Proline Derivatives for Stapled Peptide Helix Nucleation

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Solution Overview

Problem

Alpha-helical peptides are prone to unraveling into random coils, leading to reduced biological activity and susceptibility to proteolytic degradation, limiting their therapeutic potential.

Innovation Solution

The development of stabilized stapled peptides using an N-terminal proline derivative with an alkenyl or alkynyl side chain, which forms a stable alpha-helix nucleating staple through an olefin metathesis reaction.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If alpha-helical peptides are used as therapeutic agents, then biological activity is achieved through selective recognition, but the peptides are prone to unraveling into random coils and susceptibility to proteolytic degradation

Engineering Contradiction:
Improvebiological activityVSAvoidpeptide conformation stability
Core Design Contradiction:
ReliabilityVSStability of the object's composition

Solution Approach 1:

The peptide chain is segmented and reconnected through covalent crosslinks at specific positions (e.g., i to i+7) to form staple structures. This segmentation approach allows the peptide to maintain its alpha-helical conformation while introducing stability through crosslinking, resolving the contradiction between biological activity and conformational stability.

Inventive Principle:
Principle #1Segmentation

Solution Approach 2:

The invention creates composite peptide structures by combining natural amino acids with covalent crosslinks (staples) to form stapled peptides. This composite structure integrates the biological recognition capabilities of the peptide with the stabilizing effect of the crosslinked staple, simultaneously achieving high biological activity and conformational stability.

Inventive Principle:
Principle #40Composite materials

2Reliability

If covalent crosslinks are introduced to stabilize the peptide, then resistance to proteolytic cleavage increases, but the structural complexity increases

Engineering Contradiction:
Improveresistance to proteolytic cleavageVSAvoidpeptide structure complexity
Core Design Contradiction:
ReliabilityVSDevice complexity

Solution Approach 1:

Instead of uniformly modifying the entire peptide structure, the invention introduces crosslinks at specific local positions (e.g., every 7th residue) to form staples. This localized approach provides proteolytic resistance where needed while minimizing overall structural complexity and synthesis difficulty.

Inventive Principle:
Principle #3Local quality

Solution Approach 2:

The crosslinking positions are predetermined and designed in advance during peptide planning. By pre-determining the staple formation positions (e.g., at specific i and i+7 residues), the synthesis process becomes more manageable, and the structural complexity is controlled while achieving maximum proteolytic resistance.

Inventive Principle:
Principle #10Preliminary action

3Stability of the object's composition

If proline is used at the N-terminus to nucleate alpha-helix formation, then helix formation is enhanced, but proline is commonly considered an alpha-helix disrupting amino acid

Engineering Contradiction:
Improvealpha-helix formationVSAvoidbiological activity
Core Design Contradiction:
Stability of the object's compositionVSReliability

Solution Approach 1:

Instead of avoiding proline at the N-terminus as conventional wisdom suggests, the invention deliberately places proline there and uses it as a helix-nucleating residue. By inverting the conventional approach and leveraging proline's unique properties (including its ability to form hydrogen bonds that cloak amide N-H's), the invention enhances alpha-helix formation while maintaining biological activity.

Inventive Principle:
Principle #13The other way round (Inversion)

Solution Approach 2:

The invention changes the conformational parameters and hydrogen-bonding characteristics of the N-terminal proline residue to favor helix nucleation. By optimizing the local structural parameters around the proline residue and utilizing its hydrogen-bonding capabilities, the invention transforms proline from a helix-disrupting to a helix-promoting residue at the N-terminus.

Inventive Principle:
Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The stabilized peptides maintain a stable alpha-helical conformation, enhancing their resistance to proteolytic cleavage, hydrophobicity, cellular penetration, and biological activity compared to their uncrosslinked counterparts.

Implementation Method 1

Such a staple using a proline derivative may be formed with any other amino acid with an alkenyl or alkynyl side chain using an olefin metathesis reaction.

Methodology Applied
Scientific EffectOlefin metathesis:

Data Source

PatentEP3872067B1Proline derivatives
Publication Date: 2025.06.18 PRESIDENT & FELLOWS OF HARVARD COLLEGE
  • EP3872067B1 patent drawingFigure 1
  • EP3872067B1 patent drawingFigure 2
  • EP3872067B1 patent drawingFigure 3

AI summary

The present invention provides proline derivatives of the general formula (III) which have been found to be strong nucleators of alpha-helix formation. A new type of alpha-helix nucleating cross-link ("staple") formed by olefin metathesis of such a proline derivative with an alkenyl side chain and another amino acid derivative with an alkenyl side chain is provided as well.