Protein and peptide engineering for chemical exchange saturation transfer imaging in the age of synthetic biology

At the beginning of the millennium, the first chemical exchange saturation transfer (CEST) contrast agents were bio‐organic molecules. However, later, metal‐based CEST agents (paraCEST agents) took center stage. This did not last too long as paraCEST agents showed limited translational potential. By contrast, the CEST field gradually became dominated by metal‐free CEST agents. One branch of research stemming from the original work by van Zijl and colleagues is the development of CEST agents based on polypeptides. Indeed, in the last 2 decades, tremendous progress has been achieved in this field. This includes the design of novel peptides as biosensors, genetically encoded recombinant as well as synthetic reporters. This was a result of extensive characterization and elucidation of the theoretical requirements for rational designing and engineering of such agents. Here, we provide an extensive overview of the evolution of more precise protein‐based CEST agents, review the rationalization of enzyme‐substrate pairs as CEST contrast enhancers, discuss the theoretical considerations to improve peptide selectivity, specificity and enhance CEST contrast. Moreover, we discuss the strong influence of synthetic biology on the development of the next generation of protein‐based CEST contrast agents. Chemical exchange saturation transfer (CEST) contrast agents are different from other magnetic resonance imaging (MRI) agents as they do not require inorganic atoms to produce contrast. This opened up a wide range of possibilities to use peptides, proteins, and enzymes as agents. Moreover, those CEST agents can be encoded by DNA and as such can be genetically expressed in cells. This paved a new way for diagnostic agents that allow noninvasive tracking of gene delivery and stem and immune cell therapies.