Method For Solid-Phase Peptide Synthesis And Purification

• US 20100029911A1
• Filed: 11/24/2005
• Published: 02/04/2010
• Est. Priority Date: 11/24/2004
• Status: Abandoned Application

• Current Assignee: Aplagen GmbH
• Original Assignee: Aplagen GmbH
• Inventors: Casaretto, Monika, Knorr, Karsten, Frank, Hans-Georg
• Application Number:
  US11/791,342
• Publication Number:
  US 20100029911A1
• Time in Patent Office: Days
• US Class Current: 530/402
• CPC Class Codes: C07D 471/04 Ortho-condensed systems
• CPC Class Codes: C07K 1/042 characterised by the nature of the carrier
• CPC Class Codes: C07K 1/061 using protecting groups
• CPC Class Codes: C07K 1/22 Affinity chromatography or related techniques based upon selective absorption processes

Abstract

Use of an activated solid phase and a peptide-conjugated anchoring part for solid phase peptide synthesis, wherein the anchoring part is coordinatively and reversibly attached to the activated solid phase.

Claims

1. A peptide having an anchoring part for binding to an activated solid phase comprising a solid support, a metal chelating ligand covalently bound to the solid support and metal ions Mⁿ⁺ with n=1 to 3 coordinatively bound to said metal chelating ligands, for solid-phase peptide synthesis of said peptide, said activated solid phase providing coordination sites for the coordination and reversible attachment of the anchoring part of the peptide, wherein the peptide is of formula I

P—

X-L 

 

I

wherein P is the peptidyl part which optionally may comprise further non-peptide moieties or protection groups, X is a selectively cleavable linker or orthogonal amino acid protection group wherein X is not an amino acid monomer or peptide, and L is a metal chelating group.

2. The peptide of claim 1, wherein X is a linker or amino acid protection group which is base resistant and is acid-labile and/or photo-cleavable.

3. The peptide of claim 1, wherein X is an orthogonal amino acid protection group which is non-base labile and non-acid labile and which is of the Dde/Nde-type having a functional moiety of formula III

(—

CO)₂C═

C(—

R)(—

OH) 

 

III

wherein R is substituted or unsubstituted alkyl and wherein the two carbonyl groups form a cyclic structure connected by a —

CH2-CR′

R″

—

CH2-, —

NR′

—

CO—

NR″

—

or —

CR′

═

CR″

—

backbone wherein R′

,R″

are alkyl or aryl.

4. The peptide of claim 1, wherein L comprises a phenanthrolyl moiety selected from the group consisting of 5-amino- and 5-amid-[1,10]-phenantroline.

5. The peptide of claim 1, wherein the peptide is a growing peptide chain subject to peptide elongation procedures.

6. The peptide of claim 1, wherein the metal Mⁿ⁺ is selected from the group consisting of Mn²⁺, Cu²⁺, Ni²⁺, Co²⁺, Zn²⁺, Mg²⁺, Ca²⁺, Fe²⁺, Fe³⁺ and lanthanide ions.

11. The peptide of claim 1, wherein X-L is a compound of formula IV

wherein n=1-30.

12. The peptide of claim 1, wherein the metal chelating ligands of the activated solid phase are methylene-aminopyridine groups of formula V

wherein M is the solid support, Y is H or C1-C4 alkyl, Q is either (i) —

CH₂—

, —

NH—

or —

C₂H₄—

or (ii) —

(C₂H₃R′

NR′

)_x—

CH₂—

wherein each R′

is H or CH₃ and x is 1 or 2 or (iii) is eliminated such as that the nitrogen is directly bonded to the pyridyl moiety, and further wherein independent of Q, radical R is (a) H, C1-C4 alkyl or C₂-C₄ hydroxyalkyl, and wherein when Q is —

CH₂, radical R can be either (b.) allyl, benzyl or o-hydroxybenzyl or (c.) —

(C₂H₃R′

NR′

)y-CH₂-pyridyl-Y wherein each R′

is H or CH₃ and y is 0 or 1 or (d.) —

(CH₂)_m—

OY wherein m is 2 or 3 or (e.) —

C₂H₃R′

NR1R2 wherein R′

is H or CH3, R1 is H, C1-C4 alkyl, C2-C4 hydroxyalkyl, phenyl or benzyl, and R2 is H, C1-C4 alkyl or C2-C4 hydroxyalkyl, or (f.) C₃H₄SR′

″

wherein R′

″

is C1-C4 alkyl, or (g.) —

C_nH_2nCOOY with n=1 or 2, or (h.) —

C_nH_2nSO₃⁻

with n=1 or 2, or (i.) —

CH2Z and wherein Z is —

CONH₂ or —

NHCONH2.

13. The peptide of claim 12, wherein a picolylamine group of formula II is bound to the solid support M

wherein n=1 or 2 and R=H

14. The peptide of claim 6, wherein the metal Mⁿ⁺ is selected from the group consisting of Cu²⁺, Ni²⁺, and Co²⁺.

15. The peptide of claim 5, wherein the peptide is a growing peptide, bound via an anchoring part to a metal ion, which is bound to a metal chelating ligand bound to a solid support and subject to peptide elongation procedures.

16. The peptide of claim 5, wherein mono- or oligomeric amino acids are added at the C- or N-terminus to the growing peptide in a Merrifield-type sequential reaction scheme.

17. The peptide of claim 1, wherein the metal chelating ligand of the activated solid phase is selected from the group consisting of amino, hydroxyl, carboxyl, mercapto, imidazolyl, N-methylimidazolyl, aminopurinyl moieties, phenanthrolyl moieties, pyridyl moieties, bipyridyl moieties, terpyridinyl moieties, triazacyclononanonyl moieties, tetraazacyclododecanyl moieties, iminodiacetic acid moieties, nitrilotriacetic acid moieties and ethylenediaminetetraacetic acid moieties.

18. The peptide of claim 1, wherein the anchoring part of the peptide chain is located at the C-terminus and/or in at least one amino acid side chain of the peptide.

19. The peptide of claim 1, wherein after detachment of the peptide from the activated solid phase by diluting the reaction mixture with a competitive ligand, the peptide is reattached to the activated solid phase.

20. The peptide of claim 1, wherein the non-covalent and coordinative attachment of the metal ion to the metal-chelating ligand of the solid support is stronger than the attachment to the anchoring group on the peptide.

7. A peptide of formula P—

X-L, wherein P is the peptidyl part which further comprises non-peptide moieties or protection groups, X is an orthogonal amino acid protection group that is resistant to Fmoc- or to Boc-chemistry, X is not an amino acid monomer or peptide, and L is a metal chelating group.

8. The peptide of claim 7, wherein L comprises a phenanthrolyl moiety selected from the group consisting of 5-amino- and 5-amido-[1,10]-phenantroline.

9. The peptide of claim 7, wherein X is an orthogonal protection group which is both base and acid resistant and is a Dde/Nde-type protection group having a functional moiety of formula III

(—

CO)₂C═

C(—

R)(—

OH) 

 

III

wherein R is substituted or unsubstituted alkyl and wherein the two carbonyl groups form a cyclic structure connected by a —

CH2-CR′

R″

-CH2-, —

NR′

—

CO—

NR″

—

or —

CR′

═

CR″

—

backbone wherein R′

,R″

are alkyl or, taken together, aryl.

10. A compound X-L of formula IV

wherein n=1-30.

21. A method for solid phase synthesis of peptides by non-covalent attachment of a growing peptide chain to an activated solid phase, wherein the active component of the solid phase is formed by metal chelate complexes having free coordination sites for non-covalent attachment of a growing peptide chain to the activated solid phase via an anchoring part of the growing peptide chain.