Photo-activated Efficacy Against HIV-1, Multiple Microbes and Biofilms by Zinc-complexes of Combinations of Cationic Ammoniumphenyl and Methylpyridinium-Porphyrins

doi:10.21203/rs.3.rs-4321809/v1

Download PDF

Article

Photo-activated Efficacy Against HIV-1, Multiple Microbes and Biofilms by Zinc-complexes of Combinations of Cationic Ammoniumphenyl and Methylpyridinium-Porphyrins

https://doi.org/10.21203/rs.3.rs-4321809/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

This study investigates the synthesis and characterization of cationic porphyrins, namely P₃AmM, PAm₃M, and c-P₂Am₂M, and their zinc(II)-complexes, P₃AmZM, PAm₃ZM, and c-P₂Am₂ZM. These compounds were developed by strategically methylating primary amino groups in precursor porphyrins to overcome steric hindrances associated with repetitive amine alkylation. Under photodynamic therapy (PDT) conditions, these porphyrins showed significant suppression of HIV-1 synthesis and infection, along with antibacterial properties against drug-resistant Escherichia coli and Staphylococcus aureus isolates. The bacterial growth dynamics indicated restrained proliferation and reduced biofilm production in the presence of the porphyrins over five days, underscoring their efficacy as antibacterial agents. Particularly, P₃AmZM, PAm₃ZM, c-P₂Am₂ZM, c-P₂Am₂ZM, and PAm₃M exhibited heightened antibacterial activity against both strains, with Staphylococcus aureus showing greater susceptibility. Disc diffusion assays highlighted the superior antibacterial efficacy of compounds c-P₂Am₂M and c-P₂Am₂ZM, particularly against Staphylococcus aureus. Computational molecular docking simulations revealed enhanced binding energy and interaction profiles of the lead compounds, c-P₂Am₂M and c-P₂Am₂ZM, with various HIV targets. These findings suggest these compounds deserve recognition as prospective synergistic anti-HIV agents with potent antibiotic properties under PDT conditions.

Cationic porphyrins

Zn-porphyrins

ammoniumphenyl porphyrins

photosensitisers

photodynamic inactivation of viruses

anti-HIV agents

There can be no exaggeration in claiming that the future of photochemistry is vitally destined for the development of therapeutics and pharmacology, particularly in the development of antiviral and antibacterial agents using synthetic photosensitisers (PSs). Addressing the limitations of current antiviral drugs against life-threatening viruses is crucial, alongside the pressing need to develop drug candidates that are effective against drug-resistant bacterial strains.^1–12 Globally, the prevalence of drug-resistant viral and bacterial strains is on the rise,^11,13,14 which might individually or collectively result in illnesses untreatable with existing medications. Notably, bacterial species such as Staphylococcus aureus and Escherichia coli stand out as common causes of antibiotic-resistant illnesses,¹⁵ while viruses including HIV, hepatitis, and influenza pose significant challenges. Researchers worldwide have proposed various strategies to combat drug resistance, with photodynamic therapy (PDT) emerging as a promising approach.^16–18 PDT involves the synergistic action of light of a specific wavelength, a photosensitiser, and molecular oxygen to induce the ablation of cancerous tissue, as well as bactericidal and virucidal effects. The Cationic porphyrins (CPs) with nitronium derivatives of porphyrins have been at the helm of research in photodynamic therapy related to cancer (cPDT), antimicrobial (aPDT), and antiviral therapeutics.^1–12 Phosphonium and sulphonium alternatives to the popular nitronium porphyrins have also indicated specific anticancer and anti-HIV activities, respectively.^11,13,14 As a part of the synthetic strategy, alkylation of meso-(4-pyridyl) groups to produce meso-(4-methylpyridinium) or meso-(4-alkylpyridinium) moieties has been very widely explored.^3,4,6 The same strategy has also been used for obtaining pyridinium salts of 2/3- pyridyl porphyrins.⁶ Variation in the chain length of the alkyl halide employed ensured increased lipophilicity to the PSs, as did the decrease in cations.⁶ The introduction of trifluoromethyl groups has been used to impart better lipophilicity.¹⁹ CPs-bearing combinations of phosphonium and ammonium salts have also been reported.^12,13 The combinations of meso-(4-trimethylammoniumphenyl) groups along with meso-(2,4,6-trimethoxyphenyl), meso-(4-methylphenyl), and meso-(4-trifluorophenyl) substituents showed promising PDT activity against E. coli.¹² Apart from CPs having multiple substituents at meso-position, tetra-substituted PS-bearing meso-(4-trimethylammoniumphenyl) groups have demonstrated excellent photodynamic activity in vitro on Candida albicans and E.Coli.^20–22 Better cellular uptake of the tetra cationic PS was reported²³ compared to mono, di and tri-cationic PSs bearing meso-(4-trimethylammoniumphenyl) and meso-(phenyl) substituents indicating the involvement of a self-promoted uptake pathway.²⁴ Metalloporphyrins, with both light-independent and photodynamic antimicrobial properties, have gained interest as potential substitutes for existing antibiotics.²⁵ Protoporphyrin IX-derived metalloporphyrins carry anionic properties and infiltrate bacterial cells via the heme-uptake system. As a result, bacterial strains lacking heme uptake systems demonstrate resistance to these metalloporphyrin.²⁶ Manganese porphyrin-based SOD mimics and other metalloporphyrin-based antibacterials have the potential benefit of being less harmful to eukaryotic cells. Numerous porphyrin-based medicines have shown little toxicity in lab animals, including those that use manganese porphyrins as SOD substitutes. Five phase II human clinical studies are now being conducted to evaluate these drugs.²⁷ Additionally, the application of 5,10,15,20-tetra(4-trimethylammoniumphenyl)porphyrin (TMAPP) as PS in cPDT^20,21 has been researched and with great success. Though reports indicate promising photobiological applications of both TMAPP^20–22 and 5,10,15,20-tetra(4-methylpyridinium)porphyrin (TMPyP),^28–31 biological applications of PSs bearing a mixture of either of the cationic groups, meso-(4-trimethylammoniumphenyl) and meso-(4-methylpyridinium), requires further exploration. The concoction of positive charges from different chemical environments can be expected to develop into interesting photobiological outcomes.

The anti-HIV activities of the CPs are manifested through either RT inhibition,^10,32–35 or HIV protease inhibition.^35–38 Second-generation CPs with significant anti-HIV-1 activity have been reported^39,40 to evince HIV-1 inhibition through binding with G-quadruplex DNA. One of the best results concerning the entry inhibitory effect of CPs on HIV-1 was demonstrated by Sengupta et al.⁹ The meso-(4-nitrophenyl) derivatives also containing meso-(4-methylpyridinium) groups exhibited HIV-1 inhibitory capability of > 99% (at 4 µM concentration) as determined through various biological assays performed with TZM-bl cells. The CPs were also able to inhibit the entry of SIVmac (Macaque Simian immunodeficiency virus) in the same cell line. The compounds also blocked completely the entry of HIV-1 into MT4 cells. A critical aspect of their finding is that the metalloporphyrin (Compound 8) with a single meso-(4-methylpyridinium) group and three meso-(4-nitrophenyl) proved to be the best entry inhibitor, highlighting the importance of the presence of cationic moieties. Other publications have also corroborated similar findings.^10,14,39,40 The amphiphilicity rendered as a result of the presence of the cationic charge aids in enhanced therapeutic outcomes of the PSs.^6,35 Anti-HIV activity of porphyrins is also enhanced by the incorporation of metals like Au and Zn in the porphyrin core.^8–10,39

Figure 1

The synthesized water-soluble ammoniumphenyl porphyrins P₃AmM, c-P₂Am₂M, PAm₃M and their corresponding Zn (II) metal complexes P₃AmZM, c-P₂Am₂ZM, PAm₃ZM.

While the literature has indicated several structure-activity relationships (SAR) for CPs towards their roles in cPDT and aPDT, the role of CPs towards in the photodynamic inhibition of viruses and their bactericidal effects on drug-resistant strains remains inadequate.

Therefore, exploration of the possible part of CPs bearing combinations of a mixture of the cationic groups, meso-(4-trimethylammoniumphenyl) and meso-(4-methylpyridinium), as potential anti-bacterial and anti-HIV agents have been investigated in this article. The concoction of positive charges from different chemical environments can be expected to develop into interesting photobiological outcomes. We designed and synthesized porphyrins bearing combinations of the quaternary meso-(4-trimethylammoniumphenyl) and meso-(4-methylpyridinium) groups (Fig. 1) 5-(4-ammoniumphenyl)-5,10,15-tri-(4-methylpyridinium) porphyrin (P₃AmM), 5,10-bi-(4-ammoniumphenyl)-15,20 bi- (4-methylpyridinium) porphyrin (c-P₂Am₂M), 5,10,15-tri-(4-ammoniumphenyl)-20- (4-methylpyridinium) porphyrin (PAm₃M) and their Zn metal complexes 5-(4-ammoniumphenyl)-5,10,15-tri-(4-methylpyridinium) porphyrin (P₃AmZM), 5,10-bi-(4-ammoniumphenyl)-15,20 bi- (4-methylpyridinium) porphyrin (c-P₂Am₂ZM), 5,10,15-tri-(4-ammoniumphenyl)-20- (4-methylpyridinium) porphyrin (PAm₃ZM). To the best of our knowledge, this is the first example of simultaneous quaternarization of meso-(4-aminophenyl) and methylation of meso-(4-pyridyl) groups leading to the formation of meso-(4-trimethylammoniumphenyl) and meso-(4-methylpyridinium) substituted porphyrins.

2.1 Synthesis, isolation and characterization:

2.1.1 Synthesis: The cationic water-soluble porphyrins were synthesised through a multi-step methodology as outlined in Scheme S1. A one-pot three-component reaction involving 4-acetamidobenzaldehyde (1.5 eq), pyridine-4-carboxaldehyde (3.2 eq) and pyrrole (4 eq) led to the formation of the products 5,10,15-tri-(4-acetamidophenyl)-20-(4-pyridyl) porphyrin (PA₃), 5,10-di-(4-acetamidophenyl)-15,20-di-(4-pyridyl) porphyrin (c-P₂A₂), 5-(4-acetamidophenyl)-10,15,20-tri-(4-pyridyl) porphyrin (P₃A). TLC of the crude reaction mixture indicated the formation of six compounds, of which only three (PA₃, c-P₂A₂ and PA₃) were isolated and characterised. Isolation was achieved through gravity percolation chromatography, silica gel 60–200 mesh and (3%-10%) MeOH in DCM being the stationary and mobile phases, respectively. P₃A was the first to elute with 5% MeOH in DCM followed by c-P₂A₂. PA₃ eluted out in 7% MeOH in DCM. Wherever required, a second column was run for further purification. P₃A, c-P₂A₂ and PA₃ were obtained in 10%, 9% and 6% yield. The isolation of the rest of the fractions was not attempted. The compound P₃A has been reported earlier, the synthesis being achieved through a protocol either similar to the one included in this paper⁴¹ or a different one.^42,43 Compound c-P₂A₂ has been reported earlier with a different synthetic strategy.⁴⁴ To our knowledge, PA₃ has not been reported so far. The acetamidophenyl porphyrins were then hydrolysed (Scheme S1, SI) to form the corresponding meso-aminophenyl substituted porphyrin derivatives 5,10,15-tri-(4-aminophenyl)-20-(4-pyridyl) porphyrin (PAm₃), 5,10-di-(4-aminophenyl)-15,20-di-(4-pyridyl) porphyrin (c-P₂Am₂), 5-(4-aminophenyl)-10,15,20-tri-(4-pyridyl) porphyrin (P₃Am). As shown in Scheme S2, an ethanolic solution of P₃A, e.g., is reacted with HCl (5 M) under reflux conditions, the amide bond of the lone acetamidophenyl group of the compound gets cleaved, forming an aminophenyl function as in compound P₃Am. PAm₃, c-P₂Am₂ and P₃Am were obtained in 82%, 96% and 88% yield, respectively. The synthesis of P₃Am with the same reagent mixture but under different conditions of reaction and workup has been reported earlier.^41,45 In contrast compounds c-P₂Am₂ and PAm₃ have been synthesised using an entirely different methodology involving a Lossen rearrangement. Derivatives of the type CM_xPy_yP were warmed to 100°C in polyphosphoric acid, then gradually to 160°C over 3 h followed by neutralisation with NaOH to obtain c-P₂Am₂ and PAm₃ (or cis-A₂Py₂P and APy₃P) as reported in the publications.^42,43 Zn-metalation of the freebase porphyrins P₃Am, c-P₂Am₂ and PAm₃ was carried out by reacting the individual compounds with zinc acetate in CHCl₃/MeOH-9:1 v/v solution, as shown in Scheme S3. The Zn-metal complexes P₃AmZ, c-P₂Am₂Z and PAm₃Z were obtained in 87, 86 and 78% yields, respectively.

The freebase porphyrins P₃Am, c-P₂Am₂, PAm₃ and the zinc complexes P₃AmZ, c-P₂Am₂Z and PAm₃Z were then alkylated through a room temperature reaction with methyl iodide to produce the hydrophilic poly iodide salts P₃AmM, c-P₂Am₂M, PAm₃M and their Zn metal complexes P₃AmZM), c-P₂Am₂ZM and PAm₃ZM (Scheme 1). In each case, alkylation of the meso-(4-pyridyl) groups to meso-(4-methylpyridinium) and meso-(4-aminophenyl) to meso-(4-trimethylammoniumphenyl) moieties was achieved. This synthetic achievement results in the first-ever report of direct quaternarisation of meso-(4-aminophenyl) moieties in porphyrins bearing combination of meso-(4-aminophenyl) and meso-(4-pyridyl) groups. The synthesised polyiodide salts were readily hydrophilic, thus rendering them useful for biochemical explorations. Alkylation involving 5-(4-aminophenyl)-10,15,20-tri-(4-pyridyl) porphyrin attempted with a different synthetic methodology has been reported earlier.⁴⁶ Nevertheless, the authors could not achieve quaternarisation of the meso-(4-aminophenyl) group.

2.1.2 Characterisation: The synthesised compounds were characterised using ¹HNMR, HRMS (ESI)/MALDI-TOF, UV-Vis, and emission spectroscopic techniques. The proton splitting pattern of the synthesised compounds has been included in Table S1 for greater clarity. The splitting patterns are consistent with the A₃B/AB₃ and cis-A₂B₂ systems reported in the literature.^8,9,47–50 For compounds P₃A, c-P₂A₂, and PA₃, the spectral plots (Figure S5-S7) indicate that the β-pyrrole protons are split into doublet-singlet-doublet at 8.94 (d, J = 4.7 Hz)−8.85 (s)−8.82 (d, J = 4.7 Hz) ppm, 8.92 (d, J = 4.7 Hz)−8.89 (s)−8.83 (s)−8.8 (d, J = 4.7 Hz) ppm, and 8.92 (d, J = 4.2 Hz)-8.89(s)−8.84 (d, J = 4.2 Hz) ppm, respectively. The pyridyl protons resonate at 9.05 (d, J = 4.1 Hz), 8.16 (d, J = 4.1 Hz) for P₃A, 9.05 (d, J = 5 Hz), 8.16 (d, J = 5 Hz) for c-P₂A₂ and 9.04 (d, J = 4.9 Hz), 8.27 (d, J = 4.9 Hz) ppm for PA₃.

The proton splitting for the phenyl protons appeared at 7.94 (d, J = 7.9 Hz) and 7.56 (d, J = 7.9 Hz) for P₃A, 7.92 (d, J = 7.9 Hz) and 7.53 (d, J = 7.9 Hz) for c-P₂A₂, 8.15 (d, J = 8.1 Hz) and 8.06 (d, J = 8.1 Hz) ppm in the case of PA₃. The ¹HNMR spectral data of P₃A conforms to the published values.^41,45,51 For P₃Am, c-P₂Am₂ and PAm₃, the ¹HNMR spectral plots (Figure S8-S10) and tabulated spectral data (Table S1) agree with the published values^42,43 and confirm the identity of the compounds. The HRMS (ESI) data of the acetamidophenyl and aminophenyl porphyrins further indicate their purity; the mass error in each case was less than 5 ppm (Figure S20-S25). The striking feature in ¹HNMR spectral data (Table S1 and Figure S11-S13) of the Zn-complexes is the disappearance of the highly shielded inner pyrrolic proton peak, a characteristic of the successful metalation of the porphyrins. Apart from that, the β-pyrrole splitting patterns (Fig. 2) conform to that of the A₃B/AB₃ and c-A₂B₂ systems. For P₃AmZ and PAm₃Z, the β-pyrrole protons are split into doublet-singlet-doublet centred at 8.98 (d, J = 4.4 Hz)-8.81 (s)-8.79 (d, J = 4.4 Hz) and 8.9 (d, J = 4.6 Hz)-8.88 (s)-8.71 (d, J = 4.6 Hz) ppm, respectively. Proton resonances at 8.94 (d, J = 4.4 Hz)-8.91 (s)-8.77 (s)-8.74 (d, J = 4.4 Hz) ppm were observed for β pyrrolic protons of c-P₂Am₂Z. The pyridyl and phenyl protons are split into four doublets centred at 9.01 (d, J = 4.6 Hz), 8.21 (d, J = 4.6 Hz) and 7.83 (d, J = 8.1 Hz), 6.99 (d, J = 8.1 Hz) ppm, respectively

for P₃Am, 8.98 (d, J = 4.9 Hz), 8.19 (d, J = 4.9 Hz) and 7.83 (d, J = 8.1 Hz), 6.99 (d, J = 8.1 Hz) ppm, respectively for c-P₂Am₂Z and 8.97 (d, J = 4.9 Hz), 8.19 (d, J = 4.9 Hz) and 7.82 (d, J = 7.8 Hz), 6.98 (d, J = 7.8 Hz) ppm, respectively for PAm₃Z.

The amino-protons resonated as a singlet at 5.53 (s, br), 5.55 (s), and 5.48 (s) ppm for P₃AmZ, c-P₂Am₂Z, and PAm₃Z, respectively. The formation of the compounds was ably supported by MALDI-TOF data (Figure S26-28). The observed and calculated mass were in good agreement with each other.

The alkylation of the free-base compounds and their Zn-complexes was supported by the recorded MALDI-TOF (Figure S29 – S34) spectra, the mass values obtained agree well with the calculated values, and the mass error is within 5 ppm limit. ¹HNMR (Figure S14 – S19) gives further evidence in support of the alkylation, intense singlets corresponding to −CH₃ protons in methylpyridinium and ammoniumphenyl moieties were consistently observed. However, the aromatic proton splitting presented a rather complex picture. This observation may be because of the effect of the cationic charges on electrons around the porphyrin ring. Protonation of aminophenyl porphyrins forming the ammoniumphenyl counterparts has been reported to have affected ¹HNMR spectra.⁴³

The synthesised porphyrins' UV-Vis spectra (Figure S1 A, S2 A, S3 A) consistently depict the Soret and Q band absorptions. The absorption peaks, molar extinction coefficient, emission peaks and Stokes shift have been included in Table S2. Minor redshifts in the absorption peaks were observed for P₃A, c-P₂A₂ and PA₃ (Figure S1 A). Emission spectra (Figure S1 B) recorded through excitation of the same solutions of the compounds used for UV-vis measurement at the absorption maxima of the respective Soret band exhibit peaks at 644, 660, 717 nm for P₃A, 647, 664, 720 nm for c-P₂A₂ and 646, 673, 723 nm for PA₃.

For the aminophenyl porphyrins P₃Am, c-P₂Am₂ and PAm₃ redshifts in Soret band absorptions were apparent (Figure S2 A) as the number of meso-aminophenyl groups increased in the porphyrin skeleton in going from P₃Am−c-P₂Am₂−PAm₃, the magnitude of redshift being 6 nm in going from P₃Am to c-P₂Am₂ and 5 nm between c-P₂Am₂ and PAm₃. Redshifts to the tune of 20 nm can also be seen in the emission profile (Figure S2 B) of compounds P₃Am, c-P₂Am₂ and PAm₃ with a progressive increase in the number of meso-aminophenyl groups. The emission peaks corresponding to the Q(0,0) and Q(0,1) transitions are 653 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)), 718 nm for P₃Am, 673 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)), 723 nm for c-P₂Am₂ and 694 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)), 757 nm for PAm₃, although it should be stated that the Q(0,1) emission band is not well defined in c-P₂Am₂ and PAm₃ and it appears more like a shoulder peak in the spectral plots (Figure S2 B). Stokes shift of 237, 251 and 263 nm were observed for P₃Am, c-P₂Am₂ and PAm₃, the values are much lower than the acetamidophenyl porphyrins with Stokes shift of 299, 300 and 301 nm for P₃A, c-P₂A₂ and PA₃.

Evidence of Zn-metalation can be seen in the UV-Vis spectral plot (Figure S3 A) of the Zn-complexes. The incorporation of Zn is accompanied by the reduction in the number of Q-band absorptions from four in the freebase porphyrin to two in the metal complex. The reduction in the number of Q-band peaks in the Zn porphyrins is a direct consequence of the increased symmetry of the compounds relative to that of the free base porphyrins. The inner pyrrolic protons in the porphyrin core reduce the symmetry from D₄h to D₂h.⁵² Redshift in Soret band maxima can be seen with an increase in the number of appended aminophenyl groups. The λ_max of the Soret band absorption for P₃AmZ, c-P₂Am₂Z and PAm₃Z are 427, 432 and 435 nm, respectively.

The Q-band peaks appear at 560 and 603 nm for P₃AmZ, 563 and 607 nm for c-P₂Am₂Z and finally, 566 and 611 nm for PAm₃Z. A blueshift of emission maxima also accompanies complex formation (Figure S3 B); the compounds also show a blue shift in Q(0,0) emission peaks with a progressive reduction in the number of meso-aminophenyl groups from three in PAm₃Z to two in c-P₂Am₂Z and to just one in P₃AmZ. Blueshifts were also observed in the Q (0,1) emission band with a reduction in the number of meso-aminophenyl groups. However, the trend is different, with the observed blue shift being highest for c-P₂Am₂Z.

The comparative UV-Vis plot of the compounds (Fig. 3) clearly shows a blue shit in Soret band absorption of the meso-acetamidophenyl porphyrins upon hydrolysis to meso-aminophenyl porphyrins. Zn-metalation of the latter was accompanied by a significant red shift in λ_max of the Soret band. Table S2 includes the detailed UV-Vis and emission data of P₃A, c-P₂A₂, PA₃, P₃Am, c-P₂Am₂, PAm₃, P₃AmZ, c-P₂Am₂Z and PAm₃Z with calculated Stokes shift and molar extinction coefficient for better clarity. The Stokes shift values show a progressive reduction in going from the freebase acetamidophenyl porphyrins to the amino porphyrins and finally to the Zn complexes.

Such a decrease of Stokes shift in Zn-complexes relative to the freebase system was reported earlier^53,54 and can be attributed to the Zn-compounds taking on a more co-planar configuration in the first excited state than in the ground state.⁵³ Between the acetamidophenyl porphyrins and their hydrolysis products, the meso-(4-acetamidophenyl) groups probably induce more significant structural distortions on photoexcitation and, thus, the larger values of Stokes shift.

The UV-Vis spectra of the CPs were recorded with a 10 µM solution of the compounds in water. The spectral plot and data are presented in Fig. 4 and Table S3. The absorption spectra of the free-base CPs conform to the typical porphyrinic spectra exhibiting an intense Soret band and four Q-band absorptions. The Soret band peaks are redshifted in the Zn complexes compared to their freebase counterpart. The number of Q-bands in the zinc complexes is also reduced to two. The observed spectral behaviour is in line with Gouterman’s model.⁵⁵

A progressive red shift in the Soret band is also apparent in going from PAm₃ZM-c-P₂Am₂ZM-P₃AmZM-TMPyPZ (Fig. 4A). The emission spectra (Fig. 5) of the ammoniumphenyl porphyrins show variable results. The spectral data were recorded by excitation of a 10 µM solution of the compounds in water at the λ_max of their Soret bands. While emission peaks pertaining to the Q(0,0) and Q(0,1) emission bands can be observed for P₃AmZM, c-P₂Am₂ZM with emission peaks at 630 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)), 653 nm and 634, 654 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)) nm, respectively, the two peaks appear to have merged for PAm₃ZM with a maximum at 657 nm. The freebase CP c-P₂Am₂M also exhibits two emission bands at 658 and 710 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)) nm. For P₃AmM a single emission band could be observed with a maxima at 623 nm, whereas for PAm₃M emission peaks appeared at 658 (\({{\lambda }}_{\text{e}\text{m}}^{\text{m}\text{a}\text{x}}\)) and 707 nm. The UV-Vis, emission spectral data, and molar extinction coefficient have been included in Table S3. A blue shift in emission peak maxima is apparent on Zn-complexation; however, the emission maxima of P₃AmM was blue-shifted by 10 nm compared to its Zn-complex P₃AmZM. The Stokes shift calculated^56,57 presented interesting outcomes (Table S3). The large values indicate structural changes upon photoexcitation.^53,58,59 The Stokes shift values for the freebase porphyrins were more prominent than their Zn-complexes, PAm₃M and PAm₃ZM being exceptions here. This agrees with earlier reports,^53,54 and indicates that the Zn-complexes take on a more planar configuration in the first excited state than in the ground state compared to the freebase compounds.^53,59 The large values of Stokes shift also mean reduced homo-FRET and consequently minimal self-quenching.^60,61 This is desirable as far as the applications of the CPs as PSs are concerned.

2.2 Anti-HIV activity:

In the present study, the anti-HIV activities of cationic ammoniumphenyl porphyrins are reported. The anti-HIV activity was tested using several biochemical and virological assays under both non-photodynamic (non-PDT) and photodynamic conditions (PDT) as described.

2.2.1 Ammoniumphenyl Porphyrins were not cytotoxic under non-PDT or PDT conditions.

The effect of ammoniumphenyl porphyrins on the viability of HEK-293T and TZM-bl cells was determined using Cell Titre Blue Assay under non-PDT and PDT conditions as described in the methods. None of the porphyrins were toxic to the cells at the concentrations tested in non-PDT conditions from 0.1 µM to 10 µM. The half-maximal cytotoxic concentrations (CC₅₀ value) determined were greater than 10 µM (Table S4). Hence, for further experiments under non-PDT conditions, porphyrins were used at 5 µM. Under PDT conditions as described in methods, cell viability was determined in the presence of porphyrins at concentrations from 0.1 µM to 0.5 µM. Again, all the porphyrins were non-toxic to cells under PDT conditions with half-maximal cytotoxic concentrations (CC₅₀ value) of > 500 nM. Hence, porphyrins were used at 500 nM for further experiments under PDT conditions.

2.2.2 Ammoniumphenyl porphyrins reduced virus infectivity under non-PDT conditions.

The infectivity of the viruses produced in the presence of compounds was analyzed by using a TZM-bl cell-based single-cycle infectivity assay^62,63 as described in the methods. Briefly, HEK-293T cells were transfected with pNL4-3 subtype B virus and incubated with porphyrins at a concentration of 5 µM for 24 h. The amount of virus released in the supernatant was quantified by p24 ELISA. Then, TZM-bl cells were infected with 10 ng of HIV-1 p24 equivalent NL4-3 virus for 2 h. The cells were washed post-infection and incubated in fresh media further for 48 h. Relative luminescence was measured 48 h post-infection and was compared to the control (-). Control refers to the HIV-1 subtype B NL4-3 virus produced with 5% DMSO in water in the absence of porphyrins. It was observed that the porphyrins containing three ammonium phenyl moieties and single pyridinium- PAm₃M and PAm₃ZM reduced virus infectivity by 30% and 80%, respectively. Zinc metallation of PAm₃M enhanced its antiviral activity robustly. Similarly, zinc-metallated TMPyPZ exhibited a significant reduction in virus infectivity by 75% compared to its precursor TMPyP by only 30% (Fig. 6A). Hence, it was evident that the incorporation of zinc in these structures enhanced their antiviral activity. Both c-P₂Am₂M and its zinc derivative c-P₂Am₂ZM containing two ammonium phenyl moieties and two pyridinium were most effective in reducing virus infectivity by 96% irrespective of zinc metallation. Further, in the case of P₃AmM, containing three pyridinium moieties and a single ammonium phenyl group, the reduction in infectivity was 80% but was reduced to 40% inhibition on the addition of zinc in P₃AmZM. These results suggested that the presence of two ammonium phenyl moieties linked to two pyridinium groups exhibited the most potent antiviral activity.

2.2.3 Ammoniumphenyl porphyrins inhibited HIV-1 virus production post-entry under non-PDT conditions

Viral entry inhibition, an early step in the virus life cycle, was then determined in the presence of ammoniumphenyl porphyrins. Inhibition assays were performed in TZM-bl cells as described in the methods. Briefly, TZM-bl cells were infected with 10 ng of HIV-1 p24 equivalent subtype B NL4-3 virus and porphyrins at 5 µM were added during virus infection as explained in methods. The reference porphyrins- TMPyP and TMPyPZ were used as a control as they have been reported to show anti-HIV activity.^10,64 Enfuvirtide (T20), a known HIV-1 fusion inhibitor was used as a positive control.⁶⁵ c-P₂Am₂M, P₃AmM, and PAm₃ZM, restricted virus entry by 30%, when added during virus infection, whereas TMPyP and TMPyPZ restricted HIV-1 entry by just 20% (Fig. 6B, blue bars). But c-P₂Am₂ZM, P₃AmZM and PAm₃M were completely ineffective. These results show that ammoniumphenyl porphyrins exhibited weak activity inhibiting HIV entry under non-PDT conditions.

Next, we wanted to test the effect of these porphyrins on HIV-1 production if added after virus entry in cells. Briefly, TZM-bl cells were first infected with HIV-1 subtype B NL4-3 virus and then incubated with porphyrins for 48 h at 37 ºC as described in the methods.

We observed c-P₂Am₂ZM, P₃AmM, PAm₃ZM, and TMPyPZ showed 50%,36%, 36% and 42% reduction in virus production respectively (Fig. 6B, yellow bars). It was observed that the porphyrin combination c-P₂Am₂ZM, bearing two ammonium phenyl and two pyridinium moieties complexed with zinc, showed maximum reduction in virus production when added post-entry. Also, all porphyrins metallated with zinc were more effective in comparison to their precursor porphyrin except P₃AmM. These results showed that the amino-porphyrins may inhibit post-entry stages of HIV infection and reduce HIV production in infected cells under non-PDT conditions.

2.2.4 Ammoniumphenyl porphyrins strongly inhibited HIV-1 entry under PDT conditions

We next tested the effect of porphyrins on virus entry under PDT conditions as described in the methods. Briefly,10 ng of HIV-1 p24 equivalent subtype B NL4-3 virus (subtype B) was pre-incubated with the porphyrins under PDT conditions. The pre-treated virus was used to infect TZM-bl cells. Infected cells were washed and incubated for 48 h at 37 ºC. Relative luciferase activity was determined with respect to control (−). Here, control refers to cells infected with a virus treated with 5% DMSO in water in the absence of porphyrins. TMPyP and TMPyPZ were used as a reference control for porphyrins known for anti-HIV activity in PDT conditions.^10,66 Enfuvirtide (T20), a known HIV-1 fusion inhibitor, was used as a positive control.⁶⁵ It was observed that pre-treatment of HIV-1 subtype B NL4-3 virus with porphyrins before infection under PDT conditions blocked virus entry by more than 80% in TZM-bl cells (Fig. 6C, blue bars). PAm₃M and PAm₃ZM restricted virus entry by 86% and 84% respectively whereas c-P₂Am₂M and c-P₂Am₂ZM showed 80% and 82% restriction respectively. P₃AmM and P₃AmZM showed inhibition in virus entry by 83%. TMPyP and TMPyPZ reduced virus entry by 85%. Furthermore, the addition of porphyrins post-virus entry completely blocked HIV production in infected cells except PAm₃M in PDT conditions. (Fig. 6C, orange bars). These results suggested that the porphyrins were potent anti-HIV agents in the presence of light as compared to non-PDT conditions. Under PDT conditions, their activity was independent of the different combinations of ammonium phenyl group and pyridinium group or the presence/absence of zinc. Since the antiviral activity of these compounds was dependent upon light exposure, they may act as strong photosensitisers against HIV-1.

2.3 Anti-bacterial activity:

2.3.1 Assessment of anti-bacterial activity through disc diffusion experiment:

The study evaluated the antimicrobial activity of cationic ammoniumphenyl porphyrins against E. coli and S. aureus under non-photodynamic and photodynamic conditions, using three different concentrations (0.5 µM, 1 µM and 2 µM) (Supporting information: Tables S5 and S6). Bacterial strains were evenly spread on nutrient agar plates. Discs loaded with different concentrations of porphyrin compounds were placed on the plates. One set was kept in darkness, the other exposed to light for 30 mins before incubation. After 24 h at 37°C, plates were checked for inhibition zones around the discs. P₃AmZM and c-P₂Am₂ZM showed the most effective antimicrobial activity against E. coli under PDT, with 1.5 cm inhibition zones at 1 µM and 2 µM concentrations. In the absence of light, PAm₃M and PAm₃ZM displayed significant efficacy, generating 1 cm and 1.2 cm inhibition zones, respectively, at a 1 µM concentration. c-P₂Am₂M exhibited the highest antibacterial activity at 1 µM concentration under PDT conditions. Similarly, at 1 µM concentration, P₃AmM and TMPyP had inhibition zones of 1.2 cm and 1.1 cm, respectively. However, TMPyPZ showed lower antibacterial (Table S5) (Fig. 7a). In previous studies, it was found that lower concentrations of the compounds resulted in weaker associations with bacterial cells, while higher concentrations led to aggregations preventing interaction with the bacterial cell wall. Therefore, an optimal moderate concentration was recommended for maximum inhibition of bacterial cells. The susceptibility of the S. aureus against the porphyrin compounds was notably higher. c-P₂Am₂M and PAm₃ZM exhibited significant inhibition zones, measuring 3.5 cm and 3 cm, respectively, in non-PDT conditions. Under PDT conditions, c-P₂Am₂ZM and P₃AmZM displayed increased antibacterial activity at 1 µM concentrations. P₃AmM resulted in a 1.2 cm inhibition zone. However, PAm₃M, TMPyP, and TMPyPZ showed

lower antibacterial activity against S. aureus in both PDT and non-PDT conditions. At 2 µM concentrations, antibacterial activity decreased, indicating aggregation inhibition of compound activity at high concentrations (Photo-plate S1, Table S6) (Fig. 7b).

Porphyrins exhibit antimicrobial properties to catalyze peroxidase and oxidase reactions, absorb photons, generate reactive oxygen species (ROS), and incorporate into the lipids of bacterial membranes.²⁶ Batishchev et al.,⁶⁷ successfully determined the relationship between the structure and activity of PSs in terms of their antimicrobial properties for two Gram-negative bacteria, E. coli and Acinetobacter baumannii. Remarkably, with minimal light exposure of 5 J/cm², this molecule exhibited inhibition of E. coli and A. baumannii. This finding positions it as a promising candidate for combating microbial infections. Research has shown that aPDT has proven to be highly effective in eradicating a clinical isolate of Pseudomonas aeruginosa, an important pathogen often encountered in healthcare environments.⁶⁸ A study conducted by Orenstein et al.⁶⁹ demonstrated the efficacy of deuteroporphyrin in eradicating S. aureus. Nevertheless, deuteroporphyrin did not exhibit the same inhibitory effect on bacteria such as E. coli and P. aeruginosa. This issue can be addressed by applying pre-treatment to the cells using either EDTA or PMBN.⁷⁰

2.3.2 Minimal Inhibitory Concentration (MIC) of the porphyrin’s compounds:

Control bacterial isolates grew consistently within 24 h. E. coli was inhibited at 0.084 mg/mL of PAm₃ZM under light conditions, while S. aureus required lower concentrations at 0.776 mg/mL and 0.291 mg/mL in non-PDT and PDT conditions, respectively. MIC values for P₃AmZM were 0.97 mg/mL and 0.388 mg/mL for dark and light conditions, respectively, and 0.776 mg/mL and 0.291 mg/mL for S. aureus. c-P₂Am₂ZM inhibited S. aureus at 0.837 mg/mL and 0.186 mg/mL in non-PDT and PDT conditions, respectively, with MICs of 0.456 mg/mL and 0.279 mg/mL for E. coli. Similarly, c-P₂Am₂M inhibited S. aureus at 0.88 mg/mL and 0.22 mg/mL, with MICs of 1.10 mg/mL and 0.33 mg/mL for E. coli. P₃AmM showed inhibition at 192 mg/mL in PDT conditions, with higher MICs for S. aureus (0.768 mg/mL) compared to E. coli (0.480 mg/mL) in dark treatment. PAm₃M, TMPyP, and TMPyPZ exhibited MICs ranging from 0.744 mg/mL to 0.99 mg/mL in dark conditions and 0.372 mg/mL to 0.792 mg/mL in the presence of light (Table S7). Agnihotri et al.⁷¹ synthesized citrate-stabilized silver nanoparticles (AgNPs) ranging from 5 to 100 nm, demonstrating antibacterial efficacy against both E. coli MTCC 443 and S. aureus NCIM 5201) bacteria. Smaller nanoparticles (5–10 nm) exhibited increased antibacterial efficiency. According to Valduga et al.⁷² TMPyP efficiently inactivates E. coli under visible light by disrupting enzymatic and transport activities of membranes. A dose of 2.5 mg/mL of TMPyP tetratosylate achieved maximal toxicity (> 99%) against clinical strains of P. aeruginosa; higher concentrations (5 mg/mL) were required to inhibit bacteria in biofilms.⁷³

2.3.3 Effect of porphyrins on the growth profile and biofilm producing ability of the bacterial isolates:

Under PDT conditions, c-P₂Am₂ZM and c-P₂Am₂M displayed higher lethality against E. coli at concentrations of 0.46 mg/mL and 0.93 mg/mL, respectively. Similarly, compound P₃AmZM and PAm₃M inhibited bacterial growth at 0.97 mg/mL and 0.93 mg/mL, respectively. TMPyP and TMPyPZ reduced bacterial growth after a 5-day incubation at concentrations of 0.99 mg/mL and 0.98 mg/mL, respectively. PAm₃ZM showed killing activity at 0.28 mg/mL, while P₃AmM exhibited activity at 0.96 mg/mL (Fig. 8). Compounds c-P₂Am₂ZM and c-P₂Am₂M completely inhibited S. aureus growth within five days at 0.93 mg/mL and 1.10 mg/mL under PDT conditions. P₃AmZM and PAm₃M reduced growth at concentrations > 0.49 mg/mL. TMPyP and TMPyPZ efficiently killed bacteria at concentrations > 50 mg/mL. Growth of S. aureus was lower for PAm₃ZM and P₃AmM at 0.97 mg/mL and 0.93 mg/mL, respectively. Porphyrin compounds were more effective against S. aureus than E. coli in both PDT and non-PDT conditions. (Fig. 9). Utilizing porphyrins as photosensitizing molecules, photodynamic therapy offers a scientific approach to effectively eliminate microorganisms, including biofilms. This study demonstrated that in the presence of c-P₂Am₂ZM, P₃AmZM, PAm₃ZM, TMPyPZ, P₃AmM, PAm₃M and c-P₂Am₂ZM, S. aureus exhibited no biofilm formation under both PDT and non-PDT conditions. In non-PDT conditions, E. coli formed biofilms at lower concentrations of porphyrin compounds, including P₃AmZM, TMPyP, TMPyPZ, and c-P₂Am₂ZM. P₃AmZM notably reduced biofilm production in both PDT and non-PDT conditions. At 2µM concentrations, TMPyPZ and c-P₂Am₂ZM showed no biofilm formation under both conditions, indicating an inability to produce biofilm even in the presence of growth when exposed to sublethal concentrations of porphyrin-treated samples (Fig. 10).

2.4 Molecular docking Outcomes:

The docking simulations conducted on HIV target proteins indicate that compounds c-P2Am2ZM, PAm3M, P3AmZM, P3AmM, PAm3ZM, and c-P2Am2M demonstrate superior binding affinity and interaction profiles. Notably, among all the targets examined, these compounds exhibited stronger binding energy with reverse transcriptase compared to gp120 and protease. Specifically, when targeting reverse transcriptase, all compounds displayed better binding energy than stavudine and 1l4 (Fig. 11). Furthermore, c-P2Am2ZM and c-P2Am2M exhibited superior binding energy with protease compared to other targets. Interestingly, c-P2Am2ZM emerges as one of the top three compounds across all three explored HIV targets. Additionally, PAm3M stands out as a top compound with gp120 and reverse transcriptase proteins.

In the docking program's validation process, Y2E was meticulously redocked into its co-crystallized site on the HIV gp120 protein. A notably low Root Mean Square Deviation (RMSD) value of 1.53 Å was noted between the docked and native poses. This result serves as compelling evidence of the program's ability to accurately predict the binding poses of inhibitors within HIV proteins. This demonstrates the program's capability to reproduce the spatial positioning of Y2E within its designated binding site, affirming the docking algorithm's reliability and precision. The binding energies and molecular interaction profiles of the compounds were compared with those of co-crystallized ligands and drugs targeting various HIV targets. These targets play crucial roles in different stages of the viral life cycle: viral entry (gp120), production of infectious virions (protease), and replication (reverse transcriptase).

Figure 11

Binding energy details of compounds, drugs and co-crystallized ligands.

Binding energy and interaction summary of the compounds with HIV proteins.

Table 1

Molecular interaction summary of top compounds with HIV gp120.
Compounds	Binding Energy (K.cal/mol)	Interacting Amino acids	Nature of interactions
c-P₂Am₂ZM	-8.2	ILE371, MET426, GLU370, ASP368, ARG432, VAL430, SER375, ASN425, THR283	Amide- π stacked, π- sigma, π-alkyl, π- cation, π- anion, carbon hydrogen bond, van der waals
PAm₃M	-8.1	ILE371, GLY473, ASP477, ASP474, GLN428, VAL430, ASP368, GLY431, ASN425	Amide- π stacked, π- sigma, π-alkyl, π-anion, π- anion, carbon-hydrogen bond, van der Waals
P₃AmZM	-7.9	ILE371, ASP474, ASP477, ALA281, GLY473, GLN428, GLY431, VAL430, ASN425, ASP368	Amide- π stacked, π- sigma, π-alkyl, π-anion, π- anion, carbon-hydrogen bond, van der Waals

Table 2

Molecular interaction summary of top compounds with HIV protease
Compounds	Binding Energy (K.cal/mol)	Interacting Amino acids	Nature of interactions
c-P₂Am₂ZM	-7.7	ASP25, LEU23, ARG8, VAL82, PRO81	π- cation, π-alkyl, carbon hydrogen bond, van der waals
c-P₂Am₂M	-7.5	ASP25, LEU23, ARG8, VAL82, PRO81	π- π-cation, π-alkyl, carbon-hydrogen bond, van der Waals
P₃AmM	-6.1	ASP30, ASP29, ALA28, ARG8, VAL82, LEU23	π- anion, π- cation, π-sigma, π-alkyl, carbon-hydrogen bond, van der Waals

Figure 14

3D molecular representation of interactions of compounds A) c-P₂Am₂ZM, B) c-P₂Am₂ZM, and C) P₃AmM with the active site residues of HIV protease. Interactions were displayed as color-coded dashed lines.

Table 3

Molecular interaction summary of top compounds with HIV reverse transcriptase
Compounds	Binding Energy (K.cal/mol)	Interacting Amino acids	Nature of interactions
PAm₃ZM	-8.6	ARG72, LYS66, LEU74, TYR115, MET184, ALA114, ASP185, PHE160	π- π T-shaped, π-alkyl, π- cation, π-sigma, carbon hydrogen bond, van der waals
PAm₃M	-8.0	ARG72, LYS66, LEU74, GLY152, MET184, ALA114, ASP185, TYR115	π- π T-shaped, π-alkyl, π- cation, π-sigma, carbon hydrogen bond, van der waals
c-P₂Am₂ZM	-7.6	ARG72, LYS66, LEU74, ASP185, TYR115, MET184, ALA114	π- π T-shaped, π-alkyl, π- cation, π-sigma, carbon hydrogen bond, van der waals

The top compounds primarily engaged in hydrophobic and electrostatic interactions, including Amide-π stacked, π-sigma, π-alkyl, π-cation, π-anion, carbon-hydrogen bond, and van der Waals interactions. The interaction profiles, along with the involved residues and types of interactions, are summarized in Tables 1, 2, and 3. Detailed 3D representations of these interactions can be observed in Figs. 12, 14, and 16. Additionally, the binding orientations within the active site pockets can be visualized in Figs. 13, 15, and 17 for HIV gp120, protease, and reverse transcriptase, respectively.

c-P2Am2ZM displayed a distinct binding orientation compared to other top compounds by positioning its trimethyl amino group deeper within the active site pocket. In contrast, the other two compounds were situated on the outer periphery of the gp120 active site pocket. This distinction in positioning can be observed in Fig. 15. Both c-P2Am2ZM, c-P2Am2M, and P3AmM exhibited a consistent orientation within the protease active site cavity. They projected their trimethyl amino groups deeper into the pocket to establish interactions with crucial active site residues. This observation is illustrated in Fig. 15. However, all the top compounds aligned well within the reverse transcriptase active site pocket, as depicted in Fig. 17.

Across the spectrum of HIV protein targets, including gp120, protease, and RT, the porphyrin analogue t-P₂Am₂M, characterized by a trans configuration of groups, consistently demonstrated lower binding energies compared to its cis counterparts. The consistent decrease in binding efficacy observed with the trans arrangement emphasizes the critical role of the spatial orientation of molecular groups within the porphyrin structure in effectively targeting HIV protein sites.

For details regarding the binding energy and corresponding molecular interactions of t-P₂Am₂M0, please refer to the supplementary information (Table S9 & Figure S4).

In the present study, we have established the potential of newly synthesised ammoniumphenyl porphyrins to inhibit HIV infection. There is no evidence of any similar investigation being performed previously on these porphyrins. These porphyrins exerted antiviral activity in the lower nanomolar range under PDT conditions without any evident cytotoxicity. Under PDT conditions, they completely blocked virus entry inside the target cells as well as the HIV-1 production in infected cells. The antiviral activity was not affected by zinc metallation or by the presence of different combinations of ammonium phenyl or pyridinium groups. Our results suggested ammoniumphenyl porphyrins may act as active photosensitisers against HIV-1 blocking both virus entry as well as virus production in infected cells. The plausible mechanism could be their ability to absorb photons as photosensitisers generating ROS that confers photodynamic inactivation of HIV-1. Porphyrins targeting HIV-1 entry as well as production may serve as one of the promising antiviral therapeutics.

On the contrary, under non-PDT conditions, ammoniumphenyl porphyrins were not toxic up to the range of concentration tested (10 µM). However, porphyrins failed to inhibit HIV-1 entry significantly. However, virus production was modestly affected depending upon the zinc metallation, and the number of ammonium-phenyl and pyridinium groups present. In addition, the infectivity of HIV-1 produced in the presence of ammoniumphenyl porphyrin was significantly reduced. These results demonstrated antiviral activity of ammoniumphenyl porphyrins was enhanced upon exposure to light. Hence ammoniumphenyl porphyrins emerge as active photosensitisers against HIV-1.

Additionally, the effectiveness of these compounds' antibacterial properties was observed to be higher in the presence of light. As the concentration increased, there was a reduction in the growth of bacterial isolates such as E. coli and S. aureus, accompanied by a decrease in the production of biofilms.

Six hydrophilic cationic porphyrins (CPs), namely P₃AmM, c-P₂Am₂M, PAm₃M, P₃AmZM, c-P₂Am₂ZM, and PAm₃ZM, were synthesized through rigorous methylation of precursor free base aminophenyl porphyrins and their zinc-complexes. This involved quaternarization of meso-(4-aminophenyl) to meso-(4-trimethylammoniumphenyl) groups and simultaneous alkylation of meso-(4-pyridyl) to meso-(4-methylpyridinium) moieties using methyl iodide under reflux conditions. Upon investigation, these ammoniumphenyl porphyrins demonstrated minimal entry inhibitory effects on HIV-1 in the dark, yet exhibited significant inhibition upon photo-irradiation, with varying degrees of effectiveness. None of the compounds showed toxicity under test conditions. Moreover, the CPs effectively reduced HIV-1 infectivity, both in terms of entry and production, with c-P₂Am₂M and c-P₂Am₂ZM showing particularly promising results. The demonstrated effectiveness of the compounds in inhibiting HIV entry (gp120), production (reverse transcriptase), and infectivity (protease) aligns closely with their enhanced binding affinity and interaction profiles revealed in docking simulations against the pertinent HIV targets responsible for these functions. With positive outcomes observed in both in vitro studies and molecular docking evaluations, these compounds deserve further investigation as prospective candidates in the quest for anti-HIV agents The study suggests a structure-activity relationship, indicating that an equitable presence of meso-ammonium phenyl and meso-pyridinium groups contributes to enhanced photo-biological activity. In contrast, a decrease in meso-pyridinium groups correlates with reduced activity of the porphyrins. These findings underscore the potential of these compounds as anti-drug resistant antibiotics and as anti-HIV agents.

ASSOCIATED CONTENT

Supporting Information

Experimental Details: Synthetic methods, Details of biological assays employed, Characterisation Data: ¹HNMR and Mass Spectral data.

The Supporting Information is available free of charge in the publication’s website.

Data availability statement

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Author Information

Corresponding Author

*Devashish Sengupta. E-Mail: [email protected]

*Ritu Gaur. E-Mail: [email protected], *Piyush Pandey. E-mail: [email protected]

Author Contributions

D.S. carried out the synthesis, isolation, and characterization of the porphyrins and contributed to compiling the synthetic, photochemical, and photophysical aspects presented in the paper. M.R. performed the experiments and data analysis for the anti-viral studies. M.R., guided by R.G.*, wrote the antiviral section. N.D. carried out the antibacterial and antibiofilm assays under the supervision of P.P.* N.D., guided by P.P., wrote the antibacterial section presented in the manuscript. P.P.* designed the antibacterial studies and anti-biofilm assays. R.K. conducted the insilico evaluation of compounds with HIV targets. R.G.* designed and supervised the anti-HIV studies and contributed to writing the anti-HIV part of the manuscript. D.S.* supervised and coordinated the project, conceptualized the design, synthesis, and application of the compounds as anti-HIV-1 and anti-bacterial agents and contributed to data analysis, availing funds for the project, consolidation and writing of the manuscript.

Funding Sources

The support by DBT (Nanobiotechnology) Project Reg. No: BT/PR25024/NER/95/961/2017, granted to Devashish Sengupta from the Government of India, Ministry of Science and Technology; Department of Biotechnology is acknowledged.

Acknowledgement

We gratefully acknowledge STIC-SAIF, Kochi for ¹HNMR and mass spectral analysis and CDRI, Lucknow for HRMS spectra.

Pavani, C., Uchoa, A. F., Oliveira, C. S., Iamamoto, Y. & Baptista, M. S. Effect of zinc insertion and hydrophobicity on the membrane interactions and PDT activity of porphyrin photosensitizers. Photochem. Photobiol. Sci. 8, 233-240 (2009).
Deda, D. K. et al. A new micro/nanoencapsulated porphyrin formulation for PDT treatment. Int. J. Pharm. 376, 76-83 (2009).
Villanueva, A. & Jori, G. Pharmacokinetic and tumour-photosensitizing properties of the cationic porphyrin meso-tetra (4N-methylpyridyl) porphine. Cancer Lett. 73, 59-64 (1993).
McCormick, B. P. P. et al. Cationic porphyrin derivatives for application in photodynamic therapy of cancer. Laser Phys. 24, 045603 (2014).
Stallivieri, A. et al. Synthesis and photophysical properties of the photoactivatable cationic porphyrin 5-(4-N-dodecylpyridyl)-10, 15, 20-tri (4-N-methylpyridyl)-21H, 23H-porphyrin tetraiodide for anti-malaria PDT. Photochem. Photobiol. Sci. 14, 1290-1295 (2015).
Malatesti, N., Munitic, I. & Jurak, I. Porphyrin-based cationic amphiphilic photosensitisers as potential anticancer, antimicrobial and immunosuppressive agents. Biophys. Rev. 9, 149-168 (2017).
Mazumdar, Z. H. et al. meso-Thiophenium Porphyrins and Their Zn (II) Complexes: A New Category of Cationic Photosensitizers. ACS Med. Chem. Lett. 11, 2041-2047 (2020).
Sengupta, D. et al. Two cationic meso-thiophenium porphyrins and their zinc-complexes as anti-HIV-1 and antibacterial agents under non-photodynamic therapy (PDT) conditions. Bioorg. Med. Chem. Lett., 128699 (2022).
Sengupta, D. et al. Dual activity of amphiphilic Zn (II) nitroporphyrin derivatives as HIV-1 entry inhibitors and in cancer photodynamic therapy. Eur. J. Med. Chem. 174, 66-75 (2019).
Sun, R. W. Y., Yu, W. Y., Sun, H. & Che, C. M. In Vitro Inhibition of Human Immunodeficiency Virus Type‐1 (HIV‐1) Reverse Transcriptase by Gold (iii) Porphyrins. ChemBioChem 5, 1293-1298 (2004).
Vzorov, A. N., Dixon, D. W., Trommel, J. S., Marzilli, L. G. & Compans, R. W. Inactivation of human immunodeficiency virus type 1 by porphyrins. Antimicrob. Agents Chemother. 46, 3917-3925 (2002).
Spesia, M. B., Lazzeri, D., Pascual, L., Rovera, M. & Durantini, E. N. Photoinactivation of Escherichia coli using porphyrin derivatives with different number of cationic charges. FEMS Immunol. Med. Microbiol. 44, 289-295 (2005).
Modica-Napolitano, J. S. & Aprille, J. R. Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells. Adv. Drug Delivery Rev. 49, 63-70 (2001).
Kou, J., Dou, D. & Yang, L. Porphyrin photosensitizers in photodynamic therapy and its applications. Oncotarget 8, 81591 (2017).
Jones, R. N. Resistance Patterns Among Nosocomial Pathogens: Trends Over the Past Few Years. Chest 119, 397S-404S (2001).
Sabino, C. P. et al. Global priority multidrug-resistant pathogens do not resist photodynamic therapy. J. Photochem. Photobiol., B 208, 111893 (2020).
Yu, C. et al. Ti3C2Tx MXene loaded with indocyanine green for synergistic photothermal and photodynamic therapy for drug-resistant bacterium. Colloids Surf., B 217, 112663 (2022).
Boluki, E., Pourhajibagher, M. & Bahador, A. The combination of antimicrobial photocatalysis and antimicrobial photodynamic therapy to eradicate the extensively drug-resistant colistin resistant Acinetobacter baumannii. Photodiagn. Photodyn. Ther. 31, 101816 (2020).
Böhm, H. J. et al. Fluorine in medicinal chemistry. ChemBioChem 5, 637-643 (2004).
Stockert, J. C. et al. Regression of the murine LM3 tumor by repeated photodynamic therapy with meso-tetrakis-(4-N, N, N-trimethylanilinium) porphine. J. Porphyrins Phthalocyanines 13, 560-566 (2009).
Colombo, L., Juarranz, A., Cañete, M., Villanueva, A. & Stockert, J. Photodynamic therapy of the murine LM3 tumor using meso-tetra (4-N, N, N-trimethylanilinium) Porphine. Int. J. Biomed. Sci. 3, 258 (2007).
Cohen, B. A., Kaloyeros, A. E. & Bergkvist, M. Nucleotide-driven packaging of a singlet oxygen generating porphyrin in an icosahedral virus. J. Porphyrins Phthalocyanines 16, 47-54 (2012).
Hurst, A. N. The Synthesis of Photosensitizers for The Photodynamic Inactivation of Escherichia Coli., The University of North Carolina at Charlotte, (2017).
Hancock, R. E. & Bell, A. Antibiotic uptake into gram-negative bacteria. Perspectives in antiinfective therapy, 42-53 (1989).
Alenezi, K., Tovmasyan, A., Batinic-Haberle, I. & Benov, L. T. Optimizing Zn porphyrin-based photosensitizers for efficient antibacterial photodynamic therapy. Photodiagn. Photodyn. Ther. 17, 154-159 (2017).
Stojiljkovic, I., Evavold, B. D. & Kumar, V. Antimicrobial properties of porphyrins. Expert Opin Investig Drugs. 10, 309-320 (2001).
Tovmasyan, A., Batinic-Haberle, I. & Benov, L. Antibacterial Activity of Synthetic Cationic Iron Porphyrins. Antioxidants 9, 972 (2020).
Garcia-Sampedro, A., Tabero, A., Mahamed, I. & Acedo, P. Multimodal use of the porphyrin TMPyP: From cancer therapy to antimicrobial applications. PORPHYRIN SCIENCE BY WOMEN: In 3 Volumes, 11-27 (2019).
Bartoň Tománková, K. et al. Size-selected graphene oxide loaded with photosensitizer (TMPyP) for targeting photodynamic therapy in vitro. Processes 8, 251 (2020).
Acedo, P., Stockert, J., Cañete, M. & Villanueva, A. Two combined photosensitizers: a goal for more effective photodynamic therapy of cancer. Cell Death Dis. 5, e1122-e1122 (2014).
Malara, D. et al. Sensitivity of live microalgal aquaculture feed to singlet oxygen-based photodynamic therapy. J. Appl. Phycol. 31, 3593-3606 (2019).
Yang, R. et al. Scorpion-Shaped Zinc Porphyrins as Tetrafunctional TAR RNA Predators and HIV-1 Reverse Transcriptase Inhibitors. Inorg. Chem. 61, 10774-10780 (2022).
Wong, S.-Y., Sun, R. W.-Y., Chung, N. P.-Y., Lin, C.-L. & Che, C.-M. Physiologically stable vanadium (IV) porphyrins as a new class of anti-HIV agents. Chem. Commun., 3544-3546 (2005).
Dixon, D. et al. Amino-and hydroxytetraphenylporphyrins with activity against the human immunodeficiency virus. Antiviral Chem. Chemother. 3, 279-282 (1992).
Lebedeva, N. S., Gubarev, Y. A., Koifman, M. O. & Koifman, O. I. The application of porphyrins and their analogues for inactivation of viruses. Molecules 25, 4368 (2020).
Kubat, P. et al. Tetraphenylporphyrin-cobalt (III) bis (1, 2-dicarbollide) conjugates: from the solution characteristics to inhibition of HIV protease. J. Phys. Chem. B 111, 4539-4546 (2007).
DeCamp, D. L. et al. Specific inhibition of HIV-1 protease by boronated porphyrins. J. Med. Chem. 35, 3426-3428 (1992).
Schaeck, J. J. The synthesis of boronated porphyrins as inhibitors of HIV-1 protease. (University of California, San Francisco, 1997).
Rundstadler, T. et al. Gold(III) porphyrins: Synthesis and interaction with G-quadruplex DNA. J. Inorg. Biochem. 223, 111551 (2021).
Amrane, S., Andreola, M.-a., Pratviel, G. & Mergny, J.-L. Derivatives of porphyrins, their process of preparation and their use for treating viral infections. France patent US10577375B2 (2020).
Bullous, A. Development of porphyrin-antiangiogenic antibody immunoconjugates for photodynamic therapy PhD thesis, University of Hull, (2010).
Wang, C. & Wamser, C. C. Hyperporphyrin Effects in the Spectroscopy of Protonated Porphyrins with 4-Aminophenyl and 4-Pyridyl Meso Substituents. J. Phys. Chem. A 118, 3605-3615 (2014).
Wang, C. & Wamser, C. C. NMR Study of Hyperporphyrin Effects in the Protonations of Porphyrins with 4-Aminophenyl and 4-Pyridyl Meso Substituents. J. Org. Chem. 80, 7351-7359 (2015).
Szurko, A. et al. Photodynamic effects of two water soluble porphyrins evaluated on human malignant melanoma cells in vitro. Acta Biochim. Pol. 50, 1165-1174 (2003).
Boyle, R. W., Clarke, O. J., Sutton, J. M. & Greenman, J. Chromophores. US 2003O2O3888A1 (2003).
Khurana, B., Ouk, T.-S., Lucas, R., Senge, M. O. & Sol, V. Photosensitizer-hyaluronic acid complexes for antimicrobial photodynamic therapy (aPDT). J. Porphyrins Phthalocyanines 26, 585-593 (2022).
Antonangelo, A. R. et al. Synthesis, crystallographic characterization and homogeneous catalytic activity of novel unsymmetric porphyrins. RSC Adv. 7, 50610-50618 (2017).
Dechan, P., Devi Bajju, G. & Sood, P. Trans A2B2 Porphyrins: Synthesis, Crystal Structure Determinations and Hirshfeld Surface Analysis. ChemistrySelect 5, 7298-7309 (2020).
Mazumder, Z. H. Water-soluble, meso-substituted, free-base and metallated thiophenium and/or pyridinium and/or indole derivatives of porphyrin: Their Role as Photosensitizers PhD thesis, Assam University, (2017).
Sengupta, D. Synthesis, characterisation and biological activities of singly and doubly linked water-soluble porphyrin-fulleropyrrolidine dyads, University of Sydney, (2006).
Elkihel, A. et al. Cationic porphyrin–xylan conjugate hydrogels for photodynamic antimicrobial chemotherapy. J. Appl. Polym. Sci. 139, e52744 (2022).
Cheng, R.-J. et al. Symmetry and Bonding in Metalloporphyrins. A Modern Implementation for the Bonding Analyses of Five- and Six-Coordinate High-Spin Iron(III)−Porphyrin Complexes through Density Functional Calculation and NMR Spectroscopy. J. Am. Chem. Soc. 125, 6774-6783 (2003).
Fagadar-Cosma, E. et al. Syntheses, spectroscopic and AFM characterization of some manganese porphyrins and their hybrid silica nanomaterials. Molecules 14, 1370-1388 (2009).
Valicsek, Z., Horváth, O. & Stevenson, K. L. Photophysics and photochemistry of water-soluble, sitting-atop bis-thallium (I) 5, 10, 15, 20-tetrakis (4-sulfonatophenyl) porphyrin. Photochem. Photobiol. Sci. 3, 669-673 (2004).
Gouterman, M. Spectra of porphyrins. J. Mol. Spectrosc. 6, 138-163 (1961).
Myśliwa-Kurdziel, B., Solymosi, K., Kruk, J., Böddi, B. & Strzałka, K. Solvent effects on fluorescence properties of protochlorophyll and its derivatives with various porphyrin side chains. Eur. Biophys. J. 37, 1185-1193 (2008).
de Jong, M., Seijo, L., Meijerink, A. & Rabouw, F. T. Resolving the ambiguity in the relation between Stokes shift and Huang-Rhys parameter. Phys. Chem. Chem. Phys. 17, 16959-16969 (2015).
Mazumder, Z. H. et al. Photodynamic activity attained through raptured p-conjugation of pyridyl groups with a porphyrin macrocycle: Synthesis, Photophysical and Photobiological Evaluation of 5-mono-(4-nitrophenyl)-10,15,20-tris-[4-(phenoxymethyl)pyridine]porphyrin and its Zn(II) complex. Photochem. Photobiol. Sci. 19, 1776–1789 (2020).
Jaung, J.-Y. Synthesis of new porphyrins with dicyanopyrazine moiety and their optical properties. Dyes Pigm. 72, 315-321 (2007).
Zhang, L. et al. Thio-bisnaphthalimides as heavy-atom-free photosensitizers with efficient singlet oxygen generation and large stokes shifts: synthesis and properties. Org. Lett. 18, 5664-5667 (2016).
Zems, Y., Moiseev, A. G. & Perepichka, D. F. Convenient synthesis of a highly soluble and stable phosphorescent Platinum porphyrin dye. Org. Lett. 15, 5330-5333 (2013).
Platt, E. J., Wehrly, K., Kuhmann, S. E., Chesebro, B. & Kabat, D. Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1. J. Virol. 72, 2855-2864 (1998).
Checkley, M. A. et al. Reevaluation of the requirement for TIP47 in human immunodeficiency virus type 1 envelope glycoprotein incorporation. J. Virol. 87, 3561-3570 (2013).
Gonçalves, P. J. et al. Effects of environment on the photophysical characteristics of mesotetrakis methylpyridiniumyl porphyrin (TMPyP). Spectrochim. Acta, Part A 79, 1532-1539 (2011).
Matthews, T. et al. Enfuvirtide: the first therapy to inhibit the entry of HIV-1 into host CD4 lymphocytes. Nat. Rev. Drug Discovery 3, 215-225 (2004).
Zhou, Z.-X., Gao, F., Chen, X., Tian, X.-J. & Ji, L.-N. Selective Binding and Reverse Transcription Inhibition of Single-Strand poly(A) RNA by Metal TMPyP Complexes. Inorg. Chem. 53, 10015-10017 (2014).
Batishchev, O. V. et al. Antimicrobial activity of photosensitizers: arrangement in bacterial membrane matters. Front Mol Biosci 10, 1192794 (2023).
Hashimoto, M. C. et al. Antimicrobial photodynamic therapy on drug-resistant Pseudomonas aeruginosa-induced infection. An in vivo study. Photochem. Photobiol. 88, 590-595 (2012).
Orenstein, A. et al. The use of porphyrins for eradication of Staphylococcus aureus in burn wound infections. FEMS Immunol. Med. Microbiol. 19, 307-314 (1997).
Malik, Z., Ladan, H. & Nitzan, Y. Photodynamic inactivation of Gram-negative bacteria: Problems and possible solutions. J. Photochem. Photobiol., B 14, 262-266 (1992).
Agnihotri, S., Mukherji, S. & Mukherji, S. Size-controlled silver nanoparticles synthesized over the range 5–100 nm using the same protocol and their antibacterial efficacy. RSC Adv. 4, 3974-3983 (2014).
Valduga, G., Breda, B., Giacometti, G. M., Jori, G. & Reddi, E. Photosensitization of Wild and Mutant Strains ofEscherichia colibymeso-Tetra (N-methyl-4-pyridyl)porphine. Biochem. Biophys. Res. Commun. 256, 84-88 (1999).
Donnelly, R. F., McCarron, P. A., Cassidy, C. M., Elborn, J. S. & Tunney, M. M. Delivery of photosensitisers and light through mucus: Investigations into the potential use of photodynamic therapy for treatment of Pseudomonas aeruginosa cystic fibrosis pulmonary infection. J. Controlled Release 117, 217-226 (2007).

Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

Scheme1.png
Scheme 1: Synthesis of P₃AmM, c-P₂Am₂M, PAm₃M, P₃AmZM, c-P₂Am₂ZM and PAm₃ZM.

Download PDF

Version 1

posted

You are reading this latest preprint version

Photo-activated Efficacy Against HIV-1, Multiple Microbes and Biofilms by Zinc-complexes of Combinations of Cationic Ammoniumphenyl and Methylpyridinium-Porphyrins

Status:

Version 1

Abstract

Figures

Introduction

Results and Discussion

2.1 Synthesis, isolation and characterization:

2.2 Anti-HIV activity:

2.2.1 Ammoniumphenyl Porphyrins were not cytotoxic under non-PDT or PDT conditions.

2.2.2 Ammoniumphenyl porphyrins reduced virus infectivity under non-PDT conditions.

2.2.3 Ammoniumphenyl porphyrins inhibited HIV-1 virus production post-entry under non-PDT conditions

2.2.4 Ammoniumphenyl porphyrins strongly inhibited HIV-1 entry under PDT conditions

2.3 Anti-bacterial activity:

2.3.1 Assessment of anti-bacterial activity through disc diffusion experiment:

2.3.2 Minimal Inhibitory Concentration (MIC) of the porphyrin’s compounds:

2.3.3 Effect of porphyrins on the growth profile and biofilm producing ability of the bacterial isolates:

2.4 Molecular docking Outcomes:

Discussion

Conclusion

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

Status:

Version 1