Advances in MRI: Peptide and peptidomimetic‐based contrast agents

Magnetic resonance imaging (MRI) is a common medical imaging technique that provides three‐dimensional body images. MRI contrast agents improve image contrast by raising the rate of water proton relaxation in specific tissues. Peptides and peptidomimetics act as scaffolds for MRI imaging agents because of their increased size and offer the possibility to engine a higher hydration value within the design. The design of a new Gd‐based contrast agent must take into account high stability constants to avoid free Gd(III), with the subsequent nephrotoxicity, and high relaxivity values. This review analyzes various synthetic approaches, reports studies of relaxometric parameters, and focuses on the description and application of Gd(III)‐chelates based on peptide and peptidomimetic scaffolds. In addition, the X‐ray molecular structures of three DOTA complexes will be reported to emphasize the necessity of using the X‐ray diffraction analysis to identify the coordination sphere of the metals and the mechanism of action of the compounds.


| INTRODUCTION
Molecular imaging technologies provide non-invasive methods to visualize, characterize, and measure cellular and subcellular biological processes.Significant advantages for healthcare in terms of time and costs could be achieved through the creation of novel, more efficient drugs, and the acceleration of the selection of lead compounds. 1 essential imaging method for disease diagnosis and treatment is magnetic resonance imaging (MRI).In most cases, gadolinium-based contrast agents (GBCAs) have been found to be safe and useful for MRI enhancement. 2Gadolinium(III) is an optimal relaxation agents for its high paramagnetism (seven unpaired electrons), which gives a high magnetic moment (7.9 BM), and also its properties in terms of electronic relaxation time 10 À8 -10 À9 s are much longer in comparison with other lanthanides(III). 3nce Gd-approval DTPA's in 1988, MRI technology has grown significantly; currently, almost 50 tons of gadolinium are administered annually, and each year, the market for GBCAs exceeds one billion dollars. 4nce 1988, when the first GBCA was introduced to detect abnormalities of the blood brain barrier, these agents have undergone enormous development, and in addition to the drugs already approved by the United States Food and Drug Administration (FDA) 1-8 (Figure 1), the search for new and more efficient contrast agents is constantly expanding.
One of the key reasons GBCAs have been so successfull is the fact that they instantly provide crucial diagnostic information.In contrast to nuclear medicine, where the patient receives the radiopharmaceutical substance and must wait for imaging, MRI employs a contrast agent that is administered to the patient while they are in the scanner, and diagnostic images are shown within minutes.
The physical principles of MRI are based on observing the various distributions and properties of water in the tissue being examined, as well as the spatial variation of the proton longitudinal (T 1 ) and transversal (T 2 ) magnetic relaxation times in that tissue.The T 1 relaxation time of diamagnetic water solutions, in tissues, is typically five times longer than T 2 , and consequently, these agents have been explored extensively.From the physical point of view, T 1 -CAs induce a positive contrast, that is, a 1 H NMR signal of the affected tissue increases; compounds affecting the T 2 relaxation show a negative contrast because of lowering of a local proton signal.Historically, the chemistry of the T 1 -CAs has been investigated more extensively in comparison of the chemistry of the T 2 -CAs, and in this review, we focus on T 1 -CAs.
At present, the leading T 1 -contrast agents are complexes of paramagnetic Gd(III) metal ions, although, iron oxide nanoparticles and manganese(II) complexes have been approved for imaging, but they have not proven economically successful. 3nerally, GBCAs are coordination compounds in which the gadolinium is wrapped into a multidentate organic ligand 4 ; in particular, DOTA-like ones consist of an octadentate ligand, with one water molecule in the inner hydration sphere of Gd(III), as confirmed by X-ray structures.The interpretation of the longitudinal and transverse water proton relaxivities was given by Aime and coworkers 5 considering two Gd-DOTA complexes with a polyhydroxy(benzyloxy)propionamide substituent (9) and their two debenzylated derivates, as a result of the presence of only one water molecule in the complexes' inner coordination sphere.The X-ray molecular structure of one of these complexes corroborated their hypothesis (Figure 2).In particular, nine atoms participate in the coordination to the Gd 3+ ion, defining a ninecoordinate stereochemistry described as a distorted-square antiprism consisting of four backbone nitrogen atoms, four oxygen donor atoms attached to the side chains (three carboxylate and one carboxamide oxygen atom), capped with one water molecule (distances are reported below in Table 2).As reported by the authors, the nitrogen and oxygen atoms involved in the complexation are positioned in two nearly parallel planes separated by 2.33 Å, with the Gd 3+ ion at 1.65 Å from the plane defined by the four nitrogen atoms and 0.68 Å from the plane defined by the oxygen atoms.In this example, X-ray molecular structure of the Gd complex confirms the presence of one water molecule in the coordination sphere of the metal, as suggested by the NMR relaxation times.This water molecule, which directly interacts with the paramagnetic cation, plays a key role in the relaxivity of the contrast agent.
The "relaxivity" of T 1 -CAs indicates its capacity to increase the relaxation rate of the protons in the water surrounding it, indicating its efficiency. 6e efficiency is determined by the exchange rates between the water molecule in the inner hydration sphere and water molecules in the outer sphere (Figure 3).
Other parameters that affect relaxivity are τ M (residence time of water molecule in the coordination sphere) and τ R (rotational correlation time); large size and high molecular weight organic ligand can positively affect these parameters resulting in an increase of relaxivity. 7spite all the advantages of GBCAs, it is necessary to take into account that the gadolinium is extremely toxic as free ion.Therefore, for clinical use, it must be bound in a complex of high stability and exhibit a long-term resistance to Gd(III) ion transmetallation or transchelation.Although GBCAs were thought to be safe drugs for 18  Conventional contrast agents present notable limitations: lack of specificity toward pathological tissue, rapid clearance that determine the need of high administration doses, and toxic effects, particularly in terms of tissue accumulation.These limitations need to be overcome, and the development of new and more efficient contrast agents has been rapidly increased during the last decades.The possibility to monitor a disease progression with molecular imaging has naturally led to the to the advancement of targeted molecular imaging.With that in mind, many ligands have been developed such as proteins, antibodies, small molecules, aptamers, and peptides. 10Specifically, this review describes some examples of targeting peptides as T 1 -CAs.
Additionally, the advances of synthetic methodologies of gadolinium containing peptide and peptidomimetic-based contrast agents will be in-depthly examined in order to provide some perspectives for future developments.To the best of our knowledge, no crystal structures that concurrently hold both the peptide/peptidomimetic system and gadolinium are reported in literature.We report two crystal structures of the DOTA Ga 3+ and Y 3+ complexes, which aid in understanding their mechanisms of action, to demonstrate the significance of having structural information of these systems.PIERRI and SCHETTINI

| Peptide-targeted MRI contrast agents
Cancer is one of the most fatal disease responsible for a large number of deaths worldwide.According to GLOB-CAN 2022 estimates, 13 there were more than 19.3 million new cancer cases, leading to approximately 10 million deaths in 2020. 14e ongoing efforts for the development of drugs and therapeutics are more frequently devoted to the detection of early disease F I G U R E 1 Structures of somatostatin (33), octreotide (34), and complex octreotide-DOTA (35).signs and personalized medicine through non-invasive and convenient tests. 15e application of peptide-targeted MRI is in cancer diagnosis.
The growing interest for peptide in cancer is related to the overexpression of the peptide receptors in cancer tissues compared with normal tissue.Thus, based on the possibility of receptor targeting, several advances in medical imaging have been made using peptidetargeted MRI contrast agents. 9Given that peptides can operate as tumor-targeting ligands by preferentially binding particular receptors on the cell membrane, a tailored molecular imaging agent could be produced.The introduction of an imaging agent, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)ÀGd complex (2), ensures the possibility to follow the disease progression and monitor the therapeutic treatment (Figure 4).
In these perspectives, Li and coworkers focus their research on development and synthesis of four peptide Gd-DOTA conjugates for detection and diagnosis of prostate cancer, 16 which is the most common cancer in men in United States. 17tradomain B fibronectin (EDB-FN) has been identified as a molecular target for high-risk prostate cancer detection and diagnosis 18 because it is overexpressed in prostate cancer while is absent in normal tissues.Scheme 1 reports the synthetic route toward new MRI contrast agents.A typical synthetic procedure is described for ZD2-Gd-DOTA (15) as an example.The preparation of target peptide (10) was accomplished by solid-phase synthesis using Fmoc chemistry on a 2-chlototrytilic resin.The oligomerization is followed by the addition of a PEG spacer at the N-terminus of the peptide (11).Then the conjugation between the peptide (11) and DOTA isothiocyanate (12)   occurred affording the compound (13).The cleavage from the resin was performed using TFA (14).The final step was the complexation of the ligand (13) with gadolinium ions affording the targeted peptide-Gd-DOTA (15).
The same synthetic approach, starting from the corresponding peptide, was applied to obtain the contrast agents with GVK ( 16), IGK (17), and SGV (18) reported in Figure 5.
The relaxivities of all these four new contrast agents (15-18)   were measured at 1.5 T in water at 37 C, and corresponding values are reported in Table 1 and compared with commercially available Gd-DOTA (2) and Gd (HP-DO3A) (3).As shown in Table 1, the r 1 relaxivities of 15-18 [(4.1, 4.3, 4.6, 4.7) mM À1 s À1 ] were higher than that of 2 and 3 (2.9, 2.9, mM À1 s À1 respectively); the same trend was reported also for r 2 : The affinity for specific cellular receptors can also be used to treat cancers not only for diagnostic purposes.As example, we reported the synthesis of a somatostatin analog DOTA 0 -D-Phe 1 -Tyr 3 -octreotide (DOTATOC) starting from the synthesis of a DOTA prochelator and then coupling it to Tyr 3 -Lys 5 (BOC)octreotide. 19The following deprotection gives the final product DOTATOC with a yield of 65%.This novel macrocycle was successfully complexed with various radiometals to obtain 67  used 111 In-DTPA-octreotide.When the tumor absorption of the three complexes is compared, the data clearly reveal that the 67 Ga-DOTATOC outperforms the other two compounds.Given that the only variation between the three complexes is the coordinated metals, the only feasible explanation for the observed differences is that the different metals induce a different receptor binding affinity.To test this hypothesis, the authors successfully determined the X-ray molecular structure of two "simplified" DOTA-derivatised D-PheNH 2 19, complexed with Ga 3+ and Y 3+ confirming the differences in metal coordination.The Ga 3+ complex (19(Ga-DOTA-D-PheNH 2 )), in instance, exhibits a hexacoordinated metal with four backbone nitrogen atoms and two carboxylate oxygen atoms defining a cis-pseudooctahedral geometry (Figure 6A,B).The Ga 3+ ion almost lies on the equatorial plane defined by two nitrogen atoms and the two carboxylate oxygen atoms with a distance of 0.04 Å.Aside from the coordination of the Ga 3+ ion, the authors emphasize that the only free carboxylate oxygen atom is deprotonated at physiological pH.This consideration may explain why 67 Ga-DOTATOC is excreted more efficiently through the kidney.The Y 3+ ion complex X-ray molecular structure shows an octa-coordinated geometry involving (19(Y-DOTA-D-PheNH 2 )) the four nitrogen atoms, the three carboxylate oxygen atoms, and the carboxamide oxygen atom (Figure 6C,D).This coordination can be described as a distorted square-antiprismatic geometry, similar to the previously described Gd 3+ ion complex, but without the presence of a water molecule to complete the metal coordination sphere (important distances are reported in Table 2).The nitrogen and oxygen atoms are located in two almost parallel planes separated by 2.51 Å, and the Y 3+ ion is almost in the middle of these two planes, precisely at 1.22 Å from the oxygens plane and 1.29 Å from the nitrogens plane.These X-ray molecular structures allow to explain the peculiar features of the three different DOTA complexes.
In particular, the higher tumor uptake of the 67 Ga-DOTATOC could be attributed to the higher flexibility of the complex, which allows the rearrangement of the octapeptide in order to adapt to the cellular receptor.Furthermore, the free carboxylate group may be responsible for the increased kidney excretion of 67 Ga-DOTATOC.
Unfortunately, low-molecular weight MRI contrast agents present some limitations such as low sensitivity and extravasation.In order to overcome these pitfalls, some supramolecular and macromolecular gadolinium complexes have been developed.In particular, supramolecular peptide-based gadolium complexes have been created in order to exploit the potential of peptides as a new platform for the design of biomedical materials.
Starting from the first evidence of self-assembled nanostructure, 20 a large number of supramolecular peptide-based molecules have been developed, [21][22][23] and their combination with Gdcontaining contrast agent led to supramolecular structures like polymeric peptide-, amphiphile peptide-, and aromatic peptide contrast agents. 24e following sections aim to highlight that macromolecular structures, due to their size and their slow reorientation time, could enhance the relaxivity value in comparison with the low molecular weight GBCAs.

| Polymeric peptide MRI contrast agents
Since the first example of polymeric peptide contrast agent, a system bearing 114 ornithine residues functionalized with GdDO3A (Figure 5A), reported by Aime and coworkers, 25 a large number of In 2007, Caravan and coworkers reported the synthesis of a collagen specific cyclic peptide (Figure 7B) for molecular imaging of fibrosis.The peptide consisted of 16 amino acids; 10 of these amino acids are linked through disulfide bond, and a cyclic peptide has been made.The peptide was synthesized using standard solid-phase methods.
To increase the contrast agent's sensitivity, thiourea linkages were used to conjugate three Gd(DTPA) moieties to the peptide.Gadolinium complexes were linked directly to the peptides through a reactive isothiocyanate moiety.
The relaxivity of this contrast agent near the common imaging field of 1.5 T is five times higher than that of commercially [Gd(DTPA)] (Magnevist) ® (1) per Gd atom.Experimental data showed also high target affinity. 26milarly in 2010, Tei et al. reported the synthesis and application of a peptide conjugated with Gd-DOTAMA complex (Figure 7C).This peptide has been designed as an analog for Factor XIII transglutaminase, which is involved in various diseases, such as cancer, through a triggered coagulation pathway.The solid-phase synthesis allowed the preparation of a 16-term peptide followed by the coupling with chelating agent (DOTAMA).Experimental data showed MR signal enhancement and prolonged retention in tumors. 27Specifically, a relaxivity of 8.1 mM À1 s À1 was measured at 0.5 T and 298 K, two times higher than the commercially [Gd(DOTA)] (Dotarem) ® (2).
S C H E M E 5 Synthetic procedure for 51-54 with change in spacer length.
In 2015, Chaabane and coworkers reported an elegant example of a fibrin-targeting system made by a peptide conjugated with four Gd-DTPA-like chelates (Figure 7D).The peptide was selected using a phage display procedure, the synthesis was realized with a Fmoc solid phase strategy, and chelating agents were directly added to the peptide moiety.Experimental data showed high relaxivity (42 mM À1 s À1 , per molecule), in comparison with the commercially [Gd(DTPA)] (Magnevist) ® (1), and persistent signal enhancement in tumors, and it was successfully applied to visualize neuroblastoma tumors. 28ese few examples, over the abundance in literature, are examined to underline the efficacy of polymeric conjugate systems in overcoming the lack of specificity to target organs and tissues for diagnosis enhancing the relaxivity and specificity.

| Amphiphilic peptide MRI contrast agents
Another important class of molecules containing a peptide sequence and a gadolinium complex is represented by amphiphilic contrast agents.
Amphiphilic peptides consist of hydrophilic and hydrophobic regions.They not only have the advantages of peptides, but they also have self-assembly properties that are influenced by changes in temperature or pH. 29e first example of self-assembled peptide amphiphilic contrast agent was reported by Bull and coworkers (Figure 8A). 30C H E M E 6 Synthetic procedure for 55-58 with various chelating agents.
T A B L E 4 Relaxivities of Gd complexes RGD analogs.

PIERRI and SCHETTINI
In 2007, Vaccaro and coworkers reported a novel class of supramolecular aggregates based on a monomer with an upsilon shape (MonY).In the same molecule, there are three components: a hydrophobic moiety, the bioactive peptide for target recognition, and a Gdcomplex contrast agent (Figure 8B). 31 2012, Ghosh et al. 32 reported the synthesis of self-assemble smart material pH responsive characterized by three main segments: a hydrophobic alkyl tail, a β-sheet-forming peptide sequence, and a charged amino acid sequence (Figure 8C).

| Aromatic peptide MRI contrast agents
Poly-aromatic peptides are able to self-assemble generating various supramolecular aggregates like hydrogels, fibers, and vesicles.The first example of phenylalanine-based material for MRI application, its synthesis, and relaxivity values of different peptide Gd complex conjugates was reported by Diaferia et al. in 2015 33 (Figure 9A).
In 2016, Kim and coworkers made multifunctional theranostic nanoparticles, which could be used in personalized medicine (Figure 9B). 34re recently, in 2020, Gallo and coworkers 35 reported peptidebased gels containing the Gd-complexes.The synthesis and relaxometric behavior of two agents for MRI were reported (Figure 9C).
Scheme 2 reports the synthetic route toward peptide-based soft hydrogel.The preparation of target hydrogel containing a gadolinium complex was accomplished by solid phase synthesis.A typical synthetic procedure, as an example, is described for DOTA(Gd)-PEG8-(FY)3 (20).
The preparation of target peptide (20) was accomplished by solid-phase synthesis using Fmoc chemistry on a Rink amide MBHA resin.Each amino acid was coupled twice in presence of HOBt, PyBop, and DIPEA as activating agents affording the compound 21.
After each step, the Fmoc group was properly removed.Then the conjugation between the peptide 25 and Fmoc-AdOO-OH, oxoethylene spacer 26, occurred affording the compound 27.Then the coupling with the chelating agent DOTA(OtBu) 3 28 was performed affording the compound 29.The cleavage from the resin was performed using TFA/TIS/H 2 O leading the desired peptide 30.The complexation with GdCl 3 furnished the desired Gd-complex 20.
The same synthetic approach, starting from the corresponding peptide, was applied to obtain the contrast agents DTPA-PEG8-(FY)3 31 reported in Figure 10.
Both conjugates led the formation of hydrogels as a consequence of molecular self-organization into fibrillary nanostructures.
The relaxivities of two self-assembled contrast agents (20, 31)   were measured at 0.5 T, 298 K, and corresponding values are 12.1 and 12.0 mM À1 s À1 for 20 and 31, respectively.These values are really high in comparison with the commercially available contrast agents (4-5 mM À1 s À1 ).The high relaxivity values are affected by the presence of PEG spacer.This spacer is able to prevent the unfavorable steric and electronic interactions between chelating agents ensuring, simultaneously, the peptide structural organization as β-sheet self-assembled structures.
S C H E M E 7 Synthetic procedure for peptoid-based rare-earth complexes transporter.

S C H E M E 8 OBOC library synthesis.
F I G U R E 1 4 Ten primary amines on a filter plate.
This study underlines the potential of molecules that can form hydrogels for the development of peptide-based containing Gdcomplexes as injectable MRI contrast agents.

| Somatostatin analogs as peptide-based contrast agents: The first crystal structure
More recently, in 2022, Diusenova and coworkers have reported the first X-ray molecular structure of the DOTA-Phe-D-Trp-Lys-Thr-OMe (32). 36e pharmacophore sequence Phe-D-Trp-Lys-Thr, present in somatostatin (33), is responsible of specific binding with somatostatin receptors, which are overexpressed in tumor cells.The development of somatostatin analogs has been rapidly increasing during the last decades due to therapeutic applications such as the treatment of hormone-active tumors. 37Octreotide ( 34) is a cyclic octapeptide containing a disulfide bridge and was the first somatostatin analog used in clinical protocol.Generally, octreotide-derived compound contains a metal chelating moiety linear or cyclic polynitrogen structure such as DOTA; these complexes were successfully applied in clinical diagnosis and therapy if neuroendocrine tumors. 38One of the most important S C H E M E 9 Peptoid-DOTA OBOC library synthesis.
somatostatin analogs belonging to the diagnostic field is the complex octreotide-DOTA (35) (Figure 11). 39udying the X-ray molecular structure of these analogs represents an important starting point to understand the relationship between structure and activity and design novel peptide inhibitors.
The authors reported for the first time the X-ray molecular structure of somatostatin analog 32 (Figure 12).The X-ray molecular structure features an "open" conformation for the peptide chain that adopts a β-hairpin structure, like octreotide, upon complexation.
F I G U R E 1 5 Two water-soluble hexacyclic peptoids.
S C H E M E 1 0 Solid-phase synthesis using a monomer approach.

| Peptidomimetic-targeted MRI contrast agents
Many research groups have created integrin ligands based on linear or cyclic peptidomimetics to enhance MRI for cancer diagnosis. 40Peptidomimetics can be described as mimic of peptides with improved properties such as metabolic stability and high selectivity facilitating the cancer diagnosis. 41A new family of RGD (arginine-glycineaspartic acid) peptidomimetics having a bifunctional diketopiperazine (DKP) scaffold with integrin affinity has caught the interest of different research groups. 42tegrins are a family of transmembrane glycoproteins belonging to a group of cell adhesion molecules playing an important role in tumor and metastasis.In particular, α v β 3 integrin is directly involved in cancer progression, metastasis, and proliferation as it is able to bind to extracellular matrix proteins via the three amino acid sequence arginine-glycine-aspartic acid (RGD).Recently, different α v β 3 integrin inhibitors have been developed employing RGD-peptides, -peptidomimetics, or -antibodies. 43th the aim to overcome the poor stability in vivo of peptides, some researchers have been developed RGD-peptidomimetic scaffolds also useful for MRI cancer diagnosis.Kim and coworkers 44 described the synthesis of a peptidomimetic α v β 3 integrin antagonist bearing a Gd-DOTA chelating agent and The relaxivity of Gd(III)-DOTA-IAC was measured at 1.0 T at room temperature and compared with commercially Gd-DOTA (2); the corresponding values are reported in Table 3.These results demonstrated an increased relaxivity value for 42 and also an increase in Gd(III) content per cell.
Another important example of peptidomimetic-based α v β 3 integrin antagonist has been reported by Manzoni and collaborators. 45The synthesis and conjugation with an RGD sequence followed by specific tests revealed the efficiency of these novel molecules as molecular imaging probes in cancer diagnosis by MRI.Furthermore, they have performed a screening of different parameters: bifunctional derivatives of chelating agents (Figure 13A) and various spacer (Figure 13B) in order to evaluate the distance between Gd and RGD moiety.
The synthesis started from the azide (49), derived from the peptidomimetic ligand DB58, hydrogenation with palladium on carbon afforded the corresponding amine (50).The coupling with (HBTU, HOBt, DIPEA, DMF) gave the corresponding amide.For compound 51, the chelating agent was directly conjugated, while in compounds 52-54, a spacer was introduced, varying units of ethylene glycol (Scheme 5).
The authors also evaluated the effect of various chelating agents: Following the same synthetic scheme, they realized a DOTA derivative ( 55), an HP-DO3A derivative (56), and AAZTA derivatives (52-53) (Scheme 6).The final step was the complexation with Gd(III) in order to obtain the corresponding Gd-chelated compounds.After the binding affinity evaluation toward α v β 3 , the relaxivity of Gd-chelated RGD was measured.Table 4 shows that, due to their high molecular weight of Gd-chelated RGD, relaxivities were higher in comparison with the corresponding precursor Gd-DTPA (1).The higher values for compounds 57 and 58 are due to the coordination of two water molecules instead of one.
S C H E M E 1 1 Solid-phase synthesis using a monomer/sub-monomer approach.
These explorations confirm that it is possible to modulate the binding affinity for integrins and to improve relaxivity values changing the chemical structures of Gd-chelated agents.All these observations are fundamental to develop a clinically applicable integrin Gd-chelated probe.

| Peptoids as MRI contrast agents
Peptoids belong to the class of peptidomimetics; they are Nsubstituted glycine oligomers, in which the side chain is directly attached on the nitrogen atom instead on the α-carbon as in peptide. 46This important feature is responsible of an increased conformational flexibility 47 and the impossibility to form backbone hydrogen bonds that stabilize the secondary structures in peptides. 48ring the last two decades, the number of peptoids structures obtained by nuclear magnetic resonance (NMR) analysis or X-ray characterization has undergone rapid expansion, which led to the necessity of the creation of a structural database: The Peptoid Data Bank. 49e adaptability of peptoids, alongside their expansive applications, has ensured their application in various research fields 50 such as catalysis, 51,52 metal chelation, 53,54 detection of human diseases like Alzheimer 55 or Parkinson, 56 as therapeutic in cancer treatment 57 and so on.the synthesis of a combined system: DTPA ligand (1) with molecular transporters based on peptoids. 58e synthesis was carried out on solid supports using the monomer method: The monomer (59) was immobilized on a Fmocdeprotected Sieber resin using N,N 0 -diisopropylcarbodiimide (DIC) and HOBt in a microwave-assisted reaction.The iteration of this steps furnished the hexameric adduct (60).The DTPA was coupled using the anhydride (61) affording the product 62.The final cleavage with TFA afforded the desired compound 63 (Scheme 7).The complexation with various rare-earth, including gadolinium, was performed.These complexes showed low toxicity and were cell penetrable.
These peptoid-based transporters are potentially useful for MRI applications.
Napolitano and coworkers, in 2011, 59 reported a rapid and economic on-bead combinatorial synthesis (OBOC) of a library of peptoid-based DOTA derivatives.
This technique allowed a rapid screening of various imaging agents and furnished a way to recognize the most sensitive contrast agent.
After Fmoc deprotection, the peptoid portion was created using a microwave assisted reaction which furnished the product 65.Then, the coupling of Fmoc-β-Ala-OH gave the compound 66.The addition of (DO3A)-tris(tert-butyl ester) and the subsequent deprotection reaction led to the formation of 67.The treatment with N-Boc-1,-2-diaminoethane, HBTU, and NMM gave the adduct 68.After an acylation reaction with bromoacetic acid, the beads were distributed into a 96-Acrowell filter plate (Figure 14).
The first 8 amines were added to each row of the plate, then the addition of DIC and bromoacetic acid furnished the adduct 69.This step was followed by the addition of 10 amines to each column of the plate followed by the final complexation with EuCl 3 led the formation of the corresponding metal complex 70.All the amines used in this procedure are reported in Figure 14 (Scheme 8).
This combinatorial approach guaranteed many combinations to create various library of MRI contrast agent.
Later, in 2016, Singh and collaborators 60 used the same OBOC approach to synthesize a new library of peptoid-DOTA derivatives; this time, each peptoid possesses a therapeutic component.In this case, the library has been realized to identify the HIT compound.Considering the straightforward power of peptoidic synthesis, a general synthetic strategy has been developed (Scheme 9).
F I G U R E 1 6 3D representation of gadolinium complex with q = 3. Atom type: C gray, N blue, O red, and Gd magenta including three molecules of H 2 O. • B group: two consecutive Fmoc-AEEAc-OH additions and then all the steps described for A group (Scheme 9).
• C group: three consecutive Fmoc-AEEAc-OH additions and then all the steps described for A group (Scheme 9).
This easy on-bead combinatorial synthesis furnished a library of 153 600 new compounds using 10 amines and the split-pool strategy.
Each peptoid region can act as a therapeutic unit while the DOTA portion can chelate various metal for MRI (like Eu(III) or Gd(III)) or for PET ( 64 Cu, 68 Ga).
Those unique linear tripeptoid-DOTA derivatives allow a platform of new theranostic agent that permits a deeper set of different experiments.

| Macrocyclic peptoids as MRI contrast agents
Making macrocyclic peptoids is one way to reduce the conformational flexibility. 61Macrocyclic compounds, due to secondary structures stability, possess enhanced biomimetic features and can act as antimicrobials, cytotoxic agents, siderophores, glycosidases inhibitors, and so on. 62king advantage of the sub-monomeric approach of solid-phase synthesis in 2014, de Cola and coworkers 63 reported two novel cyclic hexapeptoids, 74 and 75, bearing, respectively, able to complex Gd(III) ions (Figure 15).
Starting from the preparation of Fmoc-N-(tert-butyloxycarbonylethyl)glycine (76) a solid phase synthesis was performed following a Small changes were necessary to synthesize the hexacyclic peptoid 75.A mixed monomeric/sub-monomeric approach was carried out as reported in Scheme 11.
In particular, starting from compound 78, the addition of bromoacetic acid followed by a coupling with 2-methoxyethylamine were necessary to achieve 83.
Their relaxivities resulted to be significantly higher in comparison with the commercial Gd(III) complex applied in clinical practice.
More recently, the same group reported an interesting study based on water-soluble cyclic peptoids. 64For the first time, the synthesis of chiral tetra-, hexa-, and octa-, water-soluble cyclic peptoids was reported (Figure 17).The complexation with Gd(III) ions and investigation of relaxivity properties were carried out on the cyclic hexapeptoids (89, 91, 93).
The synthetic approach was based on a Fmoc chemistry using the 2-chlorotrytilic resin as described previously.A mixed sub-monomeric approach was applied to achieve the linear peptoids incorporating L-alanine tert-butyl ester as the chiral unit and methylamine (for linear precursors of 88, 89, 90).The introduction of L-and D-alanine as benzyl esters guaranteed the synthesis of the corresponding linear oligomers of 91-94.
The complexation of 89, 91, and 93 was performed with GdCl 3 , and the corresponding relaxivity values are reported in Table 6.For all the three complexes, two water molecules were found in the inner sphere; this lower hydration number can justify the lowering of relaxivity values compared with what was previously reported. 63However, such systems in any case prove to be more efficient than the corresponding trade contrast agents.
These studies demonstrated that peptoid macrocycles are able to act as metal chelators, and it is possible to design and tune their conformational feature with the aim to enable future applications as MRI contrast agents.

| CONCLUSIONS
In this review, we have reported the most recent developments in peptide-and peptidomimetic-based contrast agent for MRI application.
The extensive use of peptide-based systems is well demonstrated by the huge number of studies in the literature and by the presence of X-ray structures; whereas, we emphasized the lack of this type of investigations in the peptidomimetics field.In the future, there will be more ligands directed toward other targets and incorporated into formulations of new complex conjugated contrast agents.
Considering the enormous diagnostic-therapeutic potentials, market values, we expect peptides and peptodomimetics-based contrast agent will affirm a long-term success in the field of MRI.Many efforts have been made to replace Gd with other metals that are less toxic, but, to date, only two manganese-based contrast agents have been approved for clinical applications by FDA: Mn-DPDP (Teslascan) and a liposomal encapsulated manganese chloride (LumenHance).Mn-based contrast agents are important candidates for replacing the gadolininium ions.
We firmly believe that the availability of X-ray molecular structures of peptide-and peptidomimetic-based contrast agents can give insights into structure-activity relationships fundamental to improve diagnostic and therapeutic tools.
Furthermore, in the era of personalized medicine, the synthesis of novel contrast agents should focus on tumor targeting issues; with new research and development leading to the discovery of more effective systems for a wide range of biological applications, as well as the possibility of engineering a higher hydration value within the design, the future of peptidomimemtic-based MRI contrast agents holds enormous promise.
This review highlights that the clinic applications of peptide-and peptidomimetic-based contrast agents require an appropriate and extensive knowledge of their structure-functional relationships, via crystallography studies, in order to expand the scope of research into this field.
years, in 2006, 8 the FDA identified a correlation between the administration of GBCAs and nephrogenic systemic fibrosis (NSF) for patients with severe kidney disease, and the long-term clinical effects are still under investigations considering restrictions or suspensions imposed by regulatory agencies (e.g., EMA/625317/2017).This potentially fatal condition, together with recent evidences that F I G U R E 1 United States Food and Drug Administration (FDA) approved gadolinium-based contrast agents (GBCAs) (NMG = meglumine).F I G U R E 2 Two different views of 9 (Gd-DOTA) complex reported by Aime. 5 For clarity, hydrogen atoms were not reported, and only the atoms with the highest occupancy factor are shown.Atom types: C gray, N blue, O red, and Gd light cyan.F I G U R E 3 Schematic representation of factors influencing relaxivity in gadolinium-based contrast agents.F I G U R E 4 Schematic representation of targeted molecular imaging agent.S C H E M E 1 Typical synthesis of a targeted contrast agent.gadolinium remaining in patients' bodies, including the brain, for months to years, 9 prompts the research toward alternative paramagnetic ion or different chelating agents.

T A B L E 2 12 F
Selected bond distances (Å) for 9(Gd-DOTA), 19(Ga-DOTA-D-PheNH 2 ), and 37(Y-DOTA-D-PheNH 2 ).Peptides are chains of several amino acids linked with amide bonds characterized by a relatively low molecular weight and less than 40 amino acid residues according to the FDA definition.Peptides have a number of advantages due to their size and their structure.In fact, the rapid growth of peptide-based contrast agents and the implementation of new technologies have provided new agents with improved metabolic stability, favorable pharmacokinetics, enhanced binding affinity and selectivity, improved imaging ability, and biosafety. 11In particular, the research on peptides is mainly focused on two different areas: the identification of peptides overexpressed in tumor cells and the development for potential clinical or diagnostic applications.I G U R E 7 Examples of polymeric peptide contrast agents.F I G U R E 8 Examples of amphiphilic peptide contrast agents.F I G U R E 9 Examples of aromatic peptide contrast agents.

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C H E M E Synthesis of DOTA-Phe-D-Trp-Lys-Thr-OMe.F I G U R E 2 DOTA-Phe-D-Trp-Lys-Thr-OMe X-ray molecular structure.For clarity, only the atoms with the highest occupancy factor are shown, and water molecules are omitted.
15-18 [(4.8, 5.0, 5.2, 5.6) mM À1 s À1 ] in comparison with 2 and 3 (3.2,3.2 mM À1 s À1 , respectively).The high relaxivity value in combination with a prolonged signal enhancement in the mouse prostate cancer model revealed the potential application of these targeted peptide-DOTA conjugate as accurate agents for detection and diagnosis with specific bind to different sites of EDB-FN.This example demonstrates unequivocally how the relaxivity of the contrast agent increases as the size of the peptide system increases in comparison with the low-molecular contrast agent such as 2.
Ga-DOTATOC,   111   In-DOTATOC, and 90 Y-DOTATOC.The biodistribution and affinity of these three novel complexes to the somatostatin-receptor were evaluated in vivo in nude mice with the rat pancreatic AR4-2J tumor.Surprisingly, all three complexes have higher tumor uptake and lower kidney retention when compared with the well-known and widely S C H E M E 4 Synthetic procedure for Gd(III)-DOTA-IAC.
Structures of chelating agent derivatives; (B) spacer groups.T A B L E 3 Relaxivities and Gd(III) per cell.Contrast agent r 1 [mM À1 s À1 ] fg Gd(III)/cell 42 (Gd(III)-DOTA-IAC) agents have been investigated.Despite the great number of data present in literature, we decided to select some examples of peptide bearing MRI contrast agents with targeting of specific cellular receptor as depicted in Figure 7.
The development of potentially therapeutic compounds with an imaging component has received interest in recent years, but the number of examples is still limited.Kӧlmel and collaborators reported

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Relaxivities of Gd-binding cyclic hexapeptoids.Contrast agent r 1 [mM À1 s À1 ] solid phase synthesis has been already described in Scheme 8. Starting from compound 68, Tentagel beads were swelled in DMF and split into three groups A, B, and C. • A group: Coupling with Fmoc-AEEAc-OH, HOBt, and DIC followed by Fmoc deprotection gave the compound 71.Subsequently coupling with bromoacetic acid and DIC, followed by Boc-Nlys.The beads were divided into 10 vessels, and 10 different amines were added (Scheme 9).
Chemical structures of targeted contrast agents.Two different views of 19 (Ga-DOTA-D-PheNH 2 ) (A, B) and 19(Y-DOTA-D-PheNH 2 ) (C, D).For clarity, hydrogen atoms and water molecules were not reported.Atom types: C gray, N blue, O red, Ga light pink, and Y orange.