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Publication Number
US-8637686-B2
Publication Date
2014-01-28
Expiration Date
Abstract
Provided herein are novel alkynyl and azide containing amino acids; kits containing these amino acids; peptides containing these amino acids; peptide macrocycles whose secondary structures are stabilized with linkers containing triazoles synthesized by reacting the side chains of the alkynyl and azide containing amino acids; and methods of making and using the alkynyl and azide containing amino acids, kits, peptides, triazole containing linkers, and peptide macrocycles.
Core Innovation
The invention relates to peptidomimetic macrocycles of Formula I and Formula (I), comprising natural and/or non-natural amino acid residues A, B, C, D, and E connected via a macrocycle-forming linker L. The linker L includes triazole-containing motifs that arise from alkyne and azide sidechains associated with the amino acid residues, and the structures include variable substituents, sequence length variables, and heteroatom linkages.
The described embodiments include macrocycles prepared by solid-phase peptide synthesis using N-alpha-Fmoc-propargylglycine building blocks and selected N-alpha-Fmoc protected amino acid building blocks containing an alkyne moiety. Macrocyclization is described using Cu and Ru macrocyclization reagents, including Cu(I) species, Cu(II) salts, Cp*RuCl(PPh3)2, and [Cp*RuCl]4, and the disclosure discusses helical stability evaluation in connection with circular dichroism.
The disclosure also provides alkynyl and azide amino acid building blocks used for forming the macrocycles, including compounds of Formula IIa and Formula IIb, N-alpha-Fmoc protected amino acid analogs, and selected azido- and alkyne-containing amino acids. The scope includes amino acid analogs that alter methylene spacing between the alpha-carbon and the terminal azide, including azido analogs of lysine and alpha-methyl-lysine and azido analogs of ornithine and alpha-methyl-ornithine.
Claims Coverage
The independent claims cover enantiomerically enriched Formula IIa/IIb compounds, kits comprising those compounds with a macrocyclization reagent, and selected enumerated alkynyl/azido amino acid building blocks including N-alpha-Fmoc-protected variants. In total, the inventive features center on compound selection, kit composition, and specific N-alpha-Fmoc or azido/alkyno amino acid derivatives.
Formula IIa or IIb enantiomerically enriched compound
A compound of Formula IIa or IIb in which R1 and R2 are independently alkyl, each being unsubstituted or substituted with halo-; each of Q and T is independently —CH2—; R7 and R8 are independently —H; R10 and R11 are independently —H; R12 is —H or alkyl; g and h are each independently integers and g+h is 3, 4, 5, or 6; the compound is enantiomerically enriched.
Kit with Formula IIa/IIb compound and macrocyclization reagent
A kit comprising at least one enantiomerically enriched compound selected from the group consisting of compounds of Formulas IIa and IIb together with a macrocyclization reagent.
Selected alkynyl and azido amino acid building blocks
A compound selected from the group consisting of (S)-2-amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, (R)-2-amino-2-methyl-8-nonynoic acid, ε-azido-alpha-methyl-L-lysine, ε-azido-alpha-methyl-D-lysine, δ-azido-alpha-methyl-L-ornithine, and δ-azido-alpha-methyl-D-ornithine.
Selected N-alpha-Fmoc alkynyl and azido amino acid building blocks
A compound selected from the group consisting of N-alpha-Fmoc-(S)-2-amino-2-methyl-4-pentynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-4-pentynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-5-hexynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-5-hexynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-6-heptynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-6-heptynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-7-octynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-7-octynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-8-nonynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-8-nonynoic acid, N-alpha-Fmoc-epsilon-azido-alpha-methyl-L-lysine, N-alpha-Fmoc-epsilon-azido-alpha-methyl-D-lysine, N-alpha-Fmoc-delta-azido-alpha-methyl-L-ornithine, and N-alpha-Fmoc-delta-azido-alpha-methyl-D-ornithine.
Kit with selected amino-acid analog and macrocyclization reagent
A kit comprising a compound selected from the group consisting of (S)-2-amino-5-hexynoic acid, (R)-2-amino-5-hexynoic acid, (S)-2-amino-6-heptynoic acid, (R)-2-amino-6-heptynoic acid, (S)-2-amino-7-octynoic acid, (R)-2-amino-7-octynoic acid, (S)-2-amino-8-nonynoic acid, (R)-2-amino-8-nonynoic acid, (S)-2-amino-2-methyl-4-pentynoic acid, (R)-2-amino-2-methyl-4-pentynoic acid, (S)-2-amino-2-methyl-5-hexynoic acid, (R)-2-amino-2-methyl-5-hexynoic acid, (S)-2-amino-2-methyl-6-heptynoic acid, (R)-2-amino-2-methyl-6-heptynoic acid, (S)-2-amino-2-methyl-7-octynoic acid, (R)-2-amino-2-methyl-7-octynoic acid, (S)-2-amino-2-methyl-8-nonynoic acid, (R)-2-amino-2-methyl-8-nonynoic acid, ε-azido-L-lysine, ε-azido-D-lysine, ε-azido-alpha-methyl-L-lysine, ε-azido-alpha-methyl-D-lysine, δ-azido-L-ornithine, δ-azido-D-ornithine, δ-azido-alpha-methyl-L-ornithine, and δ-azido-alpha-methyl-D-ornithine; and a macrocyclization reagent.
Kit with selected N-alpha-Fmoc protected compound set and macrocyclization reagent
A kit comprising a compound selected from the group consisting of N-alpha-Fmoc-(S)-2-amino-5-hexynoic acid, N-alpha-Fmoc-(R)-2-amino-5-hexynoic acid, N-alpha-Fmoc-(S)-2-amino-6-heptynoic acid, N-alpha-Fmoc-(R)-2-amino-6-heptynoic acid, N-alpha-Fmoc-(S)-2-amino-7-octynoic acid, N-alpha-Fmoc-(R)-2-amino-7-octynoic acid, N-alpha-Fmoc-(S)-2-amino-8-nonynoic acid, N-alpha-Fmoc-(R)-2-amino-8-nonynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-4-pentynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-4-pentynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-5-hexynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-5-hexynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-6-heptynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-6-heptynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-7-octynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-7-octynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-8-nonynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-8-nonynoic acid, N-alpha-Fmoc-epsilon-azido-alpha-methyl-L-lysine, N-alpha-Fmoc-epsilon-azido-alpha-methyl-D-lysine, N-alpha-Fmoc-delta-azido-alpha-methyl-L-ornithine, N-alpha-Fmoc-delta-azido-alpha-methyl-D-ornithine, N-alpha-Fmoc-epsilon-azido-L-lysine, and N-alpha-Fmoc-epsilon-azido-D-lysine; and a macrocyclization reagent.
Selected N-alpha-Fmoc 2-amino-2-methyl heptynoic to octynoic compounds
A compound selected from the group consisting of N-alpha-Fmoc-(S)-2-amino-2-methyl-6-heptynoic acid, N-alpha-Fmoc-(R)-2-amino-2-methyl-6-heptynoic acid, N-alpha-Fmoc-(S)-2-amino-2-methyl-7-octynoic acid, and N-alpha-Fmoc-(R)-2-amino-2-methyl-7-octynoic acid.
Selected N-alpha-Fmoc azido alpha-methyl lysine and ornithine compounds
A compound selected from the group consisting of N-alpha-Fmoc-epsilon-azido-alpha-methyl-L-lysine, N-alpha-Fmoc-epsilon-azido-alpha-methyl-D-lysine, N-alpha-Fmoc-delta-azido-alpha-methyl-L-ornithine, and N-alpha-Fmoc-delta-azido-alpha-methyl-D-ornithine.
Across the independent claims, coverage is directed to enantiomerically enriched Formula IIa/IIb compounds and to kits that combine those compounds or enumerated alkynyl/azido amino acid building blocks, including N-alpha-Fmoc-protected versions, with a macrocyclization reagent. Additional independent claims also cover specific enumerated compound sets for the alkyne-containing and azido-containing amino acid analogs.
Stated Advantages
Improved stability.
Improved biological properties.
Improved penetration.
Improved proteolytic resistance.
Improved cellular uptake.
Improved pharmacokinetics, including bioavailability and blood circulation.
Increased target affinity.
Improved cell penetrability.
Improved α-helical stability.
Documented Applications
The disclosed amino acid analogs are exemplary for synthesizing peptidomimetic macrocycles.
Disease-protein-derived peptide sources are provided, including BCL-2 family BH domains (including BH3) and p53 together with the MDM2-binding helix region.
Example receptor-targeting peptides are provided (including GPCR peptide ligands).
Use in the context of BCL-2 family BH3 peptides and BH domains.
Use in the context of p53 and MDM2.
Use in the context of GPCR peptide ligands.
Use in the context of other peptide ligands.
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