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1. ARCHE-NOAH: NMR supersequence with five different CEST experiments for studying protein conformational dynamics

   https://doi.org/10.1039/D3CP01580G  

   Cabrera_Allpas, Rodrigo; Hansen, Alexandar L; Brüschweiler, Rafael (June 2023, Physical Chemistry Chemical Physics)

   An NMR NOAH-supersequence is presented consisting of five CEST experiments for studying protein backbone and side-chain dynamics by¹⁵N-CEST, carbonyl-¹³CO-CEST, aromatic-¹³C_ar-CEST,¹³C_α-CEST, and methyl-¹³C_met-CEST. 

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

   https://doi.org/10.1002/nbm.4712  

   Gilad, Assaf_A; Bar‐Shir, Amnon; Bricco, Alexander_R; Mohanta, Zinia; McMahon, Michael_T (February 2022, NMR in Biomedicine)

   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. 

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3. Development of a synthetic biosensor for chemical exchange MRI utilizing in silico optimized peptides

   https://doi.org/10.1002/nbm.5007  

   Fillion, Adam_J; Bricco, Alexander_R; Lee, Harvey_D; Korenchan, David_E; Farrar, Christian_T; Gilad, Assaf_A (July 2023, NMR in Biomedicine)

   Abstract Chemical exchange saturation transfer (CEST) MRI has been identified as a novel alternative to classical diagnostic imaging. Over the last several decades, many studies have been conducted to determine possible CEST agents, such as endogenously expressed compounds or proteins, that can be utilized to produce contrast with minimally invasive procedures and reduced or non‐existent levels of toxicity. In recent years there has been an increased interest in the generation of genetically engineered CEST contrast agents, typically based on existing proteins with CEST contrast or modified to produce CEST contrast. We have developed an in silico method for the evolution of peptide sequences to optimize CEST contrast and showed that these peptides could be combined to create de novo biosensors for CEST MRI. A single protein, superCESTide, was designed to be 198 amino acids. SuperCESTide was expressed inE. coliand purified with size exclusion chromatography. The magnetic transfer ratio asymmetry generated by superCESTide was comparable to levels seen in previous CEST reporters, such as protamine sulfate (salmon protamine) and human protamine. These data show that novel peptides with sequences optimized in silico for CEST contrast that utilize a more comprehensive range of amino acids can still produce contrast when assembled into protein units expressed in complex living environments. 

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4. Co( ii ) complexes of tetraazamacrocycles appended with amide or hydroxypropyl groups as paraCEST agents

   https://doi.org/10.1039/d3dt01572f  

   Raymond, Jaclyn J.; Abozeid, Samira M.; Sokolow, Gregory E.; Bond, Christopher J.; Yap, Constance E.; Nazarenko, Alexander Y.; Morrow, Janet R. (July 2023, Dalton Transactions)

   Co( ii ) complexes of 1,4,7,10-tetraazacyclododecane (CYCLEN) or 1,4,8,11-tetraazacyclotetradecane (CYCLAM) with 2-hydroxypropyl or carbamoylmethyl (amide) pendants are studied with the goal of developing paramagnetic chemical exchange saturation transfer (paraCEST) agents. Single-crystal X-ray diffraction studies show that two of the coordination cations with hexadentate ligands, [Co(DHP)] 2+ and [Co(BABC)] 2+ , form six-coordinate complexes; whereas two CYCLEN-based complexes with potentially octadentate ligands, [Co(THP)] 2+ and [Co(HPAC)] 2+ , are seven-coordinate with only three of the four pendant groups bound to the metal center. 1 H NMR spectra of these complexes suggest that the six-coordinate complexes are present as a single isomer in aqueous solution. For the complexes which are seven-coordinate in the solid state, one is highly fluxional in aqueous solution on the NMR time scale ([Co(HPAC)] 2+ ), whereas the NMR spectrum of [Co(THP)] 2+ is consistent with an eight-coordinate complex with all pendants bound. Co( ii ) complexes of CYCLEN derivatives show CEST effects of low intensity that are assigned to NH or OH groups of the pendants. One complex, [Co(DHP)] 2+ , shows a highly-shifted CEST peak at 113 ppm versus bulk water, attributed to OH protons. However, the CEST effect is largest for two Co( ii ) CYCLAM-based complexes with coordinated amide groups that undergo NH proton exchange. All five complexes are inert towards dissociation in buffered solutions containing carbonate and phosphate and towards trans-metalation by excess Zn( ii ). These data give insight into the production of an intense CEST effect for tetraazamacrocyclic complexes with pendant groups containing NH or OH exchangeable protons. The intense and highly shifted CEST peak(s) of the CYCLAM-based complexes suggest that they are promising for further development as paraCEST agents. 

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5. Co(II) Macrocyclic Complexes with Amide‐Glycinate Pendants as ParaCEST and Liposomal CEST Agents

   https://doi.org/10.1002/chem.202401638  

   Raymond, Jaclyn_J; Chowdhury, Md_Saiful_I; Crawley, Matthew_R; Morrow, Janet_R (July 2024, Chemistry – A European Journal)

   Abstract Macrocyclic Co(II) complexes with appended amide‐glycinate groups were prepared to develop paramagnetic Co(II) chemical exchange saturation transfer (CEST) agents of reduced overall charge. Complexes with reduced charge and lowered osmolarity are important for their loading into liposomes and to provide complexes that are highly water soluble and well tolerated in animals. Co(L1) has two non‐coordinating benzyl groups and two amide‐glycinate pendants, whereas Co(L2) has two unsubstituted amide pendants and two amide‐glycinate pendants on cyclam (1,4,8,11‐tetraazacyclododecane). The¹H NMR spectrum of Co(L1) is consistent with a singlecis‐pendant isomer with both amide protons in thetrans‐configuration, as supported by an X‐ray crystal structure. Co(L2) has a mixture of different isomers in solution, including thetrans‐1,4 and 1,8 pendant isomers. The Z‐spectrum of Co(L1) shows one highly‐shifted CEST peak, whereas Co(L2) exhibits six CEST peaks. Encapsulation of 40 mM Co(L1) in a liposome with osmotically‐induced shrinking at 300 mOsm/L produces a liposomal CEST agent with saturation frequency offset of 3 ppm. Addition of the amphiphilic 1,4,7‐triazacyclononane‐based complex Co(L5) to the liposomal bilayer at 18 mM with Co(L1) encapsulated in the liposome at 50 mM changes the sign and increases the magnitude of the saturation frequency offset to −7.5 ppm at 300 mOsm/L. 

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