Natural peptides produced by living organisms are typically linear chains of amino acids, like train carriages on a track. A new class of synthetic peptides called peptide macrocycles is more like a rollercoaster: loops of amino acids are custom built with specific shapes, sizes and chemical properties.
“They’re a growing family of designer binding molecules not found in nature,” explained Zachary Gates, a Senior Scientist jointly appointed between A*STAR’s Institute of Molecular and Cell Biology (IMCB) and Institute of Sustainability for Chemicals, Energy and Environment (ISCE2).
Gates highlighted that their unique ring shapes enable them to connect with disease-related targets with high precision, making them potentially effective drug modalities for difficult-to-treat diseases such as cancer and infections.
Sequencing synthetic peptide macrocycles is essential for understanding and harnessing their biological functions, but requires innovative techniques to tackle the challenges presented by their cyclic structures.
Conventional sequencing methods like mass spectrometry break the bonds between amino acids and then measure the mass of the resulting fragments to deduce the peptide sequence. “However, for peptide macrocycles, breaking any one bond regardless of location leads to linearised ions of identical mass and hence no sequence information,” remarked Zhi’ang Chen, first author on the study.
Gates and colleagues theorised that a molecular snipping technique, called chemical linearisation, can facilitate the sequencing of even complex synthetic peptide macrocycle mixtures.
“Edman chemistry, a method to cleave the peptide at one end, efficiently converts ring structures into linear ones,” clarified Gates, adding that they applied this method to a unique group of synthetic macrocycles in which two cysteine residues are connected by a specific chemical linker (m-xylene).
Their findings indicate that they can accurately sequence the linearised macrocycles, though with a marginally lower recall rate (the percentage of peptides in a mixture that are precisely sequenced) compared to linear peptides. Moreover, the researchers identified ions bonded with sodium as a significant cause of inaccurate sequence assignments, pinpointing an area for potential refinement in automated sequencing.
This strategy paves the way for sequencing synthetic macrocycle libraries, particularly those stemming from combinatorial chemistry, to enable the discovery of novel therapeutic compounds. “Now that we’ve established a proof-of-concept, we’re looking forward to creating new methods that are applicable to a wider range of macrocycles, broadening the technique’s applicability,” commented Gates.
With key pharmaceutical entities specialising in macrocycle therapeutics establishing their presence in Singapore, Gates and his colleagues are optimistic that with support from industry partners, their contributions may ultimately transition from laboratory to commercial settings.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology (IMCB) and the Institute of Sustainability for Chemicals, Energy and Environment (ISCE2).
