Poster Presentation 6th Modern Solid Phase Peptide Synthesis & Its Applications Symposium 2017

On-resin introduction of a new fluorescent peptide constraint  (#43)

Aimee Horsfall 1 2 , Kate Wegener 1 , Kelly Keeling 1 , Andrew Abell 1 2
  1. University of Adelaide, Adelaide, SA, Australia
  2. The ARC Centre of Excellence for Nanoscale & Biophotonics, Adelaide, South Australia, Australia

Protein-protein interactions are defined by interfacial secondary structural motifs that impart a high degree of selectivity. Peptides can be designed to bind at these sites, though they are invariably unstructured, a shortcoming addressed by introducing a covalent linker to define the required geometry. Literature constraints, including those introduced by metathesis, lactamisation and ‘click’ chemistry, are non-fluorescent and thus cannot be imaged directly. This property must be introduced separately. Here we repurpose a protein cross-linker, dibromobimane, as a peptide constraint that is efficiently introduced on-resin by reaction with cysteine side-chains to define secondary structure and impart intrinsic fluorescence. 

Ac-CAC-Am, Ac-CAAAC-Am and Ac-CRAAARAC-Am were assembled on Rink Amide resin from Fmoc-Cys(Mmt)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Ala-OH under standard SPPS conditions. Resin-bound peptide was reacted with 2% TFA in DCM to remove the 2-methoxytrityl (Mmt) groups. Dibromobimane (2 equiv) and DIPEA (4 equiv) in DMF were added and after three hours the i-i+2, i-i+4 or i-i+7 cyclised peptides were isolated without evidence of linear peptide. Cyclised peptides were also obtained by solution-phase reaction of crude, fully deprotected peptides with dibromobimane in PBS. At least 10% trifluoroethanol (TFE) was required in these reactions to give the desired i-i+7 and i-i+8 cyclised products, without which many inter-peptide reactions were observed. In contrast, only desired product was observed for on-resin i-i+7 cyclisation without addition of TFE. This suggests on-resin assembly may be preferable for larger macrocycles. The secondary structure of the peptides was assessed by NMR and CD analysis to reveal helical structure for the i-i+7 constrained peptide. Constrained peptide could be detected at 10 nM via plate photometry with an excitation wavelength of 385 nm and emission at 477 nm. This new fluorescent peptide linker can be introduced both on- and off-resin and provides access to new constrained peptides that do not require further functionalization for imaging.

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