Physicochemical and photodynamic antimicrobial chemotherapy activity of morpholine-substituted phthalocyanines: Effect of point of substitution and central metal

Article abstract of DOI:10.1016/j.jphotochem.2019.01.025

In this study, novel metal-free, zinc and indium 1(4),8(11),15(18),22(25)-tetramorpholine (1a, 2a, 3a respectively) and 2(3), 9(10), 16(17),23(24)-tetramorpholine (1b, 2b, 3b respectively) phthalocyanines were synthesized and complexes 2 and 3 were quater

Full text of DOI:10.1016/j.jphotochem.2019.01.025

Accepted Manuscript
Title: Physicochemical and Photodynamic Antimicrobial
Chemotherapy Activity of Morpholine-Substituted
Phthalocyanines: Effect of Point of Substitution and Central
Metal
Authors: Azole Sindelo, Nagao Kobayashi, Mutsumi Kimura,
Tebello Nyokong
PII:
S1010-6030(18)31539-9
DOI:
Reference:
https://doi.org/10.1016/j.jphotochem.2019.01.025
JPC 11684
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
25 October 2018
21 January 2019
24 January 2019
Please cite this article as: Sindelo A, Kobayashi N, Kimura M, Nyokong
T, Physicochemical and Photodynamic Antimicrobial Chemotherapy Activity of
Morpholine-Substituted Phthalocyanines: Effect of Point of Substitution and Central
Metal, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2019),
https://doi.org/10.1016/j.jphotochem.2019.01.025
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1
Physicochemical and Photodynamic Antimicrobial Chemotherapy
Activity of Morpholine-Substituted Phthalocyanines: Effect of Point
of Substitution and Central Metal
Azole Sindelo,a Nagao Kobayashi,b Mutsumi Kimurab and Tebello Nyokonga*
aCentre for Nanotechnology Innovation, Department of Chemistry, Rhodes University,
Grahamstown 6140, South Africa.
bDepartment of Chemistry and Materials, Faculty of Textile Science and Technology, Shinshu
University, Ueda 386-8567, Japan
Graphical abstract
bacteria
3O2
Laser
inactivation
1O2
E.coli
Highlights

Zn and In tetramorpholine phthalocyanines are synthesized and then quaternized

2


Photodynamic antimicrobial chemotherapy activities towards inactivation of E. coli, S.
aureus, and C. albican were evaluated
The quaternized derivatives showed total elimination of the micro-organism with log
reductions greater than 9.
Abstract
In this study, novel metal-free, zinc and indium 1(4),8(11),15(18),22(25)-tetramorpholine (1a,
2a, 3a respectively) and 2(3), 9(10), 16(17),23(24)-tetramorpholine (1b, 2b, 3b respectively)
phthalocyanines were synthesized and complexes 2 and 3 were quaternized. The
photophysical and photochemical properties were investigated in dimethylsulfoxide. The
non-peripherally substituted phthalocyanines generated higher singlet oxygen than the
peripherally substituted phthalocyanines. Photodynamic antimicrobial chemotherapy
activities towards inactivation of Escherichia coli, Staphylococcus aureus and Candida albican
were evaluated, where all the quaternized Pcs showed total elimination of the micro-
organism with log reductions greater than 9. Though the neutral Pcs had log reductions less
than 2, for C. albican the percentage reduction was 68.5% for 2b showing the antifungal
properties of the morpholine group.
Keywords: Phthalocyanine, morpholine, photophysics, fluorescence, singlet oxygen
*Corresponding author. Tel: + 27 46 6038260; Fax: + 27 46 6225109.
1. Introduction

3
Phthalocyanines (Pcs) are tetra pyrrolic macrocyclic compounds with multiple attractive
properties such as chemical inertness, long wavelength absorption compatible with biological
window, excellent thermal and photo-stability [1-5]. Due to these properties, Pcs have been
extensively used in various applications such as in sensors, light-emitting devices, solar cells,
photodynamic therapy (photodynamic antimicrobial chemotherapy, PACT), non-linear optical
materials, and catalysis [6-13]. PACT is based on the administration of a non-toxic
photosensitizer to the microbial cells followed by activation by light of appropriate
wavelength [14-16]. The photoexcited photosensitizer produces reactive oxygen species
(ROS) such as radicals and singlet oxygen (through Type I or Type II mechanism) that are
cytotoxic to the targeted bacteria [14,15]. MPcs are efficient photosensitizers for PACT [16].
The quaternized forms of In tetra pyridyloxy phthalocyanines have been employed for the
photoinactivation of different types of bacteria and fungi [17,18]. It has been reported that
cationic phthalocyanines are more effective than neutral and anionic Pcs against the Gram
(-) bacteria such as E.coli [19]. Gram (-) bacteria have a thick cell wall, which is absent in Gram
(+) bacteria such as S. aureus [19]. Hence in this work we employ morpholine substituted Pcs
which can easily be quaternized. In addition, morpholine derivatives are well known
antimicrobial agents [20] hence the combination of a Pc and morpholine is expected to result
in improved PACT activity.
To maximise the photophysical and photochemical properties of Pcs, modification of the Pc
ring can be achieved by tuning the central atom. The introduction of central metals such as
indium has been reported to reduce aggregation and improved solubility [21,22].
Furthermore, introducing substituents at the non-peripheral (α) and peripheral (β) positions
improves solubility of phthalocyanines in common organic solvents. In this study, novel
morpholine alpha and beta tetra-substituted phthalocyanines (complexes 1a, 1b, 2a, 2b, 3a,

4
and 3b, Scheme 1, and the quaternized derivatives of the metalled complexes: Q2a, Q2b,
Q3a, and Q3b, Scheme 2) are synthesized and their PACT activity evaluated. We compare
non-peripheral to peripheral substitution on the Pc ring for PACT activity.
2. Experimental
2.1 Material
Dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dichloromethane (DCM),
tetrahydrofuran (THF), chloroform, methanol and ethyl acetate were purchased from Merck,
while deuterated chloroform (CDCl3) was purchased from Associated Chemical Enterprises.
Morpholine, 4-nitrophthalonitrile, 3-nitrophthalonitrile, zinc (II) acetate (Zn(OAc)2), indium
(III) chloride, 1,3-diphenylisobenzofuran (DPBF), anthracene-9,10-bis-methylmalonate
(ADMA) and 1,8-diazabicycloundec-7-ene (DBU) were purchased from Sigma Aldrich, while
dimethyl sulphate bought from Riedel-de Haen. All solvents were dried using molecular
sieves. Phosphate-buffered saline (PBS) solution pH 7.4 was prepared using appropriate
amounts of Na2HPO4 and NaOH in ultra-pure water from a Milli-Q Water system (Millipore
Corp, Bedford, MA, USA). Nutrient agar and agar bacteriological BBL Muller Hinton broth were
purchased from Merck, E. coli (ATCC 25922) from Microbiologic. S. aureus (ATCC 25923) and
C. albican (ATCC 24433) were from Davies Diagnostics.
2.2 Equipment

5
Electronic absorption spectra of solutions were recorded on a Shimadzu UV-2550
spectrophotometer using 3 cm quartz cell cuvette. Infrared (IR) spectra were recorded on a
Bruker Alpha IR (100 FT-IR) spectrophotometer. Fluorescence emission spectra were
recorded on a Varian Eclipse spectrofluorimeter. Mass spectra data were collected with a
Bruker AutoFLEX III Smartbeam TOF/TOF Mass spectrometer and with Bruker microTOF II
1
mass spectrometer. The spectra were acquired using dithranol as the MALDI matrix. H
nuclear magnetic resonance spectra recorded on a Bruker AMX 600 MHz NMR spectrometer.
Triplet state quantum yields were determined using a laser flash photolysis system consisting
of an LP980 spectrometer with a PMT-LP detector and an ICCD camera (Andor DH320T-
25F03). The signal from a PMT detector was recorded on a Tektronix TDS3012C digital storage
oscilloscope. The excitation pulses were produced using a tunable laser system consisting of
an Nd:YAG laser (355 nm, 135 mJ/4–6 ns) pumping an optical parametric oscillator (OPO, 30
mJ/3–5 ns) with a wavelength range of 420–2300 nm (NT-342B, Ekspla). The absorbance of
solutions of the Pcs and the ZnPc standard were ~1.5 at Q band. The solution was introduced
into a 1 cm path length UV-visible spectrophotometric cell and de-aerated using argon for 15
min.
Fluorescence decay lifetimes were measured using a time-correlated single photon counting
(TCSPC) setup (FluoTime 300, Picoquant GmbH). The excitation source was a diode laser (LDH-
P-670 driven by PDL 800-B, 670 nm (Q2a, Q2b, Q3a and Q3b), 720 nm (1b, 2a and 2b) and
760 nm (1a and 3a) 20 MHz repetition rate, 44 ps pulse width, Pico quant GmbH). Photo-
irradiations for singlet oxygen quantum yield determination and antimicrobial studies were
performed using a General Electric Quartz lamp (300 W). A 680 nm glass (Schott) and water
filters were used to filter off ultra-violet and far infrared radiations. An interference filter

6
(Intor, 700 nm with a band width of 20 nm and) was additionally placed in the light path
before the sample. A light dose of 24 J/cm2 was employed. Light intensities measured with a
POWER MAX 5100 (Molelectron detector incorporated) power meter.
2.3 Syntheses
2.3.1 General procedure for the synthesis of phthalonitriles (4 and 5).
3-Nitrophthalonitrile or 4-nitrophthalonitrile (1.97 g, 10 mmol) and morpholine (3.9 g, 45
mmol) were dissolved in dry DMF (25 ml) and reacted with anhydrous Na2CO3 (3.12 g, 30
mmol). The reaction was stirred at 100 °C, for 18 h under inert conditions, using an oil bath
to maintain the temperature. The products were precipitated in water, and the yellow
precipitates formed were filtered under vacuum and recrystallized with methanol. The crude
products were purified further by column chromatography using alumina and eluting with
ethyl acetate. Both compounds are soluble in most organic compounds (DMF, DMSO,
methanol, ethyl acetate, DCM, chloroform and THF).
3-Morpholinophthalonitrile (4). Yield: 0.56 g (22.78 %). Anal calculated for C12H11N3O, C,
67.59 %; H, 5.20 %; N, 19.71 %; found C 67.68 %; H, 4.61 %; N, 19.74 %. IR (KBr, cm-1): 2967
(C-H), 2907 (CH2), 2869 (CH2), 2218 (C≡N), 1597, 1512 (C=C),1255, 1179, 1099 (C-O), 1038,
1
957, 866, 809, 723 (C-C). H NMR (CDCl3): δ (ppm), 7.49 (d, 1H), 7.14 (d, 1H), 7.07-7.03 (m,
1H), 3.80-3.77 (d, 4H, CH2), 3.27-3.24 (d, 4H, CH2). MALDI-TOF-MS m/z: Calc: 213.09; Found:
[M+1H] = 214.10.
4-Morpholinophthalonitrile (5). Yield: 1.18 g (48.56 %). Anal calculated for C12H11N3O, C,
67.59 %; H, 5.20 %; N, 19.71 %; found C 67.61 %; H, 4.98 %; N, 19.75 %. IR (KBr, cm-1): 3109

7
(C-H), 2909 (CH2), 2864 (CH2), 2221 (C≡N), 1592, 1507 (C=C),1257, 1179, 1110 (C-O), 1037,
1
951, 863, 817, 718 (C-C). H NMR (CDCl3): δ (ppm), 7.60 (d, 1H), 7.15 (d, 1H), 7.05-7.02 (m,
1H), 3.90-3.87 (d, 4H, CH2), 3.37-3.34 (d,4H, CH2). MALDI-TOF-MS m/z: Calc: 213.09; Found:
[M+1H] = 214.11.
2.3.2 General procedure for the synthesis of metal-free phthalocyanines (1a and 1b).
Compounds 4 and 5 (0.20 g, 0.94 mmol) were each mixed with 1- pentanol and DBU (0.25 ml),
followed by stirring for 20 min at 360 °C. The products were precipitated with dilute HCl. The
green products were filtered and further washed with water, methanol and acetonitrile. The
products were further purified by column chromatography with silica gel and eluting with
chloroform. Both complexes (1a and 1b) are soluble in CHCl3, THF, DCM, DMF and partially in
DMSO
1(4),8(11),15(18),22(25)-Tetrakis-(morpholino)phthalocyanine (1a). Yield: 13 mg (7%). Anal
calculated for C48H46N12O4, C, 67.43; H, 5.42; N, 19.66; found C 66.94 %; H, 5.08 %; N, 19.51
%. IR (KBr, cm-1): 3282 (N-H), 3018 (C-H), 2949 (CH2), 2811 (CH2), 1564 (N-H), 1485 (C=C),1309,
1222, 1107 (C-O), 1022, 963, 864, 799, 736 (C-C). 1H NMR (CDCl3): δ (ppm), 8.98 (s, 2H, NH),
7.80-7.76 (s, 4H, Ar-H) 7.40-7.11 (dd, 8H, Ar-H), 4.23-4.15 (d, 16H, CH2), 3.52-3.44 (d, 16H,
CH2). Uv-vis(DMF): λmax (nm), (log ε): 323 (4.33), 644 (4.18), 682 (4.27), 745 (4.70), 775 (4.73).
MALDI-TOF-MS m/z: Calc: 854.38; Found: [M+1H] = 855.12.
2.2.2.2 2(3),9(10),16(17),23(24)-Tetrakis-(morpholino)phthalocyanine (1b). Yield: 23 mg
(12%). Anal calculated for C48H46N12O4, C, 67.43; H, 5.42; N, 19.66; found C 67.34 %; H, 5.09
%; N, 18.26 %. IR (KBr, cm-1): 3280 (N-H), 3104 (C-H), 2899 (CH2), 2804 (CH2), 1557 (N-H),
1473 (C=C),1311, 1199, 1102 (C-O), 1032, 947, 804, 781, 731 (C-C). 1H NMR (CDCl3): δ (ppm),
9.02 (s, 2H, NH), 8.24- 8.18 (s, 4H, Ar-H), 7.78-7.62 (dd, 8H, Ar-H), 3.81-3.78 (d, 16H, CH2),

8
3.28-3.24 (d, 16H, CH2). Uv-vis(DMF): λmax (nm), (log ε): 346 (4.42), 634 (4.38), 658 (4.41), 709
(4.58), 739 (4.62). MALDI-TOF-MS m/z: Calc: 854.38; Found: [M+2H] = 855.99.
2.3.3 General procedure for the synthesis of metallophthalocyanines (2a, 2b, 3a and 3b).
Compounds 4 or 5 (0.30 g, 1.4 mmol) and 0.35 mmol of metal salt (0.097 g Zn(OAc)2 and 0.12
g InCl3) were mixed in 3 ml of dry 1-pentanol and 0.1 ml DBU. The mixtures were stirred for 8
h at 138 °C. The products were precipitated with 1:1 methanol: H2O, filtered and washed with
acetonitrile. The products were further purified by column chromatography with silica gel and
eluting with chloroform. All the complexes were soluble in CHCl3, THF, DCM, DMF and DMSO.
1(4),8(11),15(18),22(25)-Tetrakis-(morpholino)phthalocyaninotozinc(II) (2a). Yield: 50 mg
(23 %). Anal calculated for C48H44N12O4Zn, C, 62.78; H, 4.83; N, 18.30; found C 63.34 %; H, 4.09
%; N, 18.26 %. IR (KBr, cm-1): 3189 (CH), 2910 (CH2), 2837 (CH2), 1601 (C=C),1309, 1220, 1094
(C-O), 1019, 946, 879, 810, 734 (C-C). 1H NMR (CDCl3): δ (ppm), 7.60 (s, 4H, Ar-H), 7.06-6.93
(m, 8H, Ar-H), 3.86-3.79 (d, 16H, CH2), 3.41-3.29 (d,16H, CH2). Uv-vis(DMSO): λmax (nm), (log
ε):340 (4.53), 682 (4.21), 747 (4.96). MALDI-TOF-MS m/z: Calc: 916.29; Found: [M] = 916.26.
2(3),9(10),16(17),23(24)-Tetrakis-(morpholino)phthalocyaninotozinc (2b). Yield: 93 mg (42.8
%). Anal calculated for C48H44N12O4Zn, C, 62.78; H, 4.83; N, 18.30; found C 62.94 %; H, 4.078
%; N, 18.81 %. IR (KBr, cm-1): 3175 (CH), 2906 (CH2), 2824 (CH2), 1608 (C=C),1298, 1214, 1102
(C-O), 1020, 952, 869, 808, 729 (C-C). 1H NMR (CDCl3): δ (ppm), 7.71 (s, 4H, Ar-H), 6.92-6.72
(m, 8H, Ar-H), 4.18-3.49 (d, 16H, CH2), 2.23-1.57 (d,16H, CH2). Uv-vis(DMSO): λmax (nm), (log
ε): 364 (4.13), 644 (3.94), 717 (4.78). MALDI-TOF-MS m/z: Calc: 916.29; Found: [M] = 916.26.

9
1(4),8(11),15(18),22(25)-Tetrakis-(morpholino)phthalocyaninotoindium (3a). Yield: 43 mg
(19.8 %). Anal calculated for C48H44ClInN12O4, C, 57.47; H, 4.42; N, 16.75; found C 56.83 %; H,
4.18 %; N, 16.32 %. IR (KBr, cm-1): 3011 (C-H), 2948 (CH2), 2849 (CH2), 1493 (C=C),1330, 1225,
1104 (C-O), 1058, 985, 892, 777, 734 (C-C). 1H NMR (CDCl3): δ (ppm), 8.92 (s, 4H, Ar-H), 8.25-
7.20 (m, 8H, Ar-H ), 4.02-3.99 (d, 16H, CH2), 3.27-3.18 (d,16H, CH2). Uv-vis(DMSO): λmax (nm),
(log ε): 366 (4.08), 396 (3.94), 765 (4.71). MALDI-TOF-MS m/z: Calc: 1002.23; Found: [M] =
1002.49.
2(3),9(10),16(17),23(24)-Tetrakis-(morpholino)phthalocyaninotoindium (3b). Yield: 87 mg
(40 %).. Anal calculated for C48H44ClInN12O4, C, 57.47; H, 4.42; N, 16.75; found C 57.27 %; H,
4.34 %; N, 16.02 %. IR (KBr, cm-1): 3023 (C-H), 2909 (CH2), 2823 (CH2), 1510 (C=C),1396, 1198,
1108 (C-O), 1078, 962, 871, 762, 727 (C-C). 1H NMR (CDCl3): δ (ppm), 7.75 (s, 4H, Ar-H), 7.62-
7.58 (m, 8H, Ar-H), 4.28-4.23 (d, 16H, CH2), 3.93-3.88 (d,16H, CH2). Uv-vis(DMSO): λmax (nm),
(log ε): 363 (3.98), 665 (3.75), 736 (4.68). MALDI-TOF-MS m/z: Calc: 1002.23; Found: [M] =
1002.89.
2.3.4 General procedure for the synthesis of phthalocyanines methylation (Q2a, Q2b, Q3a
and Q3b).
Complexes 2a, 2b (0.010 g, 0.011 mmol) or 3a and 3b (0.010 g, 0.0099 mmol) and 5.05 mmol
of dimethyl sulfate were mixed in 3 ml of dry DMF, followed by stirring for 4 h at 155 °C. The
products were precipitated with acetone, filtered and washed with chloroform and diethyl
ether. All the complexes were soluble in H2O, DMF and DMSO.
1(4),8(11),15(18),22(25)-Tetrakis-(N-methylmorpholino)phthalocyaninotozinc(II)
(Q2a).
Yield: 3.4 mg (34 %). Anal calculated for C52H56N12O20S4Zn, C, 45.83; H, 4.14; N, 12.33; S, 9.41;
found C 43.14 %; H, 4.52 %; N, 11.55%; 9.72. IR (KBr, cm-1): 3042 (CH), 2923 (CH2), 1608

10
(C=C),1329, 1173, 1100 (C-O), 1041, 912, 850, 746 (C-C). 1H NMR (D2O): δ (ppm), 9.83 (s, 4H,
Ar-H), 9.35-8.41 (m, 8H, Ar-H), 4.58-4.50 (m, 16H, CH2), 4.31-4.25 (d,16H, CH2), 2.18 (s, 12H,
CH3). Uv-vis(DMSO): λmax (nm), (log ε):352 (4.84), 614 (4.53), 678 (5.20).
2(3),9(10),16(17),23(24)-Tetrakis-(N-methylmorpholino)phthalocyaninotozinc (Q2b). Yield:
5.2 mg (52 %). Anal calculated for C52H56N12O20S4Zn, C, 45.83; H, 4.14; N, 12.33; S, 9.41; found
C 44.78 %; H, 4.07 %; N, 11.92%; S, 9.35. IR (KBr, cm-1): 3038 (CH), 2919 (CH2), 1598 (C=C),1317,
1181, 1201 (C-O), 1001, 888, 832, 768 (C-C). 1H NMR (D2O): δ (ppm), 8.89 (s, 4H, Ar-H), 8.51-
8.07 (m, 8H, Ar-H), 4.34-4.27 (m, 16H, CH2), 4.08-4.02 (d,16H, CH2), 3.88 (s, 12H, CH3). Uv-
vis(DMSO): λmax (nm), (log ε):343 (4.98), 610 (4.64), 673 (5.33).
1(4),8(11),15(18),22(25)-Tetrakis-(N-methylmorpholino)phthalocyaninotoindium
(Q3a).
Yield: 2.7 mg (27 %). Anal calculated for C52H56ClInN12O20S4, C, 43.15; H, 3.90; N, 11.61; S, 8.86;
found C 44.03 %; H, 4.02 %; N, 11.89%; S, 9.06. IR (KBr, cm-1): 3042 (CH), 2923 (CH2), 1608
(C=C),1329, 1173, 1100 (C-O), 913, 861, 745 (C-C). 1H NMR (D2O): δ (ppm), 9.95 (s, 4H, Ar-H),
9.61-9.35 (m, 4H, Ar-H), 8.74-8.51 (m, 4H, Ar-H), 4.73-4.58 (m, 16H, CH2), 4.31-4.27 (d,16H,
CH2), 3.64 (s, 12H, CH3). Uv-vis(DMSO): λmax (nm), (log ε):353 (4.51), 621 (4.17), 689 (4.90).
2(3),9(10),16(17),23(24)-Tetrakis-(N-methylmorpholino)phthalocyaninotoindium
(Q3b).
Yield: 2.7 mg (27 %). Anal calculated for C52H56ClInN12O20S4, C, 43.15; H, 3.90; N, 11.61; S, 8.86;
found C 43.65 %; H, 4.11 %; N, 11.73%; S, 9.07. IR (KBr, cm-1): 3039 (CH), 2927 (CH2), 1617
(C=C),1334, 1197, 1008 (C-O), 938, 817, 739 (C-C). 1H NMR (D2O): δ (ppm), 9.63 (s, 4H, Ar-H),
9.45-9.27 (m, 4H, Ar-H), 9.03-8.78 (m, 4H, Ar-H), 4.52-4.41 (m, 16H, CH2), 4.38-4.26 (d,16H,
CH2), 3.22 (s, 12H, CH3). Uv-vis(DMSO): λmax (nm), (log ε):356 (4.72), 616 (4.46), 684 (5.11).

11
2.4 Photophysical and photochemical parameters
2.4.1 Fluorescence quantum yields (ΦF)
Fluorescence quantum yields were determined by the comparative method (equation 1)
using ZnPc as a standard (SFtd = 0.2 in DMSO) [23,24]
F AStd n2
FStdA (nStd)2
F  SFtd
(1)
where F and FStd are the areas under the emission curves of the sample and standard,
respectively, A and AStd are the absorbances of the sample and standard, respectively, and n
and nStd are the refractive indices of the solvents used for sample and standard, respectively.
Absorbance at the excitation wavelength was ~ 0.05 in order to minimize inner filter effects.
At least three independent experiments were performed for the quantum yield
determinations. Both the sample and the standard were excited at the same relevant
wavelength.
2.4.2. Triplet quantum yields (ΦT)
The triplet quantum yields were determined in DMSO using a comparative method, and ZnPc
as a standard in DMSO (T = 0.65 [25]), using Equations 2
std
∆A ε
std
∆A
T
T
T
ΦT = ΦTstd
(2)
ε
T
where ΦTstd is the triplet quantum yield of the standard, ∆AT and ∆AsTtd are the changes in
the triplet state absorbances of the sample and the standard, respectively, T and εsTtd are the
triplet state molar extinction coefficients for the sample and the standard, respectively

12
2.4.3. Singlet oxygen quantum yields (ΦΔ)
Eq. 3 was employed for the determination of ΦΔ employing ZnPc standard (ΦΔ = 0.67 in
DMSO) [25] and AlPcSmix (containing a mixture of differently sulfonated phthalocyanines,
and synthesized as reported in literature [26]) in aqueous media (ΦΔ =0.42) [25]. DPBF and
ADMA were used as singlet oxygen quenchers in DMSO and aqueous media, respectively.
Std
W I
Δ = Std
(3)
abs
Std
Δ
W
I
abs
where 훥푆푡푑 is the singlet oxygen quantum yield for the standard, W and WStd are the DPBF
and ADMA photobleaching rates in the presence of MPc derivatives under investigation and
푆푡푑
the standard, respectively, Iabs and
I
are the rates of light absorption by the MPc
푎푏푠
derivative and standard, respectively.
2.5 Antimicrobial studies
The nutrient broth and agar were prepared using manufacturer’s specifications. Micro-
organisms were cultured in broth, placed on a rotary shaker (∼200 rpm) at 37°C until they
reached an optical density of approximately 0.6. The resultant suspension was centrifuged at
3000 rpm for 15 min, the pellet was further washed thrice with PBS. A working solution was
prepared by diluting to 1/1000 (106 colony forming unit (CFU)/mL. Photodynamic
antimicrobial chemotherapy study was performed by dissolving the compounds in 0.2 %
DMSO for all complexes (quaternized and unquaternized) and in water for quaternized which
are completely soluble in water. For S. aureus and C. albican, 0.5 μM was prepared for each
sample and for E. coli 2.5 μM was applied. In all the experiments, the bacterial/fungal
suspensions were incubated in an oven equipped with a shaker for 30 min in the dark at 37°C.
Then, half (2.5 mL) of the incubated bacterial/fungal suspensions were irradiated at the Q-

13
band maximum of the photosensitizers in 24 well plate, using the set-up described in
supporting information and the other half kept in the dark. After irradiation, 100 µL samples
were diluted with 900 µL PBS and were spotted on agar plates using micropipette. The plates
were incubated at 37 oC for 24 h for both S. aureus and E. coli and for 48 h for C. albican. All
the studies were conducted in triplicates.
3. Results and discussion
3.1 Synthesis and characterization
The synthetic route followed is shown in Scheme 1 for the unquaternized Pcs. The syntheses
of phthalonitriles 4 and 5 were achieved through nucleophilic substitution between the nitro
group of the 3/4-nitrophthalonitrile and the NH group from the morpholine at 100 °C in dry
DMF with Na2CO3 as a base catalyst for 18 h under N2 gas. IR spectra confirmed the formation
of the morpholiniphthalonitriles (4 or 5) where there was a presence of two sharp -CH2 bands
at 2907, 2869 and 2909, 2864 cm-1, as well a strong -C≡N band at 2218 and 2221 cm-1 for 4
and 5, respectively. The cyclotetramerization of 4 or 5 was performed in the presence of DBU
and dry DMF producing the metal-free phthalocyanines 1a or 1b and with the addition of
metal salts; Zn(OAc)2, InCl3 for 2a, 2b, 3a and 3b. The metallophthalocyanines were further
quaternized using dimethyl sulfate (Scheme 2). IR spectra of the all Pcs showed the
disappearance of the -C≡N band of the phthalonitriles on formation of the Pcs, with H2Pcs (1a
and 1b) showing an additional –NH peak at 3282 and 3280 cm-1, respectively. The compounds
were further characterized using elemental analysis, 1H NMR, MALDI-TOF mass spectroscopy
1
and UV-vis. The results obtained were comparable with the proposed structures. H NMR
were recorded in deuterated CDCl3 for all the unquaternized compounds and D2O for the
quaternized compounds which gave the expected signals. For all the phthalocyanine

14
complexes, the aromatic protons appeared between 6.72 and 9.95 ppm, and the CH2 protons
between 1.59 and 4.58 ppm. The quaternized compounds had an additional sharp singlet
upfield (δ = 3.88-2.18 ppm) from the additional –CH3 protons. Compound 1a and 1b) had an
additional singlet signal at 8.98 and 9.02 ppm, respectively, attributed to the presence of –
NH. In mass spectroscopy, all the proposed structures were obtained with peaks at 855.15 as
[M+1H], 916.26 [M] and 1002.46 [M] for 1a, 2a and 3a, respectively.
3.2 Ground state electronic absorption properties
The UV-Vis spectra of complexes 2a, 2b, 3a, 3b, Q2a, Q2b, Q3a, Q3b) showed a single sharp
Q band typical of monomeric phthalocyanines [27]. The UV-Vis of the H2Pcs 1a and 1b (Fig.
1(i)) showed characteristic split Q band at 775 and 745 nm for 1a and 739 and 709 nm in DMF
for 1b (Table 1), respectively, confirming the structure to be of D2h symmetry. However,
Compounds 1a and 1b showed a single Q bands in DMSO (Fig. 1, insert), suggesting
deprotonation of the inner pyrrolic H atoms in the Pc core and thus, symmetry change from
D2h to D4h occurs due to basicity of these solvents [28].
Compounds 2a and 2b (Fig. 1(ii)) showed a Q-band at 747 and 717 nm and compounds 3a and
3b (Fig. 1(iii)) showed the characteristic Q-band peak at 765 and 736 nm in DMSO (Table 1).
The non-peripherally substituted compound 1a, 2a and 3a were red shifted when compared
to the peripherally substituted compounds 1b, 2b and 3b. This large-scale red shift can be
attributed to the linear combinations of the atomic orbitals coefficients at the non-peripheral
positions of the highest occupied molecular orbital (HOMO) being greater than that of
peripheral positions [29]. As a result, the HOMO level is destabilized more at the non-
peripheral position that it is at the peripheral position, resulting in the red shifting of the Q
band in the former.

15
For all Pcs there was an additional peak observed between 400 nm and 500 nm attributed to
charge transfer [30]. The Pcs were dissolved in THF, DCM and DMSO to examine the solvent
effect as well the aggregation behaviour of the newly synthesized Pcs, Fig. 2. All Pcs show no
aggregation in the above mentioned solvents. Lambert-Beer’s Law was obeyed in the
concentration range 5.5 × 10-6 to 3.5 ×10-5 mol/L (Fig. S1A for 2b used as an example) in DMSO.
With increase polarity of the solvent (THF < DCM < DMSO) there was a spectral shift as shown
in Fig. 2. The MPcs upon quaternization became blue shifted (Fig. 3) in DMSO and in aqueous
media containing 0.2% DMSO, Table 1. This is due to the lowering of the electron donating
ability of the nitrogen groups upon quaternization. There were slight red shifts in the Q band
maxima for the quaternized in aqueous media compared to in DMSO, Table 1, due to the
increase in polarity. Spectra were recorded in water containing 0.2% DMSO since this was the
media used for bacterial work. Phthalocyanines are known to aggregate in aqueous solution
[31], mainly through the formation of the so called H aggregates. This type of aggregation is
evidenced by broadening and splitting of the Q band, with the high energy band being due to
the aggregate and the low energy band to the monomer. The quaternized derivatives (Q2a,
Q2b, Q3a and Q3b) were soluble in aqueous solution and showed no sign of aggregation in
water alone or with 0.2% DMSO (Fig. 3 for Q3b used as an example for water plus 0.2%
DMSO). The spectra in water with or without 0.2% DMSO were similar. Water solubility and
lack of aggregation are imperative for the PACT applications. Lambert-Beer’s Law was obeyed
in water in the concentration ranging from 6.23 × 10-6 to 8.90 ×10-6 mol/L (Fig. S1B for Q2b as
an example). However, the neutral unquaternized derivatives (using complex 3b as an
example in Fig. 3), were highly aggregated as evidenced by the split Q band. This will affect
their PACT activity as will be discussed below.

16
3.3 Physicochemical parameters
3.3.1. Fluorescence quantum yields and lifetimes
The fluorescence absorption, excitation, and emission spectra of 2b is shown in Fig. 4. There
was splitting of the excitation spectrum showing change in symmetry following excitation.
Both indium Pcs exhibited very low intensity due to the heavy atomic effect of the central
metal hence not shown.
The fluorescence quantum yields were performed in DMSO and aqueous media using ZnPc
and AlPcSmix, respectively, as standards. Generally, all compounds (1a, 1b, 2a, 2b, 3a and
3b) had a weak fluorescence emissions and low ΦF values common to morpholine containing
Pcs [32].
Compounds 1a and 1b showed larger ΦF values of 0.08 and 0.09 respectively, compared to
the metalled Pcs. The presence of metals encourage the population of the triplet via
intersystem crossing for complexes 2a,b and 3a,b. The quaternized compounds Q2a and Q2b,
had an intense fluorescence emission, with higher ΦF values of 0.19 and 0.24, respectively.
The ΦF values for the quaternized complexes 3 are lower than for quaternized complexes 2,
since In is heavier than Zn, hence the former results in enhanced heavy atom effect. The ΦF
values in water decreased to 0.09, 0.11 for Q2a and Q2b. For Q3a and Q3b the values were
below 0.01 in aqueous media due to the quenching effect of water.
Time-correlated single photon counting was used to determine the fluorescence lifetime
(Table 1). All Pcs showed mono-exponential decay shown in Fig. 5 with their corresponding
residual graph in DMSO. The metal-free Pc 1a and 1b showed higher ns lifetimes of 2.08 and
5.48 due to the absence of a metal, while the metalled Pc showed a general decrease in

17
lifetimes. All the peripheral Pcs had a higher lifetime compared to the non-peripheral Pcs
highlighting the effects of substituent position.
3.3.2. Triplet quantum yields and lifetimes
Laser flash photolysis was used to determine the triplet quantum yields and triplet lifetimes
in DMSO using ZnPc as a standard. Transient absorption of the ZnPc (Fig. 6) (following
excitation at 682 nm for Q3a, 679 nm for Q3b, 680 nm for Q2a and 670 for Q2b) showed a
broad T1- Tn triplet absorption peak at 480 nm [33]. The unquaternized complexes (1a, 1b, 2a,
2b, 3a, 3b) gave a very weak signal. It has been shown before that nitrogen containing
substituents bound directly to the Pcs ring quench the triplet state [34], hence no triplet
quantum yield values in Table 1. In contrast, the introduction of the methyl groups to the
nitrogen atoms improved the signal (Fig. 6) with the ΦT values of Q2a, Q2b, Q3a, Q3b being
0.71, 0.66, 0.92, 0.87, respectively, Table 1. The non-peripheral Pcs had higher values than
peripheral Pcs, corresponding to the low fluorescence quantum yields of the former. The
heavier central atom, indium yielded a greater conversion to the triplet state than the
corresponding zinc complexes.
Triplet state lifetimes (τT) were studied to confirm the population of the triplet state, the
decay curve (Fig. 6, insert) showed the expected mono-exponential decay with heavy indium
Pcs (Q3a and Q3b) with lower lifetime of 76 and 92 μs when compared to 112 and 292 μs of
Q2a and Q2b, respectively. The peripheral Pcs had longer lifetime compared to their non-
peripheral counterparts due to the reasons stated above, corresponding to the large ΦT
values. Efficient spin orbit coupling (heavy atom effect) is known to result in shortening of
triplet lifetimes and increase in triplet quantum yields [35]. There were no signals in aqueous
media.

18
3.3.3. Photostability and singlet oxygen quantum yields
Prior to their use for PACT, the photostabilities of the compounds were evaluated. The
spectral changes of each compound were observed in DMSO after 30 seconds of irradiating
with light. Predominantly, all Pcs showed no to low significant changes upon the introduction
of light over time as shown in Fig. S2 (ESI). Both the Q-band and the B-band spectra maintain
their original shape and intensity, therefore our compounds were stable. Singlet oxygen
quantum yields (Φ) of the metal-free and metallated Pcs were determined in DMSO using
1,3-diphenylisobenzofuran (DPBF) and anthracene-9,10-bis-methylmalonate (ADMA in water
containing 0.2% DMSO) as a quenchers. The solutions were irradiated in 10 seconds intervals.
A similar trend to the photostability studies was observed where there was a minimal Q-band
change, Fig. 7. The degradations of the DPBF band at 417 nm and ADMA at 380 nm were
monitored (Fig. 7). Akin to the previous properties, the indium Pcs generated the highest
values of Φ, 0.68, 0.63, 0.66 and 0.62 for 3a, 3b, Q3a and Q3b compared to 0.54, 0.47, 0.58
and 0.50 of 2a, 2b, Q2a and Q2b, respectively in DMSO. The importance of metalation was
evident as the metal-free Pcs 1a and 1b illustrated considerably lower Φ than the metallated
Pcs due to the absence of a heavy metal. The Φ values decrease in water since water is a
known quencher [36]. This could also be due to the fact that oxygen has higher solubility in
many organic solvents used in this study compared to water [37], which could be responsible
for low singlet oxygen generation in water. Non-peripheral substituted Pcs (1a, 2a, 3a, Q2a
and Q3a) had larger Φ values compared to the corresponding peripherally substituted (1b,
2b, 3b, Q2b and Q3b) corresponding to the higher triplet quantum yields for Q2a and Q3a
compared to respective Q2b and Q3b.
3.4 Bacterial studies

19
The antimicrobial activities of 2a, 2b, 3a, 3b, Q2a, Q2b, Q3a and Q3b were studied against
gram positive S. aureus, gram negative E. coli and fungi C. albican. The phthalocyanines were
dissolved in 0.2 % DMSO (diluted with PBS) for all complexes and in water only for the
quaternized derivatives. A control using the same amount of DMSO in the culture was
assessed and the organic solvent had negligible effects hence used (Fig. 8(i)). For all Pcs the
optimum concentration was determined to be 0.5 μM for S. aureus and C. albican studies and
2.5 μM for E. coli. The bacterial suspension of 106 CFU/mL was used for all micro-organisms.
Log reductions and reduction percentage were used to quantify the results, Fig. 8, Table 2.
The cationic Pcs (Q2a, Q2b, Q3a and Q3b) had higher log reductions (9.90, 9.84, 9.92, 9.87)
than the neutral derivatives (1.17, 1.36, 1.03, 1.21) against E. coli in aqueous media containing
0.2% DMSO. The cationic Pcs are hydrophilic which encourages interaction between the dyes
and cytoplasm of the micro-organisms. For C. albican, the log reduction values for neutral Pcs
were below 2. But when evaluating the results using percentage reduction of the viable cells
(Fig. 8 (ii)), there was an observed increase of the reduction percentage for 2a, 2b, 3a, 3b of
60.2%, 68.5 %, 35.6 %, 40.3 % for C. albican, compared to 32.8 %, 48.6 %, 27.1%, 30.4 % for
S. aureus, respectively. This increase of the hydrophobic compounds was due to the
antifungal property of the morpholine group. Morpholines are known ergosterol synthesis
inhibitors, which is a hormone that stimulates the growth in fungi [38]. Moreover, the
methylated Pcs took less than 45 min to reduce C. albican colonies, which is faster than E. coli
and S. aureus. Generally, there was a moderate difference between the non-peripheral,
peripheral, zinc and indium Pcs. Additionally, the solubility improved the engagement
between the dye and the micro-organism hence the cationic compounds had very high log
reductions. The inability to penetrating or interacting with cell limits the singlet oxygen’s
potential to terminate the micro-organism. The low log reduction for the unquartenized

20
phthalocyanines could be a result of aggregation (shown in Fig. 3), which would affect the
penetration of dyes into the cell wall and membrane [39]. The log reduction values in the
presence of DMSO are not too different from the values in aqueous solution only for the
quaternized derivatives, Table 2.
4. Conclusions
The syntheses of novel non-peripherally and peripherally morpholine tetra-substituted metal
free phthalocyanines, Zn(II), In(III) and the quaternized metallated Pcs are presented. The
compounds were characterized using various methods such as elemental analysis, infrared,
ultra-violet visible, 1H nuclear magnetic resonance and MALDI-TOF spectroscopy. Generally,
the Pcs were soluble in most organic solvents such as DCM, THF, chloroform, DMF and DMSO.
The photophysical and photochemical parameters were studied in DMSO and singlet oxygen
quantum yields in water for the quaternized derivatives. The non-peripherally substituted Pcs
showed red shifted spectra when compared to the peripheral Pcs, with the H2Pcs showing a
split Q-band in DMF. The fluorescence quantum yield values decreased with the introduction
of metals and increased with quaternization. In turn, the singlet oxygen quantum yields were
significantly higher for the indium Pcs than the zinc and metal-free Pcs. All the non-peripheral
Pcs had higher Φ values compared to the peripheral value in DMSO. The Φ values of these

21
compounds range from 0.20 to 0.68 in DMSO. The Pcs were applied for PACT of E. coli, S.
aureus and C. albican where the cationic Pcs were far superior than the neutral Pcs.
Acknowledgements
This work was supported by the Department of Science and Technology (DST) Innovation and
National Research Foundation (NRF), South Africa through DST/NRF South African Research
Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology (UID 62620) as well
as Rhodes University. This work was also partially supported by a bilateral program between
the Japan Society for the Promotion of Science (JSPS) and NRF of South Africa (uid: 92425),
JSPS KAKENHI grant numbers 15H00910 and18K05076.

22
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Scheme 1: Synthetic route of phthalonitriles (a or b) using (i) N2, dry DMF, Na2CO3, 18 h and
phthalocyanines (1a – 3b), (ii) N2, n-pentanol, DBU, 138 °C, 8h; (iii) N2, Zn(OAc)2 or n-
pentanol, DBU, 138 °C, 8h.

29
Scheme 2: Synthetic route of the quaternized phthalocyanines using dimethyl sulfate, dry
DMF, 155 o C, 4 h.