UV–vis absorpition: It is known that absorption of free-base porphyrins and as synthesized metal complexes (metalloporphyrins) featured by two characteristic types of bands and are shown in Fig. 9. The λmax values of these species are reported in Table 1 and compared to Co and Cu metalloporphyrins
Figure 1. UV–Vis absorption spectra of a) H2TPP and CuTPP b) H2TPPCOOMe and
TPPCOOMe C) H2TPPNH2 and CuTPPNH2
In all cases the absorption wavelength of copper metalloporphyrin is lower than that of the free base porphyrins. The number of Q bands decreases from four to a single band when going from the free base porphyrin to their metal complexes, CuTPP and CuTPPCOOMe, as well as four to two for the metal complex CuTPPNH2. The decreases in number of Q bands and a shift on wavelength indicate the insertion of the copper in to the porphyrin cores. The Soret band is blue shifted up to 8 nm for CoTPPOMe and 7 nm for CoTPPCOOH, as well as 26 and 27 nm Hypsochromic shift for the Q band of CoTPPOMe and CoTPPCOOH from their free base porphyrins respectively. As the same as to copper metalloporphyrin, the decreases in the number of Q bands from four to a single band when going from the free base porphyrin to its cobalt complex indicates the insertion of the cobalt in the porphyrin core of H2TPPCOOH and H2TPPOMe and forms CoTPPCOOH and CoTPPOMe respectively. The Soret band is blue shifted up to 8 nm for CoTPPOMe and 7 nm for CoTPPCOOH, as well as 26 and 27 nm Hypsochromic shift for the Q band of CoTPPOMe and CoTPPCOOH from their free base porphyrins respectively.
Figure 2.UV–Vis absorption spectra of: a) H2TPPOMe and CoTPPOMe; b) H2TPPCOOH and CoTPPCOOH
Role of ligand substitution on UV–vis spectra: The absorption band of para-substituted CuTPPNH2 shows red-shift when compared with Cu-TPP and CuTPPCOOMe, suggesting the electronic effects from the electron releasing group NH2. CuTPPNH2 shows higher red-shift than Cu-TPP but CuTPPCOOMe shows lower blue shift than Cu-TPP due to the steric effect of methoxycarbonyl group in CuTPPCOOMe. CoTPPOMe shows a red shift when compared to CoTPPCOOH by 8 nm at the sort band and 12 nm at the Q band, which was also similar result in the free base porphyrins(shown in appendix A), suggesting the electronic effects from the electron releasing -OMe (methoxy) group. For clarity the UV-Vis data of porphyrins and metalloporphyrins are shown in supporting information (Table SI-1).
Figure 3.UV–Vis absorption spectra of: a) Copper complexes, b) Cobalt complexes
Mass spectrometry
The expected structure of all free base porphyrins had been confirmed by using mass spectrometry and the data is shown in Table SI-2. The H2TPPNH2 exhibited the molecular ion peak at m/z 676.54, as a major peak. The mass spectra of other porphyrin ligands are shown in Appendix. The mass spectra for all show very intense molecular ion peak due to a great deal of energy requirement for fragmentation of porphyrins. Therefore, fragmentation of porphyrin structure was not observed at all and the molecular ion peak [M+H]+ were observed. The ESI-Mass of CoTPPCOOH is displayed in Figure 11 and Appendix. The strong molecular ion peak [M+H]+ at m/z 848.91 is assigned for [CoTPPCOOH+H]+. In similar fashion, the mass spectra of all metalloporphyrins showed very intense molecular ion peaks due to the high energy from the delocalized electrons in the molecule. The mass spectra have been successfully received to confirm the expected corresponding structure of porphyrins as well as metal complexes.
Figure 4. The mass spectrum of cobalt-5,10,15,20-tetrakis(4-carboxyphenyl) porphyrin
Antimicrobial Test Results
The compounds were tested for their in vitro antibacterial activity and were compared with the commercially available drug, gentamicin. They were tested against two Gram-positive (S. aureus and S. pyogenes) and two Gram-negative (E. coliandK. pneumoniae) bacteria. All the tasted metalloporphyrins were found active against all the tested pathogens. The commercial antibiotic drug( gentamicin) exhibited highest activities with inhibition zones ranging from 25 mm to 28 mm in all the four pathogens. The results of antimicrobial activities of the study compounds are reported as inhibition zone of diameter (mm) are showed in Table 1.
Table1. Antibacterial activity of Metalloporphyrins, Metal salt, and reference antibiotic drug gentamicin
Compounds
|
Antibacterial activity ( mean IZ diameter(mm) ± SD)
|
S. aureus
|
S. pyogenes
|
E. coli
|
K. pneumonia
|
CoTPPCOOH
|
16.5
|
15
|
16
|
16
|
CoTPPOMe
|
12
|
14
|
10
|
12
|
CuTPPCOOMe
|
16
|
15
|
13
|
13.5
|
CuTPPNH2
|
13
|
13.5
|
12
|
12.5
|
Co acetate
|
6.5
|
7
|
6.5
|
6.5
|
CuCl2. 2H2O
|
6.75
|
7
|
6.25
|
6.5
|
DMSO
|
0
|
0
|
0
|
0
|
Gentamicin
|
25
|
27
|
26
|
25
|
The synthesized compounds (metalloporphyrin) showed higher activities than free base porphyrins (ligands) against all bacteria’s. This indicates that reaction of metal ions with the ligands plays an important role in antibacterial activity. This increased activity of metal complex can be explained on the basis of the overtone concept and chelation theory. According to the overtone concept of cell permeability, the lipid membrane that surrounds the cell favors the passage of only lipid-soluble materials in which lipo-solubility is an important factor that controls the antimicrobial activity.24 On chelation, the polarity of the metal ion will be reduced to a greater extent due to overlap of ligand orbital and partial sharing of the positive charge of the metal ion with donor groups. Furthermore, it increases the delocalization of π-electrons over the whole chelate ring and enhances the lipophilicity of complexes. It is likely that the increased lipo solubility of the ligand upon metal complexation may contribute to its facile transport into the bacterial cell which blocks the metal binding sites in enzymes of microorganisms. These complexes also disturb the respiration process of the cell and thus block the synthesis of proteins, which restricts further growth of the organism.25
Figure 5. Bar graph of CuTPPCOOMe and CuTPPNH2 on the same concentrations
Figure 6. Bar graph of CoTPPCOOH and CoTPPOMe on the same concentrations
As we saw in the table and the bar graphs, the antibacterial results shows that the complexes were active and inhibit bacteria’s even at the lowest concentration (31.25 mg/L).The complex CoTPPCOOH exhibited the greater antimicrobial activities than other metallporphyrins with inhibition zones 16.5 mm for S. aureus.
Figure 7. Gram positive Bacteria’s, S. aureus and S. pyogenesin CoTPPCOOH molecule
Figure 8. Gram negative Bacteria’s, E.coli andK. pneumoniain CoTPPCOOH molecule
Generally, as we saw in the bar graphs the metal complexs containing electro withdrawal group, CuTPPCOOMe and CoTPPCOOH showed better activities than the metal complex containing electro donating groups namely CuTPPNH2 and CoTPPOMe.