**4.2 Radiopeptides**

In clinical practice, radiolabeled receptor ligands are used routinely for diagnostic imaging of overexpressed receptors and PRRT. Causes of clinical success of radiopeptidic receptor ligands are the following:


The radioiodine which is used for radiolabeling of biologically active molecules is frequently preferred in PET imaging (124I), SPECT imaging (123/125I), treatment of different cancer types (131I), and biodistribution and kinetic investigation of novel peptide radiopharmaceuticals (125I). Direct radioiodination is based on the substitution of an aromatic proton with \*I<sup>+</sup> (electrophilic radioiodide) and is successful only in electron-rich aromatic systems including activating substituents such as ▬OH, ▬NH2, ▬OR, ▬NHCOR, or ▬SR. For in situ production of \*I<sup>+</sup> , chloramine-T (sodium tosylchloroamide) and iodogen (1, 3, 4, 6-tetrachloro-3a, 6a-diphenylglycoluril) are utilized. Chloramine-T is added to the reaction medium with sodium metabisulfite to terminate the labeling reaction and prevent oxidative damage. Oxidative enzymes (lactoperoxidase) are used for substitution of peptide sequences that have high sensitivity to oxidation. Tyr- and also His-containing peptides are readily radioiodinated in buffers such as phosphate buffered saline (PBS) or Tris (hydroxymethyl)-aminomethane (TRIS) at pH 7–8. Pre-radioiodination should be carried out by prosthetic groups. Selective prosthetic group conjugation is provided to the thiols with the help of pre-radioiodinated maleimides, and prelabeling of the corresponding peptide is carried out by the use of stannylated vinyl alkylating agents. In this way, tissue deiodinase and unfavorable structural conditions for radioiodination can be overcome [90].

**15**

*Synthesis and Applications of Synthetic Peptides DOI: http://dx.doi.org/10.5772/intechopen.85486*

Complex biomolecules such as peptides or proteins cannot be directly labeled with a highly basic [18F] fluoride by nucleophilic substitution and cannot tolerate labeling conditions. Activated aromatic precursors (NO2, CN, CI, etc.) are substituents bound to the leaving group in the ortho- or para-position. Many receptor binding peptides such as octreotide, bombesin, neurotensin, and RGD analogues have been labeled using [18F]FP-NP(4-nitrophenyl-2-[18F]fluoropropionate) or [18F]SFB(N-succinimidyl-4-[18F]fluorobenzoate) [91]. Chemoselective strategies provide a one-step prosthetic group labeling reaction by unprotected precursors. Reaction of a 18F-labeled aldehyde with aminooxy- or hydrazino-functionalized peptides so-called click chemistry has recently found most popular application [89]. A suitable chelating agent is required for the radio metallization of the peptides. When a chelating agent is conjugated with a receptor binding peptide, it can affect both the binding affinity of the peptidic ligand and peptide pharmacokinetics [89].

out by using peptide-bound tetradentate 99Tcm chelators (N3S or N4 scaffold). The peptides are coordinated with donor groups such as amine, carboxylate, or hydroxyl of the HYNIC (hydrazinonicotinic acid) chelator. In order to initiate the labeling reaction, a generator eluate (99Tcm-saline) should be added into a vial containing

boronocarbonate, Na2[HBCO2] (which also serves as an in situ CO source); and stannous ion, SnCI2 (reducing agent) are used [89]. 68Gallium labeling reaction is initiated by eluting the 68Ge/68Ga generator using hydrochloric acid (HCl) (0.05–0.60 M). Approximately 2 mL of the eluent is transferred to the reaction vial. Reaction vial containing a mixture of the lyophilized DOTA-peptide conjugate and sodium acetate buffer with the eluent is heated for 15 minutes at 100°C (25 min at 90–95°C). The intermediate product is pushed through an extraction cartridge. The

final product is analyzed to determine the labeling efficacy and purity [92].

The selective permeability and hydrophobic profile of the cellular membranes provide strict control of the molecular changes between the cytosol and the extracellular environment [93–95]. Generally, peptides are selective and effective signaling molecules which bind to specific cell surface receptors that are involved in physiological mechanisms such as peptides, hormones, neurotransmitters, growth factors, G-cell receptors (GPCRs), and ion channel ligands [96]. This characteristic of the peptides mentioned above and their attractive pharmacological profile represent a new starting point in the redesign and in-cell recruitment of molecules for therapeutic purposes [93–97]. Prior to the discovery of cell-penetrating peptides (CPPs), various methods have been used for cellular uptake of therapeutic agents and drugs, such as microinjection, electroporation, and liposome- and viral-based vectors, but these have disadvantages such as restricted bioavailability, low productivity, high toxicity, and low specificity [95]. After all these developments, in the late 1980s, a group of short peptides, such as the protein translocation site, membrane translocation sequence, Trojan peptide, or most commonly CPP, which serve as cellular uptake and delivery vectors of large molecules for therapeutic purposes, were identified [94, 96].

CPPs are mostly defined as the short (containing less than 40 amino acid residues)

partially hydrophobic and/or polybasic natural and synthetic peptides [94, 97]. With the discovery of CPPs, it has emerged as a new tool that allows cell membrane

**5. Cell-penetrating peptides as molecular carries**

**5.1 Definition and classification of CPP**

+ ,TcO+

) is carried

; disodium

+

Radiolabeling of peptides with the oxo-technetium ions (TcO2

all the mixtures. For the labeling with 99Tcm, the [99Tcm(CO)3(H2O)3]

*Synthesis and Applications of Synthetic Peptides DOI: http://dx.doi.org/10.5772/intechopen.85486*

*Peptide Synthesis*

patients [72].

**4.2 Radiopeptides**

ratios.

radiopeptidic receptor ligands are the following:

clinical purposes (SPECT, PET, PRRT).

the substitution of an aromatic proton with \*I<sup>+</sup>

tions for radioiodination can be overcome [90].

*4.1.3.5 αvβ3 integrin-targeting peptides*

αvβ3 integrins are a transmembrane protein that can be expressed in proliferative endothelial cells and overexpressed in newly formed blood vessels where tumors are fed. The arginine-glycine-aspartic acid (RGD) tripeptide is essential for the interaction of extracellular matrix proteins to αvβ3 receptors. The cyclic RGD analogue containing these amino acids has the highest binding affinity. Many radiolabeled DTPA and DOTA-RGD conjugates with 111In, 90Y, 177Lu, 68Ga, and 64Cu which provide SPECT and PET imaging and PRRT have been discovered in recent years. Monomeric, dimeric, and tetrameric RGD peptides are bound to DOTA for developing receptor binding affinity and then radiolabeled with 111In. Although the monomeric and dimeric analogues have higher in vitro receptor affinity, the in vivo tumor uptake of the tetrameric analogue is higher. Also, it has been shown that multimeric RGD peptides are effective clinical molecules for in vivo determination of tumor angiogenesis in cancer

In clinical practice, radiolabeled receptor ligands are used routinely for diagnostic imaging of overexpressed receptors and PRRT. Causes of clinical success of

1.First, the presence of different radionuclides, having similar chemical properties, enables to label the same peptide with different radionuclides for different

2.Second, the influence of the high hydrophilic radiometal complex on peptide pharmacokinetics leads to rapid renal excretion and good target/background

3.Third, one-step in-house labeling methodology that facilitates the preparation

The radioiodine which is used for radiolabeling of biologically active molecules is frequently preferred in PET imaging (124I), SPECT imaging (123/125I), treatment of different cancer types (131I), and biodistribution and kinetic investigation of novel peptide radiopharmaceuticals (125I). Direct radioiodination is based on

successful only in electron-rich aromatic systems including activating substituents such as ▬OH, ▬NH2, ▬OR, ▬NHCOR, or ▬SR. For in situ production of \*I<sup>+</sup>

chloramine-T (sodium tosylchloroamide) and iodogen (1, 3, 4, 6-tetrachloro-3a, 6a-diphenylglycoluril) are utilized. Chloramine-T is added to the reaction medium with sodium metabisulfite to terminate the labeling reaction and prevent oxidative damage. Oxidative enzymes (lactoperoxidase) are used for substitution of peptide sequences that have high sensitivity to oxidation. Tyr- and also His-containing peptides are readily radioiodinated in buffers such as phosphate buffered saline (PBS) or Tris (hydroxymethyl)-aminomethane (TRIS) at pH 7–8. Pre-radioiodination should be carried out by prosthetic groups. Selective prosthetic group conjugation is provided to the thiols with the help of pre-radioiodinated maleimides, and prelabeling of the corresponding peptide is carried out by the use of stannylated vinyl alkylating agents. In this way, tissue deiodinase and unfavorable structural condi-

(electrophilic radioiodide) and is

,

of peptide radiopharmaceuticals in clinical routine [89].

**14**

Complex biomolecules such as peptides or proteins cannot be directly labeled with a highly basic [18F] fluoride by nucleophilic substitution and cannot tolerate labeling conditions. Activated aromatic precursors (NO2, CN, CI, etc.) are substituents bound to the leaving group in the ortho- or para-position. Many receptor binding peptides such as octreotide, bombesin, neurotensin, and RGD analogues have been labeled using [18F]FP-NP(4-nitrophenyl-2-[18F]fluoropropionate) or [18F]SFB(N-succinimidyl-4-[18F]fluorobenzoate) [91]. Chemoselective strategies provide a one-step prosthetic group labeling reaction by unprotected precursors. Reaction of a 18F-labeled aldehyde with aminooxy- or hydrazino-functionalized peptides so-called click chemistry has recently found most popular application [89]. A suitable chelating agent is required for the radio metallization of the peptides. When a chelating agent is conjugated with a receptor binding peptide, it can affect both the binding affinity of the peptidic ligand and peptide pharmacokinetics [89]. Radiolabeling of peptides with the oxo-technetium ions (TcO2 + ,TcO+ ) is carried out by using peptide-bound tetradentate 99Tcm chelators (N3S or N4 scaffold). The peptides are coordinated with donor groups such as amine, carboxylate, or hydroxyl of the HYNIC (hydrazinonicotinic acid) chelator. In order to initiate the labeling reaction, a generator eluate (99Tcm-saline) should be added into a vial containing all the mixtures. For the labeling with 99Tcm, the [99Tcm(CO)3(H2O)3] + ; disodium boronocarbonate, Na2[HBCO2] (which also serves as an in situ CO source); and stannous ion, SnCI2 (reducing agent) are used [89]. 68Gallium labeling reaction is initiated by eluting the 68Ge/68Ga generator using hydrochloric acid (HCl) (0.05–0.60 M). Approximately 2 mL of the eluent is transferred to the reaction vial. Reaction vial containing a mixture of the lyophilized DOTA-peptide conjugate and sodium acetate buffer with the eluent is heated for 15 minutes at 100°C (25 min at 90–95°C). The intermediate product is pushed through an extraction cartridge. The final product is analyzed to determine the labeling efficacy and purity [92].
