*2.2. Peptide*

John reported a self-assembled amphiphilic peptide (PA) that was used to produce the CO [142]. Thereby, a covalent combination of a hydrophobic alkyl chain and a hydrophilic short sequence peptide endures the self-assembled peptide chain material. Amphiphilic peptides can spontaneously release CO and are prone to toxicity themselves. In that perspective they first designed the amphiphilic peptide PA1; which contains a β-aspartate residue to generate the NH2-CH-RCOOH unit closely resembling the CORM-3 fragment. Next, PA1 and [Ru(CO)3Cl2]2 were synthesized in the presence of sodium methoxide at room temperature to synthesize the CO-releasing peptide PA2 (Figure 9). The CO kinetic release curve proved that peptide P2 was synthesized in an aqueous solution like the first-order rate constant of CORM-3. The half-life of the CO released from both sources is quite identical. The half-life of CO released from CORM-3 is 2.14 ± 0.17 min, while the half-life of the CO released peptide P2 is 2.16 ± 0.05 min. In order to increase the half-life of CO released from P2, the incorporation of nanofiber gel PA2 and a strong gel PA was made. The half-life of the CO released from this nanofiber gel was significantly increased (~17.8 min) compared to PA2 and CORM-3 in an aqueous solution.

**Figure 9.** Synthesis of PA2 having CO-moiety for spontaneous release CO.

Rather than encapsulating a mere CO segmen<sup>t</sup> inside the transport materials, the CORMs entity incorporates with different functional groups of parent CORM's ligand commodity. In that scenario, peptide is linked with the Manganese-based Photo-CORM [Mn(CO)3]+ ligand tpm (tris(pyrazolyl)methane) using a Pd-catalyzed based Songashira cross-coupling mechanism and click reaction at N-terminal (azide-) and side-chain (iodoarene-) functionalization [140].

Ulrich Schatzschneider exposed the peptide linkage for the CORMats development [144]. They introduced the Mo-carbonyl [Mo(CO)4(bpyCH3,CHO)] associated with aldehyde functional groups at the peripheral position. The bioactive β-target peptide ligand 2,20-bipyridine (bpy) attached with molybdenum-carbonyl by *N*-terminal bonds of aminoxy acetic through catalyst-free and bio-orthogonal oxime ligation (Figure 10). The photo-activated CORMats gets activated upon 468 nm photons lights irradiations.

**Figure 10.** [Mo(CO)4(bpyCH3,CHO)] complex has been constructed through bio-orthogonal peptide conjugate.

Radacki and Ulrich Schatzschneider jointly synthesized the Manganese carbonyl complexes [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6 [139]. The peptides ligand 2,2-bis(pyrazolyl)ethylamine (bpea) is bearing aminoxy, azide and N-terminal alkyne residues (Figure 11). The researchers applied the transforming growth factor β-recognizing (TGF-β) peptide sequence for developing the photo-activated delivery agent. This peptide conjugation could be utilized for further development of new CORMats.

**Figure 11.** The synthesis route of [Mn(bpeaNHCH2C6H4CHO)(CO)3]PF6 for Photo-CORMats.

The JJ Kodanko group successfully synthesized the ionic water-soluble compound [FeII(CO)(N4Py)] by a method of continuous CO bubbling through a ligand N4Py and one equivalent of FeII(ClO4)2 under the action of the organic solvent acetone (ClO4)2 (Figure 12A) [141]. A myoglobin experiment shows that the compound is stable under dark conditions and its releasing half-life is more than one day. When it was irradiated with 365 nm ultraviolet light, the CO can be quickly released. According to MTT experimental studies, [FeII(CO)(N4Py)](ClO4)2 exhibited the effective cytotoxicity against human prostate cancer cell line (PC-3) under light-induced conditions. When the concentration reaches 10 μM the cell survival rate was monitored as 63% of the control group. In order to further investigate the CO release behavior, the carbonyl segmen<sup>t</sup> with acetonitrile was replaced. The UV-vis analysis found that the substitution process is very slow [FeII-(MeCN)(N4Py)]2+ and the concentration of acetonitrile was a quick step to replace CO. Moreover, it was also found that the N4Py ligand can be modified with a peptide (Ac-Ala-Gly-OBn) to obtain a peptide-conjugated photo-induced release molecule (Figure 12B). Biological experiments have showed that the peptide chain conjugation might be evaluated for the improvement of cell-specific itself or tissue-specific CO transport properties.

**Figure 12.** The ionic water-soluble Iron complexes [FeII(CO)(N4Py)] (**A**) could be modified into Photo-CORMats by the replacement of the N4Py ligand with peptide china Ac-Ala-Gly-OBn (**B**) for the improvement of the cell-specific itself or tissue-specific therapeutic properties.

In the development of Photo-CORMats, metal-coligand plays a vital role in the photoexcitation at a prescribed wavelength. This allows the photons energy to penetrate and push the CO molecules to pull out these molecules from the metal-ligand fragment. In other words, these wavelengths are providing extra energy that enables the CO molecules excitation from its parent location. The CORM-2 and CORM-3 are hydrolytically active. Synthetically, these CORMs could be transformed into the photo-activated reagent. Leone Spiccia and Ulrich Schatzschneider described the ruthenium (II) dicarbonyl complexes functionalized with 2-(2-pyridyl)pyrimidine-4-carboxylic acid (CppH) (Figure 13A) [143]. They were able to successfully construct the monomeric PNA backbone with ruthenium (II) di-carbonyl complexes to produce ruthenium (II) dicarbonyl dichloride-based PNA-like monomer [RuCl2(Cpp-L-PNA)CO2] (where PNA= peptide nucleic acid) (Figure 13B).

**Figure 13.** The hydrolytically activated Ruthenium dicarbonyl complexes CORM-2 and CORM-3 could be transformed into Photo-CORMats by peptide ligands through different functionalization: (**A**) Polypyridyl ligand of 2-(2-pyridyl)pyrimidine-4-carboxylic acide (CppH); (**B**) monomeric PNA backbone.
