Development of Bifunctional, Raman Active Diyne-Girder Stapled α-Helical Peptides

Abstract

Stapled peptides are a unique class of cyclic α-helical peptides that are conformationally constrained via their amino acid side-chains. They have been transformative to the field of chemical biology and peptide drug discovery through addressing many of the physicochemical limitations of linear peptides. However, there are several issues with current chemical strategies to produce stapled peptides. For example, two distinct unnatural amino acids are required to synthesize i, i+7 alkene stapled peptides, leading to high production costs. Furthermore, low purified yields are obtained due to cis/trans isomers produced during ring-closing metathesis macrocyclisation. Here we report the development of a new i, i+7 diyne-girder stapling strategy that addresses these issues. The asymmetric synthesis of nine unnatural Fmoc-protected alkyne-amino acids facilitated a systematic study to determine the optimal (S,S)-stereochemistry and 14-carbon diyne-girder bridge length. Diyne-girder stapled T-STAR peptide 29 was demonstrated to have excellent helicity, cell permeability and stability to protease degradation. Finally, we demonstrate that the diyne-girder constraint is a Raman chromophore with potential use in Raman cell microscopy. Development of this highly effective, bifunctional diyne-girder stapling strategy leads us to believe that it can be used to produce other stapled peptide probes and therapeutics.

Keywords: Raman active; bifunctional; protease stable; stapled peptide; α-helical.

Figure 1

Previous all‐hydrocarbon i, i+7 stapling technique requires two distinct amino acids and results in a mix of cis/trans isomers. This work demonstrates the use of one amino acid type to create a symmetrical, rigid diyne constraint, resulting in improved yield, cheaper starting material costs, improved α‐helicity and protease stability, and is a Raman chromophore with potential for cell imaging.

Scheme 1

The synthesis of Fmoc‐alkyne amino acids with varying stereochemistry and length using the Ni(II) Schiff Base method.

Scheme 2

Solid phase peptide synthesis of diyne‐girder stapled peptides. Nle=norleucine. Ahx=6‐aminohexanoic acid. FTU=Fluorescein thiourea label. n=number of carbons.

Figure 2

Percentage Glaser cyclisation conversion for 1,3‐diyne‐girder stapled peptides with different bridge lengths and alkyne amino acid stereochemistries.

Figure 3

Circular dichroism spectra of native peptide 29 and stapled peptides 27–39. A) CD shown as MRE, B) % peptide helicity at 222 nm. Conditions: peptides 50 μM in PBS, pH 7.4. Spectra recorded between 185 and 260 nm.

Figure 4

Stability comparison of native T‐STAR‐1 26, T‐STAR‐HCS‐2‐cis 37, T‐STAR‐HCS‐2‐trans 38, T‐STAR‐S ₇ S ₇ 29 and T‐STAR‐S _7H S _7H 39 in the presence of chymotrypsin (0.01 mg/mL) from t=0 to t=4 hours. Points are the mean of three independent experiments ±SEM. Calculated half‐life for native T‐STAR 26=t _1/2 109 min.

Figure 5

Fluorescence imaging showing A) the diyne‐girder T‐STAR S₇S₇ stapled peptide 29, B and C) alkene stapled peptides 37 & 38 and D) diyne‐girder T‐STAR S_7HS_7H stapled peptide 39 internalised in cells. E) The native T‐STAR peptide 26 showed no internalisation. F) DMSO negative control. Microscopy images were acquired with a custom‐built multi‐modal microscope setup. Fluorescein excitation was conducted at 495 nm. Scale bars=40 μm. Images were processed using MetaMorph software.

Figure 6

Solid‐state Raman spectra of A) diyne‐girder stapled T‐STAR S₇S₇ 41. The diyne functionality gives a peak at ∼2,255 cm⁻¹, in the cell‐silent region and B) the acetylated native T‐STAR 40 showing the absence of the diyne functionality peak.