A hypervalent Bismuth(III) derivative of Tetrakis-4-methoxyphenyl porphyrin: Synthesis, spectroscopic characterisation and morphological investigations

Article abstract of DOI:10.1016/j.molstruc.2019.127320

In this study, we aimed to synthesize as well as spectroscopically characterize a sitting-atop (SAT) macro molecule of Tetrakis-4-methoxyphenylporphyrinato Bi(III). The bismuth porphyrin was prepared by the reaction of free base macrocycle, 5, 10, 15, 20-

Full text of DOI:10.1016/j.molstruc.2019.127320

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A hypervalent Bismuth(III) derivative of Tetrakis-4-methoxyphenyl porphyrin:
Synthesis, spectroscopic characterisation and morphological investigations
Padma Dechan, Gauri Devi Bajju
PII:
S0022-2860(19)31429-2
DOI:
https://doi.org/10.1016/j.molstruc.2019.127320
MOLSTR 127320
Reference:
To appear in: Journal of Molecular Structure
Received Date: 7 September 2019
Revised Date: 29 October 2019
Accepted Date: 30 October 2019
Please cite this article as: P. Dechan, G.D. Bajju, A hypervalent Bismuth(III) derivative of Tetrakis-4-
methoxyphenyl porphyrin: Synthesis, spectroscopic characterisation and morphological investigations,
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A Hypervalent Bismuth(III) Derivative of Tetrakis-4-Methoxyphenyl porphyrin:
Synthesis, Spectroscopic Characterisation and Morphological Investigations
Padma Dechan and Gauri Devi Bajju*
Postgraduate department of chemistry, University of Jammu, Jammu-180006, India
Abstract
In this study, we aimed to synthesize as well as spectroscopically characterize a sitting-atop
(SAT) macro molecule of Tetrakis-4-methoxyphenylporphyrinato Bi(III). The bismuth
porphyrin was prepared by the reaction of free base macrocycle, 5, 10, 15, 20-Tetrakis-4-
methoxyphenylporphyrin H2TMP (I) with bismuth nitrate pentahydrate, Bi(NO3)3·5H2O in
dry pyridine. The spectroscopic data demonstrates the existence of a binuclear moiety
[Bi(TMP)NO3]2 in the solid state and even in solution in which two bismuth porphyrins are
interlinked by a bridging bidentate nitrate. Self dimerisation of bismuth porphyrin is expected
to arise due to strong tendency of Bi3+ ions to become hypervalent. The electronic absorption
as well emission bands in complex (II) are bathochromically shifted relative to compound (I)
with emission bands significantly quenched in the metal chelate. At pH ~5, complex (II) is
demetallized to form the dicationic analogs of I which tend to undergo prominent J-type
supramolecular aggregation. Demetallation is also followed when a dichloromethane solution
of metal chelate (II) is kept for a long period at room temperature. The macro rings were also
studied by nano technology like SEM, TEM, EDX and powder XRD. Thermal analysis and
invitro cytotoxicity screening of the macro rings against leukaemia K562 strains have been
further demonstrated.
Keywords: sitting-atop (SAT), binuclear moiety, bismuth porphyrin, bidentate nitrate,
hypervalent
1. Introduction
The first bismuth porphyrins, mesoporphyrin IX dimethyl ester and etioporphyrin bismuth
(III) was reported back in 1969 [1]. However, the UV-visible and elemental analysis data
alone could not establish the complete structure of these complexes. Later some additional
spectroscopic measurement data like IR, 1H NMR and mass spectrometry aided the enhanced
characterisation of third bismuth porphyrin i.e. bismuth octaethylporphyrin (OEP),
Bi(OEP)(O)NO2 [2]. When the size of a central metal ion is higher than that of porphyrinic
core, the metal ion sits above the N4 core plane of ligand thereby distorting it. The first
evidence for out of plane coordination of Bi3+ ion was reported by Tanya Barbour and co-
1
workers on the basis of variable-temperature H NMR investigation of [Bi(TTP)]NO3 [3].
The out of plane coordination mode of bismuth porphyrin owing to size mismatch between

the central metal ion and porphyrin core was later supported by the first single crystal XRD
report of another bismuth porphyrin, Bi(OEP)SO3CF3 [4]. In the solid state, this complex
exists as dimer with two [(OEP)Bi]+ cations inter linked via three oxygen atoms belonging to
-
two symmetrically related SO3CF3 anions [4]. Solid state dinuclear bismuth porphyrins
containing halide anion, Bi(tpClpp)(X), X = Cl, Br and nitrate anion, Bi(tpClpp)(NO3) were
also reported by Bernard and co-workers [5]. The halide complexes Bi(tpClpp)(X), X = Cl,
Br crystallizes as centrosymmetric dimers doubly bridged through Cl or Br, whereas the
nitrate complexes, Bi(tpClpp)(NO3) crystallises as a less symmetric dimer in which one NO3
ligand bridges the two bismuth centres and the second NO3 ligand interacts with only one
bismuth atom. In its dinuclear form, bismuth centre acts as strong Lewis-acid and hence
become ‘hypervalent’, a tendency satisfied by self association and sometimes by solvent
coordination [6-9]
.
In this report we present similar hypervalent interaction of a Bi3+ derivative of Tetrakis-(4-
methoxyphenyl) porphyrin and on the basis of our findings we suggest that the judicious
choice of chelating ligands (very less sterically demanding) may allow identification of
‘hypervalency’ even in those systems for which crystallographic analyses are not
forthcoming. The remarkable history of medicinal use of bismuth compounds [10-14] led us
investigate the antitumor activity of the synthesized macro ring II against Leukaemia K562
strains and compare the results with its free base analog I. Thermal stability of both the
macrocycles has been further demonstrated. The proposed structure of synthesized complex
II is given in Scheme 1. The scheme explains that the Lewis acidity of Bi3+ centre is satisfied
by interactions with two oxygen atoms of a bridging bidentate nitrate from neighbouring Bi
atom, giving a dimer moiety.
Scheme 1 Proposed structure of II

2. Experimental
2.1. Materials and methods
The analytical reagent grades chemicals were used for the synthesis of I and II. The AR
grade Chloroform, dichloromethane, pyridine, pyrrole, 4-methoxybenzaldehyde, propionic
acid and conc. HCl were purchased from Sigma-Aldrich and were used as received. The
metal carrier, Bi(NO3).5H2O and K562 cell lines established from human chronic
myelogenous leukaemia (CML) cells in blastic crisis were also purchased from Sigma
Aldrich.
2.2. Analytical methods
FT-IR spectra of the compounds were recorded between 4000 cm-1 to 400 cm-1 using KBr
1
pellets on Perkin Elmer Spectrum. H NMR spectra were recorded on Bruker spectrometer,
model AV 400 N (400 MHZ), using CDCl3 as solvent and TMS as internal reference at room
temperature. UV-Visible spectra were recorded on PG spectrophotometer in dichloromethane
ranging from 300 to 700 nm. To introduce acidic conditions in the organic solvent containing
porphyrinic dye, stock aqueous solutions of 0.1M HCl were used. The pH values of the
solution were measured with a pH paper. Photoluminescence emission studies were recorded
on Cary eclipse Agilent using dichloromethane as solvent. Mass spectra were acquired on
Bruker Compass Data Analysis 4.1. TGA were recorded on Perkin Elmer under nitrogen
atmosphere. The phase purity and crystallite sizes of the as-prepared samples were
determined from powder X-ray diffraction (PXRD) using XPERT-PRO Diffractometer in the
2θ range of 4° to 80°, with CuKα radiation (λ = 1.540 Å). Scanning electron microscopy
(SEM) micrographs of the samples were recorded on FEI Nova Nano SEM 450.
Transmission electron microscopy (TEM) was recorded on Tecnai G2 20 S-TWIN
transmission electron microscope with a field emission gun operating at 200 kV. The samples
for TEM measurements were prepared by evaporating a drop of the colloid onto a carbon-
coated copper grid.
2.3. Cytotoxicity assay against leukaemia K562 cells
Cell culture: The cells were cultured in RPMI-1640 medium supplemented with 10% fetal
bovine serum, 100µg/ml penicillin, 10µg/ml streptomycin and 2mmol/l L- glutamine. The
cells were maintained in log phase growth at 37 ˚c in a humidified atmosphere containing 5%
CO2. The leukaemia K562 cells were treated with I and II at different concentrations of 1, 5,
10, 20, 30, 40 and 50 µM for 48h. In vitro cytotoxicity against leukaemia K562 cells was
determined using sulforhodamine B dye assay [15, 16].
Synthetic procedure

2.4. Synthesis of I
The starting free base porphyrin H2TMP, I was synthesized according to modified Adler’s
method [17]. Equivalent moles of pyrrole (2.08 mL, 20mmol) and 4-methoxybenzaldehyde
(3.65 mL, 20mmol) were added to the propionic acid solution (50 mL) in a 250 mL round
bottomed flask fitted with water condenser (scheme 2). The reaction mixture was then
refluxed at 60˚C for half an hour in dark. At the end of the reaction, the mixture was allowed
to cool at room temperature and then filtered. The residues were allowed to dry in vaccum
dessicator overnight and the crude product was chromatographed on a silica gel column using
pure methanol as eluent. The porphyrin elutes from the column as purple band. The solid
compound obtained after evaporating the organic solvent was finally recrystallized from
ethylacetate/petroleum ether mixtures (8:2).
I was obtained as purple solid with 60% yield. FT-IR (KBr, νmax, cm-1): 3317.66 ν(N-
H)pyrrole, 730.46 γ(C-H)ph, 800.04 γ(C-H)pyrrole, 1341.22 ν(C-N), 1595.22 ν(C=N),
2913.18 ν(C-H) OCH3, 1023.84 ν(C-O-C)sym, 1233.98 ν(C-O-C)asym.1H NMR (CDCl3,
400MHz, δ, ppm): -2.74 (s., 2H, NH), 4.12 (s., 12H, OCH3), 7.31-7.33 (d., 8H, ArHm), 8.14-
8.16 (d., 8H, ArHo), 8.89 (s., 8H, βH). UV-Vis (λmax, nm, CH2Cl2): 421 (B band), 519, 555,
594, 651 (Q bands).
2.5. Synthesis of II
Bismuth porphyrin was synthesized according to the method provided in the literature [4]. In
a typical reaction, 0.0813 mmol of H2TMP and 0.813 mmol of Bi(NO3)·5H2O were added to
a basic pyridine solution (30 mL) in a 250 mL round bottomed flask fitted with water
condenser (scheme 2). The mixture was then refluxed at 65˚C for 1 hour in dark under inert
atmospheric conditions. As the reaction progresses the solution color changed from dark
purple to dark green. The progress of the reaction was also determined
spectrophotometrically (by noting the position of B and Q bands). As the reaction completed,
the mixture was allowed to cool at room temperature and the solvent was completely
removed using rotary vapour. The crude green residues were then chromatographed on silica
gel column. Elution with dichloromethane first removed a faint purple fraction containing
unreacted H2TMP. The desired compound II eluted later from the column as a dark green
band using methanol/dichloromethane fractions (80:20).
Complex II was obtained as green solid with 40% yield. FT-IR (KBr, νmax, cm-1): 1349.92
ν(C-N), 1602.55 ν(C=N), 2834.84 ν(C-H), 1035.58 ν(C-O-C)sym, 1245.78 ν(C-O-C)asym
,
1
989.30 (Bi-N). H NMR (CDCl3, 400MHz, δ, ppm): 4.15/4.19 (s., 12H, OCH3), 7.35-
7.37/7.55-7.58 (d., 8H, ArH3,5), 8.57-8.59/8.73-8.75 (d., 8H, Ar2,6), 9.28 (s., 8H, βH). UV-Vis

(λmax, nm, CH2Cl2): 454, 471 (soret bands) and 605, 680 (Q bands). MS (intensity): m/z
941.27 [Bi(III)(TMP)]+.
3. Results and Discussions
3.1. Syntheses
The starting material I was prepared according to the modified Adler’s method [17]. For the
preparation of complex II, we followed the same procedure as described in the literature [4].
However, in our method we have used only 30 mL of basic solvent i.e. pyridine against 100
mL used in the previous method for the same molar ratios (~ 1:10) of the reactants i.e. free
base and Bi(NO3).5H2O mixtures. Also in the previous method, the free ligand,
Tetramesitylporphyrin and Bi(NO3).5H2O were needed to reflux for 5 days. However, in the
present method, the chelating ligand I was subjected to Bi3+ insertion by refluxing the two
reactants i.e. Tetra-para-methoxyphenylporphyrin and Bi(NO3).5H2O for just one hour under
dark in an inert atmospheric conditions. Inert atmosphere was used since bismuth is highly
sensitive towards reactive oxygen [18]. Dark environment during the entire refluxing period
was maintained to avoid the effect of light on the highly conjugated π system. The crude
metal porphyrin like in previous method was purified on silica gel column using
CH2Cl2/CH3OH mixtures. However in our case, we had to slightly increase the polarity of the
eluents as the metal complex was reluctant to elute from the column using low polar eleunts.
As a result, we were needed to reconsider the CH2Cl2/CH3OH ratio as 20:80.
Scheme 2 Synthetic route for I and II

FT-IR spectroscopy, 1H NMR spectroscopy, Mass spectrometry, Powder XRD data and UV–
Vis spectroscopy aided the full characterization of Tetra-para-methoxy phenyl porphyrin and
its Bi3+ analogue.
3.2. FT-IR spectroscopy
The selected regions of the FT-IR spectra of the synthesized porphyrins are presented in Fig.
2. The full spectrum is provided in the supplementary file as Fig. S1. The free base porphyrin
I exhibit bands occurring due to N–H stretching and bending vibrations at 3315.03 and
981.17 cm-1 [Fig. 2b]. These bands are however absent in the bismuth porphyrin due to the
replacement of two imino protons by the metal ion [19]. The bismuth porphyrin in fact
exhibits characteristics bands due to Bi–N stretching [Fig. 2b] at 989.30 cm-1. A band due to
aromatic ring vibration at 964.23 cm-1 in I appear as diminished broad band in II. FT-IR
spectroscopy also helped in determining the mode in which the nitrate anions coordinate with
the Bi3+ ion in complex II. Nitrate ion can coordinate to a metal in three different modes as
shown in Fig. 1. In particular the ability of nitrate to act as a bridge between two metal ions
plays important role in the formation of unique multinuclear coordination complexes [20-22].
(a)
(b)
(c)
Fig. 1 Different coordination modes of the nitrate ion: (a) monodentate, (b) bidentate
bridging and (c) bidentate chelating.
The FT-IR spectra of II indicate the presence of bidendate nitrates. Peak due to N=O
stretching mode (ν1) appear at 1465.63 cm-1 [Fig. 2c]. The asymmetric stretching mode (ν5)
of the –NO2 group in the complex appear at 1286.28 cm-1 [Fig. 2c]. The symmetric stretching
mode (ν2) of the –NO2 group in the complex appear at 1018.23 cm-1 [Fig. 2b]. The separation
between the two high frequency bands is ~180 cm-1 indicating bidendate mode of
coordination of nitrate group with the metal ions. The symmetrical in plane bending (ν4) of
NO2 coordinated with the metal ion is split into two bands [Fig. 2b] and can be assigned at
711.60 and 725.10 cm-1 or 740.53 and 755.95 cm-1 [23]. The FT-IR spectroscopy also helps
in determining the dinuclear bismuth porphyrin. The new bands ~560-580 cm-1 [Fig. 2a] and
~636-680 cm-1 (Fig. 2b) in the metal complex II are attributed to symmetric and asymmetric

stretching vibrations of Bi-O-Bi bonds [24, 25]. A small peak ~850 cm-1 [Fig. 2b] may be due
to Bi-O bending vibration in the metal complex [26].
Fig. 2 The FT-IR spectra of I (magenta) and II (blue).
3.3. 1H NMR spectroscopy
Fig. 3 and Fig. 4 represent the selected regions of the 1H NMR spectra of I and II in CDCl3 at
room temperature. The complete spectra are given in the supplementary file as Fig. S2 and S3
respectively. The pmr spectra of I consists of singlets due to β-pyrrole proton (Ha), para
phenyl methoxy proton (Hd) and imino proton (He) resonances accompanied by a pair of

doublets each consistent with ortho phenyl proton (Hc) and meta phenyl proton (Hb)
resonances. Upon Bi3+ insertion, the signal due to highly shielded imino protons in I
disappear due to the replacement of pyrrolic hydrogens by the metal ion confirming the
successful metallation of the chelating ligand [Fig. S3]. The characteristic feature is the
splitting of the signals due to (Hb) and (Hc) in bismuth porphyrin, II. This splitting of signals
may be ascribed to magnetic inequivalence of ortho and meta aryl protons due to out of plane
coordination of metal ion with ligand molecule I [3]. Complex II further shows splitting of
methoxy proton (Hd) signal which are not originated due to out of plane coordination of
metal ion and in fact may be attributed to the existence of a binuclear moiety with the two
bismuth (III) centres having different coordination numbers. The split signal is the pretence
of ‘hypervalent’ interaction in action which leads to self dimerisation even in solution. The
molecular complexation of I with Bi3+ ion causes downfield shifts of resonance signals. This
is due to increase in ring current produced by extension of the aryl π system.
Fig. 3 The selected regions of the 1H NMR spectra (400 MHz pulse FT) I ( 4.0 X 10-5 M) in
̴
CDCl3.

1
Fig. 4 The selected regions of the H NMR spectra (400 MHz pulse FT) II (
̴
4.0 X 10-5 M)
in CDCl3.
3.4. Mass spectrometry
Electrospray ionization mass spectrometry (ESI/MS) also allowed the characterisation of
bismuth porphyrin dimer. Fig. 5 represents the mass spectrum of metalloporphyrin II. The
dissociation of [(BiTMP)NO3]2 (molecular weight 2007.64g/mol) produce (BiTMP)+ and
[Bi(TMP)(NO3)2]+ ions with m/z 941.27 and 1065.46 (calculated value 940.82, 1064.82).
The bismuth porphyrin [(BiTMP)NO3]2 also dissociates to produce ions with m/z 1470.62
+
and 561.11 (calculated value 1465.68 and 540.96) which may correspond to (TMP)2 and
+
(BiNO3)2 species. The peak with m/z 1676.58 (calculated value 1673.66) may be due to the
formation of [Bi(TMP)2]+. The highest intensity peak with m/z 735.31 (calculated value
733.84) correspond to (TMP)+.

Fig. 5 Mass spectrum of II
4. Powder XRD analysis
The structure and phases of the resulting macro rings I and II were measured using powder
X-ray diffraction (PXRD). Fig. 6 shows the X-ray diffraction patterns of I and II. The peaks
at 2θ 6.35˚, 12.57˚, 14.71˚, 19.06, 25.24˚ and 28.38˚ in I [Fig. 6a] corresponds to diffraction
from (111), (310), (321), (422), (620) and (711) planes respectively. Similarly the XRD peaks
at 2θ 6.45˚, 10˚, 12.72˚ 17.72˚, 19.66˚, 32.88˚ and 47.08˚ in II [Fig. 6b] are consistent with
diffraction from (211), (321), (422), (622), (633), (1055) and (1297) planes respectively. The
diffraction peaks corresponding to metal complex II [Fig. 6b] are significantly different from
that of I suggesting appreciable change introduced in the crystal structure of I upon
incorporation of metal ion. The XRD patterns indicate that I crystallize in face centred cubic
geometry (JCPCDS card number 77-0437) whereas II crystallizes in the body centred cubic
geometry (JCPCDS card number 45-0109). The high intensities of the diffraction peaks in I
indicate high crystalline nature of the compound. The diffraction peaks in II are broader
especially at larger 2θ values. The average crystallite sizes of the macro rings were measured
from the characteristic peaks using Scherer’s equation (1)
D=0.9λ/βcosθ
(1)

Where D is the crystallite size, λ is the wavelength of the X- ray, β is the FWHM (full width
at half maximum) of the diffraction peak, θ is the diffraction angle and k is the Scherer’s
constant of the order of unity. The average crystallite sizes calculated using three higher
intensity peaks at 2θ 6.35˚/18.85˚/25.18˚ (I) and 6.42˚/17.72˚/32.88˚ (II) was found to be
60.45 (I) and 31.11 nm (II). The calculated result indicates that metal coordinated porphyrin
particles are of very small crystallite size. The smaller interplanar spacing (Table S1) in II
may ascribe to close intermolecular interaction induced by bridging bidentate nitrate group.
The scattering angles and calculated d-spacings of the most intense scattering peaks are
summarised in Table S1.
Fig. 6 X-ray powder diffraction patterns of (a) I and (b) II.
5. Absorption spectroscopy

The UV-Visible absorption and excitation spectra of I in CH2Cl2 are given in Fig. S4. The
absorption spectrum of I manifest five characteristic bands [Fig. S4 (left)]. The most intense
band at 422 nm is the B band and the four weak bands between 518 to 652 nm are the Q
bands. The B band originates because of π, π∗ transition from the ground singlet state to the
second excited singlet state (S0 → S2), while the Q bands arise due to π, π∗ transitions from
the ground singlet state to the first excited singlet state (S0 → S1) [27]. Both B and Q bands
arise from π−π electronic transitions. In comparison to the spectra of tetraphenyl porphyrin
(B band at 416 nm) under the same solvent [28], the absorption bands in tetra para
methoxyphenyl porphyrin are shifted towards longer wavelengths. This is attributed to
increased π conjugation and reduction of the HOMO–LUMO energy gap induced by the
electron donating para methoxy substituents in I [29, 30]. The excitation spectra of I [Fig. S4
(right)] is in good agreement with its corresponding absorption spectra.
The coordination of Bi3+ ion to the nitrogen atoms of I results in the marked splitting of B
band followed by reduction in the number of Q bands to only two [Fig. 7]. The split B bands
appear at 453 and 472 nm, while as the two Q bands are centred at 605 and 680 nm
respectively. The splitting of B band and whole spectral broadening reflects the existence of
binuclear motifs in the solution. The splitting occurs due to the exciton coupling between the
two porphyrin macrocycles in the ground state [31, 32, 33]. The split bands are separated by
λmax ~ 18 nm [Fig. 7 (right)]. The reduced number of Q bands in metal porphyrin is due to the
increase in the symmetry (D4h) of the macro ring. The presence of two diagonally positioned
hydrogens within the porphyrin core of free base ligand decreases the symmetry of macro
ring to D2h [34].
The bathochromic shift of absorption bands in II relative to I is ascribed to electronic as well
as steric effect induced by complexation of I with heavy metal ion (Bi3+). These effects can
be explained as the atomic orbitals of weakly bonded metal centre affects the unoccupied
metal orbitals of porphyrin more than the occupied ones resulting in the reduction of the
HOMO-LUMO energy gap (electronic factor) while as distortion of ligand plane due to
sitting atop coordination of metal ion results in twisting of phenyl rings from almost
perpendicular orientation to the planar ones favouring strong interaction to occur between the
peripheral aryl rings and porphyrin π system (steric factor) [35, 36, 37]. In addition to B and
Q bands, the metalloporphyrin II manifests an extra absorption band at 328 nm characteristic
of a hyper p type porphyrin [Fig. 8 (left)]. This band appear due to charge transfer electronic
transition from filled 6p orbitals of Bi3+ centre to empty π∗ orbitals of porphyrin [38].

Out of plane (OOP) metalloporphyrins are kinetically labile in nature due to the fact that the
weakly bonded metal ion in OOP complexes lies out of ligand plane and can be easily
demetallized under certain conditions like presence of acid [39]. Similar behaviour is also
reflected by the bismuth porphyrin in the present case. At pH ~ 5, notably a split B band
appears at 422 and 454 nm accompanied by a high energy red shifted QI band centred at 691
nm which is forbidden otherwise [Fig. 8 (left)]. The splitting of B band into two individual
Lorentzian bands and appearance of high energy red shifted QI band signifies metal removal
followed by formation of dications which tend to undergo J-type (head to tail) intermolecular
aggregation [40, 41, 42]. The dications are generated due to the replacement of Bi3+ ion from
the porphyrin ring of I by the protons of the acid. The lower energy band at 422 nm is
consistent with that of electrically neutral free base monomer, whereas the higher energy
band at 454 nm (J-band) corresponds to the J aggregated dications. The small bands at 517
and 557 nm further correspond to the Q bands of neutral free base monomer. The band at 333
nm (marked with star) is attributed to charge transfer transition from peripheral para methoxy
phenyl rings (π) to protonated porphyrin core (π*) [42].
A dichloromethane solution of macro ring II is initially green in color characteristic of
bismuth porphyrins. However, when the solution is kept for a long period at room
temperature, the solution becomes acidic (turns litmus paper to pink) followed by
precipitation of a highly shine blue crystals. When the blue crystals separated by filteration
method were redissolved in dichloromethane, notably the solution becomes deep green. The
UV-Visible spectrum of solid blue precipitates measured in dichloromethane [Fig. 8 (right)]
is consistent with the absorption spectrum of II measured in acidic media [Fig. 8 (left)]. This
entire phenomenon may be attributed to dichloromethane induced demetallation of II
followed by self association of the demetallized porphyrins. Bismuth with vacant 6p and 4d
orbitals acts as strong lewis acid and accepts the lone pair electrons of porphyrin N4 core.
When the metal complex is kept dissolved in dichloromethane for a longer time, it is possible
that the out of plane and weakly coordinated metal ion with vacant 6p and 4d orbitals
interacts with the lone electron pair of chlorine atoms of CH2Cl2 moiety more strongly than
that of nitrogen atoms of porphyrin core, leading to demetallation followed by protonation of
the inner nitrogen atoms of porphyrinic core. The protonated analog of I then undergoes
aggregation in J-type fashion as reflected by the appearance of J and QI band in the
absorption spectrum (Scheme 3).

Fig. 7 [left] The UV/Vis absorption spectra of II in CH2Cl2 at increasing concentration [right]
enhanced view of B bands.
Fig. 8 [left] The UV/Vis absorption spectra of II in CH2Cl2 at pH ~ 5 [right] absorption
spectra of blue precipitates.

Scheme 3 Demetallation pathway of II in CH2Cl2.
6. Emission spectroscopy
Fig. 9 represents the emission spectra of macro rings at two different excitation wavelengths
i.e. 390 and 454 nm. At 390 nm excitation [Fig. 9 (a)], the macro rings reflects the usual S1
emissions i.e. Q(0,0) and Q(0,1) in the visible region and the less common S2 fluorescence
(with a small shoulder band) designated as B(0,0) in the near UV region marked with star.
The Q(0,0)/Q(0,1) bands for the macro rings appear at 655/724 (I) and 660/728 nm (II)
whereas, B(0,0) bands are centred at 436 nm with the shoulder bands appearing at 458 (I) and
471 nm (II) respectively. The shoulder peak in II is bathochromically shifted by λmax 13 nm
relative to I. The presence of less common S2–S0 emission in both the macro rings indicates
good mirror image relationship between their corresponding absorption and emission bands.
The intensity distributions of S1 emissions in I and II are consistent with the intensity
distributions of their corresponding S1 electronic absorption bands.
At 454 nm excitation [Fig. 9 (b)], the S1 emissions in I occurs at 656 and 720 nm whereas S2
emission (marked with star) appears at 480 nm. The Q(0,0) in II split into two bands
appearing at 725 and 743 nm. The split bands are separated by λmax ~ 18 nm. The other bands
i.e. Q (0,1) and B(0,0) bands are centred at 779 and 487 nm respectively. It can be observed
from Fig. 10 that the emission peaks in bismuth porphyrin are bathochromically shifted
relative to free base ligand. The bathochromic shift is more pronounced at higher excitation
wavelength. The phenomenon is attributed to distortion of macrocyclic ring due to out of
plane coordination of Bi3+ ion with the ligand molecule.

Fig. 9 The emission spectra of I and II at (a) 390 and (b) 454 nm excitations in CH2Cl2.
In case of compound I on absorbing the energy from UV-Visible radiation, the π electrons of
the conjugated system undergo excitation from singlet ground state S0 to first excited singlet

state S1 or second excited singlet state S2 [Fig. 10]. After a very short time span in the excited
state, the π electrons return to the ground state by emitting the fluorescence ~ 400 and 600–
750 nm respectively. In case of metalloporphyrin II, Bi3+ ion (heavy metal ion) induces orbit
coupling which results in the acceleration of the intersystem crossing S1 →T1 [43]. Thus the
density of electrons accumulated in the triple state increases and since many electrons hop to
the excited triplet state, the emitting fluorescence drop dramatically. As a result due to
combination of non planarity, heavy atom effect and efficient energy transfer between the
two porphyrin molecules in the dimer moiety due to strong intermolecular interactions, the
emission bands in II are quenched relative to that in I [44].
Fig. 10 The energy level diagram of fluorescence of porphyrins
The lifetime of an excited state is the average time a fluorophore spends in the excited state
before returning to the ground state by emitting a photon. The fluorescence lifetimes for the
macro ring at 390 and 454 nm excitations (Fig. S5-S6) were found to be 7.56 (I)/7.0(II) and
7.58(I)/3.03(II) ns respectively. Since the lifetime measurements of the fluorophores were
recorded in uniform solvent (CH2Cl2), the decay was found to be monoexponential in nature.
The coordination of Bi3+ ion to the nitrogen atoms of I reduces the excited state lifetime of
the ligand by ~ 0.5 ns (λexc 390 nm) and ~ 4.5 ns (λexc 454 nm) which is again attributed to
heavy atom effect generated by metal ion of large ionic radius.
7. SEM and TEM analysis
The morphological features of the synthesized porphyrins were investigated by SEM and
TEM analysis. Fig. 11 depicts the typical SEM micrographs of I and II. The SEM image of I
[Fig. 11 (left)] shows plate-like morphology with rough surface. After metallation with Bi3+
ion an appreciable change in surface morphology takes place [Fig. 11 (right)]. The surface
morphology in II is fairly uniform and smoother relative to I.

Fig. 11 SEM micrographs of I (a) and II (b).
TEM image of I [Fig. 12 (left)] shows that the monomers are stacked one above another in a
linear fashion leaving significant interplanar distance and imparting leave shaped morphology
or one can simply assume a linear extended aggregations of monomers of I as represented in
[Fig. 12 (right)]. The TEM micrographs of bismuth porphyrin [Fig. 13 (left)] clearly reflect
parallel displaced monomeric sub units of binuclear motifs forming nanosheet like structures.
A representation of parallel displaced mononuclear rings of II is illustrated by [Fig. 13
(right)]. Fig. 14 shows another TEM micrograph of II in which the monomers are involved in
the formation of number of slipped stacked multilayers. Some other TEM images showing
layered structures of II have been included in supplementary file as Fig. S7.
Fig. 12 TEM micrograph of I (a) and representation of supramolecular aggregation of
mononuclear rings of I (b).

Fig. 13 TEM micrograph of II (a) and representation of head to tail aligned monomeric
subunits of binuclear motifs of II (nitrate groups have been omitted for clarity) (b).
Fig. 14 TEM micrograph of II (a) representation of J-type aggregation of mononuclear rings
of II (nitrate groups have been omitted for clarity) (b).
8. EDS analysis
EDS analysis (Fig. S8-S9) allows determining the chemical compositions of I and II. In the
EDX profiles of porphyrins, the peaks of essential elements like C, N, O [I] and C, N, O, Bi
[II] which constitute the molecules of chelating ligand I and its metal chelate II are clearly
identified supporting the proposed structures.
9. Thermal analysis

In order to determine the temperature stability of the free base porphyrin I and its metal
derivative II, thermogravimetric analyses (TGA) have been carried out. Compound I is stable
up to ~250˚C [Fig. 15 (left)]. After this, the graph shows constant mass loss till temperature
reaches around ~ 450˚C. A drastic weight loss associated with the decomposition of
peripheral phenyl rings occurs at ~ 501.87 ˚C. The residual mass percent (45.929 %) at ~
800˚c is in agreement with the mass percent of highly stable tetrapyrrole macro ring
(42.78%). Compound II is stable up to ~ 150˚C [Fig. 15 (right)]. The compound then
undergoes constant weight loss up to ~ 400˚C. A sharp weight loss due to demetallation
leading to the decomposition of entire porphyrin scaffold takes place ~ 480˚C. The residual
mass (27.929%) of the compound at ~ 550˚c is comparable with the calculated weight of BiN
(23.69%). A comparison especially of the onset temperatures (OT) of decomposition of the
two macrocycles reflects that macro ring I is thermally more stable than II. The poor thermal
stability of II can be attributed to weaker bonds in the out of plane coordinated metal
porphyrin. Hence the obtained results confirm that the insertion of heavy metals like Bi3+
within the porphyrin ring system of I greatly affects their thermal robustness.
Fig. 15 Thermogravimetric analysis curves of I (red) and II (blue)
10. Cytotoxicity against leukaemia K562 cell lines
The invitro cytotoxicity assay of I and II was investigated against leukaemia K562 cell lines
for 48 h. Bismuth porphyrin exhibited much higher potency (IC50 = 40.08) as compared to
free base porphyrin having (IC50 > 100) classifying the former as chemotherapeutically
significant dye against leukaemia K562 strains [Fig. 16]. The greater antitumor activity of II

might be attributed to an increased cellular uptake of Bi3+ centre. The invitro cytotoxicity
data of porphyrins are summarised in Table S4.
60
(I)
(II)
50
40
30
20
10
0
% Growth
inhibition
0
1
5
10
20
30
40
50 IC50
Concentrations (µM)
Fig. 16 Cell inhibition at different concentrations of I (green) and II (dark blue) against
leukaemia K562 cell lines.
11. Conclusion
A Bi3+ derivative of Tetra-para-methoxyphenylporphyrin has been synthesized and
characterised with the aid of spectroscopic measurement and nanotechnology data. Due to
strong hypervalent nature of the central metal ion, Bismuth porphyrin tends to undergo
dimerisation in the solid state and to certain extend in solution. A dichloromethane solution
of II when kept for long time causes remarkable demetallation followed by simultaneous
protonation of the demetallized porphyrin. Demetallation also occurs when the media is
acidic in nature. The XRD result confirms that I and II belongs face centred and body centred
cubic geometries. SEM images reflect plate like morphology of the macro rings whereas,
TEM micrographs reveal the linear associations of monomers of I and slipped (head to tail)
stacked layers of particles of II. The EDX data confirms the chemical compositions of
porphyrins. TGA analysis reveals weak thermal stability of II. In the end, the incorporation
of Bi3+ ion into the porphyrinic core of I remarkably enhances the biological activity of I
against Leukaemia K562 strains.
Acknowledgements
This research was supported by Council of Scientific and Industrial Research (CSIR) India
(grant No. 09/100(0190)/2016-EMR-I).
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•
•
A sitting-atop (SAT) macro molecule of Tetrakis-4-methoxyphenylporphyrinato
Bi(III) has been synthesized.
Bismuth porphyrin exists as binuclear moiety owing to hypervalent nature of Bi3+
centre
•
•
•
•
Dinuclear bismuth porphyrin lie in head to tail stacking fashion
TEM images reflects mutilayered nanosheets of synthesized complex
Bismuth complex exhibit high antitumor activity against leukaemia K562 strains
Due to out of plane coordination mode, the synthesized complex have low thermal
stability

Declaration of interests
ꢀ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
ꢁ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: