSu)-OtBu building block and purified according to the above described method. Results are shown in
Example 10: Formation of Lipidated Peptide P16 and P17a Bound to Purification Solid Support Palm-S Building Block
[0508] The inventive method for the modification and purification of peptides was applied to
TABLE-US-00023 (SEQ ID NO: 16) H-SGGKWSKSSVIGWPAVRER-OH
is a sequence 1-19 (S1M) and
TABLE-US-00024 (SEQ ID NO: 17) H-GGKWSKSSVIGWPAVRER-OH
is a sequence 2-19 both of the Nef protein what bears an N-terminal myristoyl group.
[0509] During the SPPS of
TABLE-US-00025 H-GGKWSKSSVIGWPAVRER-OH
N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine building block was used for Lys.sup.3 and Boc-Gly-OH for Gly.sup.1. After peptide was assembled the N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protection group was removed by treatment of 2% N.sub.2H.sub.4*H.sub.2O in DMF 3×3 min. Thereafter the linker molecule X1 was coupled as described in the general methods.
[0510] During the SPPS of
TABLE-US-00026 H-SGGKWSKSSVIGWPAVRER-OH
N-alpha-(9-Fmoc)-O-(t-butyl-dimethyl-silyl)-L-serine building block was used for Ser.sup.1 and Boc-Gly-OH for Gly.sup.1. After peptide was assembled the t-butyl-dimethyl-silyl protection group was removed by treatment of AcOH/THF/H.sub.2O 3:1:1 for 2 h. Thereafter Fmoc was removed by treatment with 2×5 min piperidine/DMF 1:4, the resin was washed and the linker molecule X1 was coupled as described in the general methods.
[0511] The peptides were immobilized and washed according to the General Methods in a 25 μmol scale. After washing according to the General Methods, it was washed 3×DMF. Followed by the addition of 8 eq Palm-S-(4,6-Dimethyl-2-pyrimidinyl) thioate (Palm-S) building block with 2 eq DIPEA in 600 μL N-methylpyrrolidone. After addition of this solution beads were shaken for 1 hours and afterwards the supernatant the filtered of and the solid support was washed with 3×DMSO, 3×DMF, 3× and the modified peptide was released under the conditions given at General Methods\Release of Modified Peptides.
[0512] Palm-SGGK(Palm)WSK(Palm)SSVIGWPAVRER-NH.sub.2: The peptide precursor of P16 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with Palm-S building block and purified according to the above described method. Results are shown in
[0513] Palm-GGKWSK(Palm)SSVIGWPAVRER-NH.sub.2: The peptide precursor of P17a has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with Palm-S building block and purified according to the above described method. Results are shown in
Example 11: Formation of Lipidated Peptide P17b Bound to Purification Solid Support Palm-S Building Block
[0514] The inventive method for the modification and purification of peptides was applied to
TABLE-US-00027 (SEQ ID NO: 17) H-GGKWSKSSVIGWPAVRER-OH
is a sequence 2-19 of the Nef protein what bears an N-terminal myristoyl group.
[0515] During the SPPS of
TABLE-US-00028 H-GGKWSKSSVIGWPAVRER-OH
N-α-Fmoc-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine building block was used for Lys.sup.3 and Boc-Gly-OH for Gly.sup.1. After peptide was assembled the N-ε-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) protection group was removed by treatment of 2% N.sub.2H.sub.4*H.sub.2O in DMF 3×3 min. Thereafter the linker molecule X1 was coupled as described in the general methods.
[0516] The peptides were immobilized and washed according to the General Methods in a 25 μmol scale. After washing according to the General Methods, it was washed 3×DMF. Followed by the addition of 8 eq Myr-OSu building block with 2 eq DIPEA in 600 μL N-methylpyrrolidone. After addition of this solution beads were shaken for 1 hours and afterwards the supernatant the filtered of and the solid support was washed with 3×DMSO, 3×DMF, 3× and the modified peptide was released under the conditions given at General Methods\Release of Modified Peptides.
[0517] Myr-GGKWSK(Myr)SSVIGWPAVRER-NH.sub.2: The peptide precursor of P17b has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with Myr-OSu building block and purified according to the above described method. Results are shown in
Example 12: Formation of Macrocycles of Peptides P6 & P8 Bound to Purification Solid Support (aldehyde-modified agarose) with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TATB) (intramolecular bicyclization)
[0518] The inventive method for the modification and purification of peptides was applied to two peptides of different length, cysteine positions and polarity, these were
TABLE-US-00029 (SEQ ID NO: X) H-ACSWPARCLHQDLCA-NH.sub.2 (P6) and (SEQ ID NO: X) H-ACYNEFGCEDFYDICA-NH.sub.2 (P8).
[0519] The crude Linker-tagged peptide was immobilized for 16 h and washed according to the General Methods, as a deviation from the general method, after immobilization and removal of the immobilization supernatant, an O-methylhydroxylamine hydrochloride solution (5 w % in H.sub.2O, 250 μL) was added and the immobilized peptide was shaken for 15 min. After washing according to the General Methods, Linker was reduced, and StBu-protection groups were removed by addition of 12.5 mg DTT in 250 μL 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) and shaking for 60 min. Subsequently, 1.9 mg TCEP dissolved in 30 μL 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) was added and shaking was continued for 15 min. Thereafter, the supernatant was filtered of and the solid support was washed with 3×H.sub.2O, 2×MeCN and 3×5 w % aq. NaHCO.sub.3 (0.6 M; pH 8). 125 μL of 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) was added to the solid support followed by TATB (1-2 eq) in 125 μL MeCN. The beads were shaken for 60 min and afterwards washed with 3×H.sub.2O and 3×MeCN. The modified peptide was released following the conditions of General Methods.
TABLE-US-00030 H-ACSWPARCLHQDLCA-NH.sub.2:
The peptide precursor P6 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.3 μmol scale. Results are shown in Table LuS1 and
TABLE-US-00031 H-ACYNEFGCEDFYDICA-NH.sub.2:
The peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.0 μmol scale. Results are shown in Table LuS1 and
Example 13: Formation of Macrocycles of Peptides P6 & P8 Bound to Purification Solid Support (aldehyde-modified poly (methyl acrylate)) with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TA TB) (intramolecular bicyclization)
[0520] The inventive method for the modification and purification of peptides was applied to two peptides of different length, cysteine positions and polarity, these were
TABLE-US-00032 (SEQ ID NO: X) H-ACSWPARCLHQDLCA-NH.sub.2 (P6) and (SEQ ID NO: X) H-ACYNEFGCEDFYDICA-NH.sub.2 (P8).
[0521] The crude Linker-tagged peptide was dissolved in pure HFIP and immobilized for 16 h and washed on aldehyde-modified poly (methyl acrylate) as solid support. After immobilization and removal of the immobilization supernatant, an O-methylhydroxylamine hydrochloride solution (5 w % in H.sub.2O, 250 μL) was added and the immobilized peptide was shaken for 15 min. After washing according to the General Methods, Linker was reduced, and StBu-protection groups were removed by addition of 12.5 mg DTT in 250 μL 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) and shaking for 60 min. Subsequently, 1.9 mg TCEP dissolved in 30 μL 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) was added and shaking was continued for 15 min. Thereafter, the supernatant was filtered of and the solid support was washed with 3×H.sub.2O, 2×MeCN and 3×5 w % aq. NaHCO.sub.3 (0.6 M; pH 8). 125 μL of 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) was added to the solid support followed by TATB (1-2 eq) in 125 μL MeCN. The beads were shaken for 60 min and afterwards washed with 3×H.sub.2O and 3×MeCN. The modified peptide was released by treatment of the solid support with 0.4 mL TFA/H.sub.2O (95:5) for 1 h, subsequently adding 0.4 mL TFA and eluting the peptide with 2×0.4 mL TFA/H.sub.2O (95:5). The released modified peptide was precipitated with 10 mL cold Et.sub.2O and after cooling the mixture down in freezer for 15-30 min, it was centrifuged, and the supernatant was decanted.
TABLE-US-00033 H-ACSWPARCLHQDLCA-NH.sub.2:
The peptide precursor P6 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.3 μmol scale. Results are shown in Table LuS1 and
TABLE-US-00034 H-ACYNEFGCEDFYDICA-NH.sub.2:
The peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.0 μmol scale. Results are shown in Table LuS1 and
Example 14: Formation of macrocycles of peptides P6 & P8 bound to purification solid support (aldehyde-modified aqarose) with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TA TB) on with sodium dodecyl sulfate as additive for peptide dissolution (intramolecular bicyclization)
[0522] The inventive method for the modification and purification of peptides was applied to two peptides of different length, cysteine positions and polarity, these were
TABLE-US-00035 (P6) (SEQ ID NO: X) H-ACSWPARCLHQDLCA-NH.sub.2 and (P8) (SEQ ID NO: X) H-ACYNEFGCEDFYDICA-NH.sub.2.
[0523] The crude Linker-tagged peptide was immobilized for 16 h and washed according to the General Methods, as deviations from the general method, peptide dissolution was performed with 1 M SDS in DMSO+10 vol. % Immobilization Buffer 2 and after immobilization and removal of the immobilization supernatant, an O-methylhydroxylamine hydrochloride solution (5 w % in H.sub.2O, 250 μL) was added and the immobilized peptide was shaken for 15 min. After washing according to the General Methods with an additional wash with 3×8 M Urea in H.sub.2O/MeOH (9:1), Linker was reduced, and StBu-protection groups were removed by addition of 12.5 mg DTT in 250 μL 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) and shaking for 60 min. Subsequently, 1.9 mg TCEP dissolved in 30 μL 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) was added and shaking was continued for 15 min. Thereafter, the supernatant was filtered of and the solid support was washed with 3×H.sub.2O, 2×MeCN and 3×5 w % aq. NaHCO.sub.3 (0.6 M; pH 8). 125 μL of 5 w % aq. NaHCO.sub.3 (0.6 M; pH 8) was added to the solid support followed by TATB (1-2 eq) in 125 μL MeCN. The beads were shaken for 60 min and afterwards washed with 3×H.sub.2O and 3×MeCN. The modified peptide was released following the conditions of General Methods.
TABLE-US-00036 H-ACSWPARCLHQDLCA-NH.sub.2:
The peptide precursor P6 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.3 μmol scale. Results are shown in Table LuS1 and
TABLE-US-00037 H-ACYNEFGCEDFYDICA-NH.sub.2:
The peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.0 μmol scale. Results are shown in Table LuS1 and
Example 15: Formation of macrocycles of peptides P6 & P8 bound to purification solid support (aldehyde-modified poly (methyl acrylate)) with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TA TB) on with sodium dodecyl sulfate as additive for peptide dissolution (intramolecular bicyclization)
[0524] As claimed in Example 13 the crude Linker-tagged peptide was immobilized on aldehyde-modified poly (methyl acrylate) as solid support, whereas, as claimed in Example 14, 1 M SDS in DMSO+10 vol. % Immobilization Buffer 2 was used for dissolution of the peptide instead of pure HFIP. As a deviation, 40 vol. % of beads-volume of an aniline-buffer-solution (0.46 vol. % Aniline in 0.79 w % NH.sub.4OAc in H.sub.2O/AcOH (9:1)) was added to the solid support prior to addition of dissolved peptide. The immobilized peptide was washed, modified with TATB and released as claimed in Example 13. As a deviation, after immobilization and wash of the peptide, it was additionally washed with 3×8 M Urea in H.sub.2O/MeOH (9:1).
TABLE-US-00038 H-ACSWPARCLHQDLCA-NH.sub.2:
The peptide precursor P6 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.3 μmol scale. Similar results as in Example 3 were achieved and are shown in Table LuS1. 3.9 mg of modified peptide with a purity of 55% were obtained as white powder. ESI-MS: (calculated MH.sup.2+: 941.1 m/z, found: 940.7 m/z).
TABLE-US-00039 H-ACYNEFGCEDFYDICA-NH.sub.2:
The peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized and modified with TATB according to the above described method in a 1.0 μmol scale. Similar results as in Example 3 were achieved and are shown in Table LuS1. 3.7 mg of modified peptide with a purity of 48% were obtained as white powder. ESI-MS: (calculated MH.sup.2+: 1035.6 m/z, found: 1035.3 m/z).
Example 16: Formation of Macrocycles of Peptides P6 & P8 Bound to Purification Solid Support (aldehyde-modified agarose or poly (methyl acrylate)) with 1,1′,1″-(1,3,5-Triazinan-1,3,5-triyl)tris(2-bromoethanon) (TATB) while cooling (intramolecular bicyclization)
[0525] The inventive method for the modification and purification of peptides was applied to two peptides of different length, cysteine positions and polarity, these were
TABLE-US-00040 (P6) (SEQ ID NO: X) H-ACSWPARCLHQDLCA-NH.sub.2 and (P8) (SEQ ID NO: X) H-ACYNEFGCEDFYDICA-NH.sub.2.
[0526] The crude Linker-tagged peptide was immobilized, washed, modified with TATB and released as claimed in Examples LuS1 or LuS2 on aldehyde-modified agarose or poly (methyl acrylate) as solid support respectively, as a deviation from this methods, the modification with TATB was performed at a temperature of 1° C. by keeping the reaction container in an ice-water-bath.
TABLE-US-00041 H-ACSWPARCLHQDLCA-NH.sub.2:
The peptide precursor P6 has been synthesized as described in the SPPS paragraph above. It has been immobilized on aldehyde-modified poly (methyl acrylate) and modified with TATB according to the above described method in a 1.3 μmol scale. Results are shown in Table LuS1 and
TABLE-US-00042 H-ACYNEFGCEDFYDICA-NH.sub.2:
ne peptide precursor P8 has been synthesized as described in the SPPS paragraph above. It has been immobilized on aldehyde-modified poly (methyl acrylate) and modified with TATB according to the above described method in a 1.0 μmol scale. Results are shown in Table LuS1 and
Example 17: Formation of Macrocycle of Peptide P3 Bound to Purification Solid Support with by Disulfide (Intramolecular Cyclization)
[0527] The inventive method for the immobilization, modification and purification of peptides was applied to the peptide
TABLE-US-00043 (P3) (SEQ ID NO: 3) H-CRVPGDAHHADSLC-NH.sub.2, (P4) (SEQ ID NO: ?) H-VRCPGAAHHADSLC-NH2, (P9) (SEQ ID NO: 9) H-CYFQNCPRG-NH.sub.2, (P10) (SEQ ID NO: 10) H-KCNTATCATQRLANFLVHSSNFGPILPPTNVGSNTY-NH.sub.2, (P11) (SEQ ID NO: ?) H-CCEYCCNPACTGCY-NH.sub.2.
TABLE-US-00044 H-CRVPGDAHHADSLC-NH.sub.2
(underlined C residues are connected by the scaffold): The linker-tagged peptide is immobilized on 1.5 times the amount of aldehyde-modified polymethacrylate loaded with 152.6 μmol/1 gm beads. After transferring, the beads material is washed with 3× milli-Q water and 3× immobilization buffer 1 (0.1 M citric Acid/Na2CO3 pH 4.5). 113 μl of DMSO was added to the beads and soaked for 5 min. The crude linker-tagged peptide was dissolved in 223 μl of DMSO and 26 μl Buffer2 and the mixture was then added to the polymethacrylate beads and shaken. Of note, the beads should have a free and good fluctuation in the immobilization solution. After a reaction time of 72 h, the immobilization solution is filtered off with suction. The immobilization worked equally efficiently, with various amounts of beads. With 1.5 times-15 times (To the linker tagged peptide) of the amount of aldehyde-modified polymethacrylate loaded with 152.6 μmol/1 gm, beads successfully immobilized linker-tagged peptide quantitatively (1.5 times-72 hours, 3 times-48 h, 7.5 times requires 300 minutes, whereas 15 times requires 180 minutes).
[0528] Washing: After immobilization, the beads linker-connected to the peptides were washed each, three-times 5 mL (for 2.5 μmol scale) with the following solvents and solutions [0529] 1) 0.9 M guanidinium chloride in DMSO [0530] 3) 70% ethanol in with 0.1 M NaCl Milli-Q water
[0531] Reduction: To the immobilized peptide was added 5 ml of Acetonitrile. A fresh solution of DTT in 5 wt % of NaHCO.sub.3 solution was added (33.6 eq, 0.26 M) and the reactor was shaken for 60 min. After the removal of the DTT solution by filtration, the bead material was washed with 3×H.sub.2O and 3×MeCN.
[0532] oxidation: Thereafter 500 μL of H.sub.2O/MeCN (1:1) was added to the beads followed several additions of 20 μL of iodine (10 mg/mL) till which the solutions stayed yellowish and the cartridges were shaken for a total of 10 min. Beads were washed 3× with ascorbic acid (15 mg/mL) in water, 3× water and twice with MeCN.
[0533] Release of the peptide: To this vial 166.6 μl 95% TFA was added and after 30 min and eluted with 2×166.6 μl of 95% TFA in water in pre-weighed cold ether vials precipitate the peptide by centrifuging and decanting the ether the pure peptide.
[0534] The release also worked equally efficient by treating the oxidized beads with 166 μl of 80:10:10 (H.sub.2O:MeCN:TFA) or 90:05:05 (H.sub.2O:MeCN:TFA) for 18 hours and then eluting with 2.5× volume H2O/MeCN (7:3)+0.1% TFA and 1.5× volume H2O/MeCN (3:7)+0.1% TFA. The reuslting soltion was lyophilized to get the desired peptide with 95% purity. ESI-MS: (calculated MH.sup.2+: 774.13 g/mol, found: 773.91 m/z).
TABLE-US-00045 H-VRCPGAAHHADSLC-NH.sub.2
(underlined C residues are connected by the scaffold): The linker-tagged peptide is immobilized on 3 times the amount of aldehyde-modified polymethacrylate loaded with 152.6 μmol/1 g beads. After transferring, the beads material is washed with 3× milli-Q water and 3× immobilization buffer 1 (0.1 M citric Acid/Na2CO3 pH 4.5). 226 μl of 83% TFA was added to the beads. The crude linker-tagged peptide was dissolved in 498 μl 83% TFA and the mixture was then added to the polymethacrylate beads and shaken. Of note, the beads should have a free and good fluctuation in the immobilization solution. After a reaction time of 150 minutes, the immobilization solution is filtered off with suction. The immobilization worked equally efficiently, with of Solvents like HFIP (150 minutes). Immobilization was also performed in DMSO as descriped in the P3 peptide with various amounts of beads corresponding to the peptide (1.5, 3 & 5 aldehyde groups to the peptide). After 16 hours of immobilization, in the case of 1.5 times of aldehydes 55% peptide immobilized, 3 times of aldehyde 70% peptide and 5 times of aldehyde case peptide immobilized quantitively. Adding catalyst (50 mM aniline) to the 3 times of the aldehyde increased the immobilization rate 4 times.
[0535] Washing: In case of TFA as immobilization solvent, after immobilization, the beads linker-connected to the peptides were washed each three-times 3 mL (for 2.5 μmol scale) with the following solvents and solutions [0536] 1) Milli-Q water [0537] 2) 0.9 M guanidinium chloride in DMSO [0538] 3) 70% ethanol in with 0.1 M NaCl Milli-Q water
[0539] In case of HFIP as immobilization solvent, after immobilization, the beads linker-connected to the peptides were washed each three-times 3 mL (for 2.5 μmol scale) with the following solvents and solutions [0540] 1) HFIP [0541] 2) Milli-Q water
[0542] In all other cases, the peptide was washed according to the method mentioned P3 peptide case.
[0543] Reduction: Reduction procedure was followed according to the method mentioned P3 peptide case.
[0544] oxidation: Thereafter 1000 μL of DMSO was added to the beads followed purging of air to the bead's solutions (5 min) and shaken for 24 hours, filtered off, washed (3×H.sub.2O, 1×CH.sub.3CN) and dried.
[0545] Release of the peptide: Release of the peptide procedure was followed according to the method mentioned P3 peptide case to get desired peptide with 79% purity. ESI-MS: (calculated MH.sup.2+: 717.43 g/mol, found: 717.71 m/z).
[0546] H-CYFQNCPRG-NH.sub.2 (underlined C residues are connected by the scaffold): TFA immobilization, washings and Reduction procedures was followed according to the method mentioned P4 peptide.
[0547] oxidation: Thereafter 1000 μL of 20% DMSO in 0.1 M solution of disodium hydrogen phosphate was added to the beads followed purging of air to the bead's solutions (5 min) and shaken for 4 hours, filtered off, washed (3×H.sub.2O, 1×CH.sub.3CN) and dried.
[0548] Release of the peptide: Release of the peptide procedure was followed according to the method mentioned P3 peptide case to get desired peptide with 85% purity (calculated MH.sup.2+: 542.72 g/mol, found: 543.12 m/z).
[0549] H-KCNTATCATQRLANFLVHSSNFGPILPPTNVGSNTY-NH.sub.2 (underlined C residues are connected by the scaffold): TFA Immobilization, washings and Reduction, Oxidation, Release of the peptide was followed according to the method mentioned P9 peptide to get desired peptide with 10% purity (calculated MH.sup.4+.sub.calc: 959.48 m/z, found: MH.sup.4+.sub.found: 959.84 m/z)
TABLE-US-00046 H-CCEYCCNPACTGCY-NH.sub.2
(underlined C residues are connected by the scaffold): TFA Immobilization, washings and Reduction, Oxidation, Release of the peptide was followed according to the method mentioned P9 peptide to get desired peptide with 10% purity (calculated MH.sup.2+.sub.calc: 763.21 m/z, found: MH.sup.2+.sub.found: 763.85 m/z).
TABLE-US-00047 TABLE 1 crude UV.sub.210 purity/ purity after Organic modifi- molecules cation or *purity yield calculated sequence conditions of of vs. after for linker- modifi- found No. modification Example modification peptide cation ESI masses P1 H-AK(Palm-Glu-OH) Example 1 Palm-(Glu(OH)- 62%/ 95% MH.sup.2+.sub.calc.: 935.09 m/z EFIAWLVRGRG-NH.sub.2 OtBu, HCTU 83% MH.sup.2+.sub.found.: 935.31 m/z P2 H-HAEGTFTSDVSSYLEG Example 1 Palm-(Glu(OH)- 30%/ 88% MH.sup.4+.sub.calc.: 937.75 m/z QAAK(Palm-γE-OH) OtBu, HCTU 54% MH.sup.4+.sub.found.: 937.95 m/z EFIAWLVRGRG-NH.sub.2 P3a S------------S Example 2 m-Xylylene 68%/ >99% MH.sup.2+.sub.calc.: 791.38 m/z H-CRVPGDAHHADSLC- dibromide 64% MH.sup.2+.sub.found.: 791.45 m/z NH.sub.2 (mDBX) P4a S----------S Example 2 mDBX 58%/ >99% MH.sup.2+.sub.calc.: 769.38 m/z H-VRCPGAAHHADSLC- 54% MH.sup.2+.sub.found.: 769.49 m/z NH.sub.2 P5 S--S Example 2 mDBX 56%/ >99% MH.sup.2+.sub.calc.: 796.40 m/z H-VRVPGCAHCADSLY- 74% MH.sup.2+.sub.found.: 796.55 m/z NH.sub.2 P6a S-----S-----S Example 3 1,3,5-Tris 60%/ >99% MH.sup.2+.sub.calc.: 893.92 m/z H-ACSWPARCLHQDLCA- (bromomethyl) 72% MH.sup.2+.sub.found.: 894.16 m/z NH.sub.2 benzene (TBMB) P7 S------S------S Example 3 TBMB 60%/ >99% MH.sup.2+.sub.calc.: 1016.02 m/z H-ACREGFLRCLHRPTVC 67% MH.sup.2+.sub.found.: 1016.30 m/z NG-H.sub.2 P8a S-----S------S Example 4 1,1′,1″- 49%/ >99% MH.sup.2+.sub.calc.: 1034.89 m/z H-ACYNEFGCEDFYDICA- (1,3,5- 71% MH.sup.2+.sub.found.: 1034.51 m/z NH.sub.2 Triazinan- 1,3,5-triyl) tris(2- bromoethanon) (TATB) P8b S-----S------S Example 5 1,3,5- 49%/ >99% MH.sup.2+.sub.calc.: 1055.98 m/z H-ACYNEFGCEDFYDICA- Triacryloyl- 81% MH.sup.2+.sub.found.: 1056.02 m/z NH.sub.2 1,3,5- triazinan (TATA) P3b S------------S Example 6 Disulfide, 68%/ 22% MH.sup.2+.sub.calc.: 739.32 m/z H-CRVPGDAHHADSLC- DMSOand 10% MH.sup.2+.sub.found.: 739.52 m/z NH.sub.2 0.4 M (NH.sub.4).sub.2CO.sub.3, 6 M GdnCl, pH8.7 P3b S------------S Example 7 none 69%*/ >99% MH.sup.2+.sub.calc.: 739.32 m/z H-CRVPGDAHHADSLC- (Disulfide) 95% MH.sup.2+.sub.found.: 739.62 m/z NH.sub.2 P4b S----------S Example 7 none 67%/ >99% MH.sup.2+.sub.calc.: 717.34 m/z H-VRCPGAAHHADSLC- (Disulfide) 79% MH.sup.2+.sub.found.: 717.71 m/z NH.sub.2 P9 S----S Example 17 none 60%*/ >95% MH.sup.2+.sub.calc.: 542.72 m/z H-CYFQNCPRG-NH.sub.2 (Disulfide) 85% MH.sup.2+.sub.found.: 543.12 m/z P10 S----S Example 17 none traces*/ >99% MH.sup.2+.sub.calc.: 959.48 m/z H-KCNTATCATQRLANF (Disulfide) 10% MH.sup.2+.sub.found.: 959.84 m/z LVHSSNFGPILPPTNVG SNTY-NH.sub.2 P11 S-S-SS---S--S Example 17 none not >75% MH.sup.2+.sub.calc.: 763.21 m/z H-CCEYCCNPACTGCY- (Disulfide) determined*/ MH.sup.2+.sub.found.: 763.85 m/z NH.sub.2 10% P12a H-KKKKSC(C.sub.3H.sub.5O.sub.2- Example 7 1-bromopropyl- 67%*/ 47% MH.sup.2+.sub.calc.: 635.98 m/z diPalm)-NH.sub.2 2,3- not MH.sup.2+.sub.found.: 636.39 m/z dipalmitate determined (Pam.sub.2-Br) P12b H-KKKKSC(C.sub.3H.sub.5O.sub.2- Example 7 1-iodopropyl- 67%*/ 73% MH.sup.2+.sub.calc.: 635.98 m/z diPalm)-NH.sub.2 2,3- not MH.sup.2+.sub.found.: 636.42 m/z dipalmitate determined (Pam.sub.2-I) P12d H-KKKKSC(C.sub.2H.sub.4O- Example 7 Palm-OC.sub.3H.sub.4I 67%*/ 39% MH.sup.+.sub.calc.: 1002.70 m/z Palm)-NH.sub.2 (Pam-Et-I) not MH.sup.+.sub.found.: 1002.80 m/z determined P13a H-KEKFSINSEDNNGC Example 7 1-iodopropyl- 67%*/ — MH.sup.3+.sub.calc.: 712.41 m/z (C.sub.3H.sub.5O.sub.2-diPalm)-NH.sub.2 2,3-dipalmitate not MH.sup.3+.sub.found.: 712.40 m/z (Pam.sub.2-Br) determined P13b H-KEKFSINSEDNNGC Example 7 1-iodopropyl- 67%*/ — MH.sup.3+.sub.calc.: 712.41 m/z (C.sub.3H.sub.5O.sub.2-diPalm)-NH.sub.2 2,3-dipalmitate not MH.sup.3+.sub.found.: 712.40 m/z (Pam.sub.2-I) determined P13c H-KEKFSINSEDNNGC Example 7 1-tosylpropyl- 55%*/ >98% MH.sup.3+.sub.calc.: 712.41 m/z (C.sub.2H.sub.40-Palm)-NH.sub.2 2,3-dipalmitate not MH.sup.3+.sub.found.: 712.11 m/z (Pam.sub.2-OTs) determined P13d H-KEKFSINSEDNNGC Example 7 Palm-OC.sub.2H.sub.4I 55%*/ 46% MH.sup.2+.sub.calc.: 933.49 m/z (C.sub.2H.sub.4O-Palm)-NH.sub.2 (Pam-Et-I) not MH.sup.2+.sub.found.: 933.89 m/z determined P14b H-C(C.sub.3H.sub.5O.sub.2-diPalm) Example 7 1-iodopropyl- 84%*/ — MH.sup.+.sub.calc.: 884.66, m/z RG-NH.sub.2 2,3-dipalmitate not MH.sup.+.sub.found.: 884.66 m/z (Pam2-I) determined P14c H-C(C.sub.3H.sub.5O.sub.2-diPalm) Example 7 1-tosylpropyl- 84%*/ >97% MH.sup.+.sub.calc.: 884.66 m/z RG-NH.sub.2 2,3-dipalmitate not MH.sup.+.sub.found.: 884.75 m/z (Pam.sub.2-OTs) determined P14d H-C(C.sub.2H.sub.4O-Palm) Example 7 Palm-OC.sub.2H.sub.4I 84%*/ 66% MH.sup.+.sub.calc.: 616.42 m/z RG-NH.sub.2 (Pam-Et-I) not MH.sup.+.sub.found.: 616.58 m/z determined P2b H-HAEGTFTSDVSSYLE Example 8 Palm-Glu(OSu)- 7%/ >99% MH.sup.4+.sub.calc.: 938.25 m/z GQAAK(Palm-Glu- OtBu 72% MH.sup.4+.sub.found.: 938.73 m/z OH)EFIAWLVRGRG-OH P15 H-HAibFGTFTSDVSSYL Example 9 tBuOC(O)-Ste- 6%/ >99% MH.sup.4+.sub.calc.: 1029.29 m/z EGQAAK(CO.sub.2H-Ste- Glu(aeea-aeea- 67% MH.sup.4+.sub.found.: 1029.20 m/z Glu(aeea-aeea-OH)- OSu)-OtBu OH)EFIAWLVRGRG-OH P16 Palm-SGGK(Palm)WSK Example 10 Palm-S-(4,6- traces*/ >99% MH.sup.3+.sub.calc.: 934.61 m/z (Palm)SSVIGWPAVRE Dimethyl-2- 53% MH.sup.3+.sub.found.: 934.71 m/z R-NH.sub.2 pyrimidinyl) thioate (Palm-S) P17a Palm-GGKWSK(Palm) Example 10 Palm-S traces*/ >99% MH.sup.3+.sub.calc.: 826.19 m/z SSVIGWPAVRER-NH.sub.2 6% MH.sup.3+.sub.found.: 826.17 m/z P17b Myr-GGKWSK(Myr)SSV Example 11 Myr-OSu traces*/ >99% MH.sup.3+.sub.calc.: 807.50 m/z IGWPAVRER-NH.sub.2 3% MH.sup.3+.sub.found.: 807.65 m/z P6b S-----S-----S Example 12 1,1’,1″-(1,3,5- 51%*/ >99% 941.1 m/z H-ACSWPARCLHQDLC Triazinan-1,3,5- 69% 940.7 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB) P6b S-----S-----S Example 13 1,1’,1″-(1,3,5- 51%*/ >99% 941.1 m/z H-ACSWPARCLHQDLC Triazinan-1,3,5- 70% 941.0 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB) P6b S-----S-----S Example 14 1,1’,1″-{1,3,5- 51%*/ >99% 941.1 m/z H-ACSWPARCLHQDLC Triazinan-1,3,5- 61% 940.7 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB) P6b S-----S-----S Example 15 1,1’,1″-(1,3,5- 51%*/ >99% 941.1 m/z H-ACSWPARCLHQDLC Triazinan-1,3,5- 55% 940.7 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB) P6b S-----S-----S Example 16 1,1’,1″-(1,3,5- 51%*/ >99% 941.1 m/z H-ACSWPARCLHQDLC Triazinan-1,3,5- 69% 940.5 m/z A-NH.sub.2 triyl)tris(2- bromocthanon) (TATB) P8a S-----S------S Example 12 1,1’,1″-(1,3,5- 44%*/ >99% 1035.6 m/z H-ACYNEFGCEDFYDIC Triazinan-1,3,5- 45% 1035.5 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB) P8a S-----S------S Example 13 1,1’,1″-(1,3,5- 44%*/ >99% 1035.6 m/z H-ACYNEFGCEDFYDIC Triazinan-1,3,5- 44% 1035.3 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB) P8a S-----S------S Example 14 1,1’,1″- 44%/ >99% 1035.6 m/z H-ACYNEFGCEDFYDIC (1,3,5-Triazinan- 43% 1034.9 m/z A-NH.sub.2 1,3,5-triyl) tris(2- bromoethanon) (TATB) P8a S-----S------S Example 15 1,1’,1″- 44%*/ >99% 1035.6 m/z H-ACYNEFGCEDFYDIC (1,3,5-Triazinan- 48% 1035.3 m/z A-NH.sub.2 1,3,5-triyl) tris(2- bromoethanon) (TATB) P8a S-----S------S Example 16 1,1’,1″-(1,3,5- 44%*/ >99% 1035.6 m/z H-ACYNEFGCEDFYDIC Triazinan-1,3,5- 44% 1035.2 m/z A-NH.sub.2 triyl)tris(2- bromoethanon) (TATB)