Synthesis of hydrophilic conjugated porphyrin dimers for one-photon and two-photon photodynamic therapy at NIR wavelengths

Article abstract of DOI:10.1039/b814789b

We report the synthesis of a series of hydrophilic butadiyne-linked conjugated zinc porphyrin dimers (1-7), designed as photodynamic therapy (PDT) agents. These porphyrin dimers exhibit exceptionally high two-photon absorption cross sections (δ_max≈ 8,000-17,000 GM) and red-shifted linear absorption spectra (λ_max≈ 700-800 nm) making them ideal candidates for one-photon and two-photon excited photodynamic therapy. Four polar triethyleneglycol substituents are positioned along the sides of each dimer, but, on their own, these TEG chains do not confer sufficient solubility in aqueous physiological media for reproducible delivery into live cells. Charged cationic (methylpyridinium and trimethylammonium) and anionic (sulfonate and carboxylate) substituents have been appended to the meso-positions of porphyrin dimers using three synthetic strategies: 1) Suzuki coupling, 2) Sonogashira coupling, and 3) nucleophilic Senge arylation. Approaches 1 and 3 both allow attachment of aromatic substituents directly to the meso-positions of porphyrins. Approach 2 provides a route to hydrophilic porphyrin dimers with an ethyne link between the porphyrin and the polar aromatic substituent. The palladium-catalysed approaches 1 and 2 allow the synthesis of a broader range of meso-capped porphyrins, as many aryl halides are available. However the synthesis of the intermediate required for these routes necessitates a statistical reaction step, which decreases the overall yield. On the other hand, Senge-arylation provides highly regioselective nucleophilic aromatic substitution, and offers higher overall yield than the other routes. All these charged dimers exhibit good solubility in polar solvents (e.g. methanol) and aqueous solvent mixtures (aqueous DMSO or DMF).

Full text of DOI:10.1039/b814789b

Supplementary Material (ESI) for Organic & Biomolecular Chemistry
This journal is (c) The Royal Society of Chemistry 2009
Electronic Supplementary Information for
Synthesis of hydrophilic conjugated porphyrin dimers for one-photon and
two-photon photodynamic therapy at NIR wavelengths
Milan Balaz, Hazel A. Collins, Emma Dahlstedt and Harry L. Anderson*
Department of Chemistry, Oxford University, Chemistry Research Laboratory, Mansfield
Road, Oxford OX1 3TA, UK.
Fax: +44 1865 285002; Tel: +44 1865 275704; E-mail: harry.anderson@chem.ox.ac.uk
List of Contents
Material and Methods
2
3-4
HPLC, NMR and MALDI data of conjugated porphyrin dimer 2 (P2-NMeI)
HPLC, NMR and MALDI data of conjugated porphyrin dimer 5 (P2-SO3NH4)
HPLC, NMR and MALDI data of conjugated porphyrin dimer 3 (P2C2-NMeI)
HPLC, NMR and MALDI data of conjugated porphyrin dimer 6 (P2C2-CO2NH4)
HPLC, NMR and MALDI data of conjugated porphyrin dimer 4 (P2-NMe3OAc)
HPLC, NMR and MALDI data of conjugated porphyrin dimer 7 (P2-Suc)
HPLC method E (linear gradient)
5-6
7-8
9-10
11-12
13-14
15
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Materials and Methods
HPLC purification
The HPLC purification of conjugated porphyrin dimers was done on a VWR Hitachi ELITE
LaChrom HPLC system equipped with L-2130 quaternary pump, L-2455 diode array
detector, L-2200 autosampler and FoxyJr fraction collector. The chromatographic separations
were monitored in the range 260 nm – 800 nm.
Analytical HPLC were carried out using C8 5 µm, 3.9 × 150 mm Eclipse XDB-C8 column
o
(Agilent) using 1 mL / min flow and stepwise gradient. The column was heated to 40 C.
Semipreparative HPLC separations were carried out using C8 5 µm, 9.4 × 250 mm Eclipse
XDB-C8 column from Agilent using 4 ml / min flow rate and a stepwise gradient. The
column was heated to 40 oC.
NMR spectra
NMR spectra were recorded at ambient probe temperature using either a Brucker DPX400
(400 MHz), or Brucker AVANCE AV400 (400 MHz). Chemical shifts are quoted as parts per
million (ppm) relative to tetramethylsilane and coupling constants (J) are quoted in Hertz
(Hz). UV/Vis spectra were recorded on a Perkin Elmer Lambda 20 UV-Vis.
MS spectra
Mass spectra were carried out using Matrix Assisted Laser Desorption Ionization-Time of
Flight (MALDI-TOF) only molecular ions and major peaks are reported. Melting points are
reported uncorrected and boiling points were taken from the vapor temperature of the
distilling product.
malononitrile (DCTB).
Matrix:
2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]
Purity of porphyrin dimers
Proton NMR spectra and MALDI-TOF spectra of RP-HPLC purified dimers did not show
presence of any impurities. The purity of all conjugated porphyrin dimers was further
confirmed by the RP-HPLC. A small amount of the each purified dimer was dissolved in THF
or DMF and injected into the HPLC system. The HPLC profile was monitored between 280
nm and 800 nm (DAD detector). The HPLC chromatographs monitored at two different
wavelengths (440 nm and 695 nm) can be found in the ESI section of this manuscript. HPLC
profiles did not show presence of any impurities and confirmed the high purity of all
hydrophilic porphyrin dimers required for in vitro or in vivo biological testing.
- 2 -

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Conjugated porphyrin dimer 2 (P2-NMeI)
Figure S1. HPLC chromatograph of the porphyrin dimer 2 monitored 440 nm (Soret band).
Chromatographic condition: T = 40oC, flow = 4 mL / min, gradient solvent system: water /
MeOH / THF / 1% acetic acid in water, Table S1. The porphyrin dimer was dissolved in THF
and 20 µL was injected each time.
Table S1. Solvent gradient used for HPLC purification of the porphyrin dimer 2 (Method A)
Time (min)
Water
Methanol
THF
2% aqueous
acetic acid
60 %
0.0
9.0
11.0
12.0
14.0
10 %
17 %
12 %
5 %
15 %
50 %
24 %
10 %
15 %
15 %
25 %
52 %
80 %
80 %
8 %
12 %
5 %
2.5 %
2.5 %
4
4
4 4 4 4
9.0
8 4
4
6
8
8 8 8 8 8 12
4.0 3.5
10.0
9.5
8.5
8.0
7.5
7.0
6.5
Chemical Shift (ppm)
6.0
5.5
5.0
4.5
Figure S2. 1H NMR spectrum of porphyrin dimer 2 (CDCl3 / 5% pyridine-d5, 400MHz).
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Conjugated porphyrin dimer 2
Observed data
Theoretical [M-2I]2+
Figure S3. ESI-TOF of the porphyrin dimer 2 m/z ESI-TOF 963.32
2+
(C108H106N10O16Zn2 , [M]2+, requires 963.32)
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Conjugated porphyrin dimer 5 (P2-SO3NH4)
Figure S4. HPLC chromatograph of the porphyrin dimer 5 monitored 440 nm (Soret band).
Chromatographic condition: T = 40oC, flow = 4 mL / min, gradient solvent system: MeOH /
DMF / ammonium acetate in water (1g/L), Table S2. The porphyrin dimer 5 was dissolved in
DMF and 20 µL was injected each time.
Table S2. Solvent gradient used for HPLC purification of the porphyrin dimer 5 (Method B)
Time (min)
Methanol
DMF
aqueous ammonium
acetate (1g/L)
40 %
0.0
3.0
8.0
10.0
14.0
20 %
6.7 %
6.7 %
0 %
40 %
80 %
80 %
100 %
100 %
13.3 %
13.3 %
0 %
0 %
0 %
4
2
4
2
4 6 2
2
8
4
4
10.0
9.5
9.0 8.5
Chemical Shift (ppm)
8.0
7.5
4
2
4 2 10 2 2
9.0 8.5
8 4
4
8
8
8 8 8 8 12
10.0
9.5
8.0
7.5
7.0
6.5
Chemical Shift (ppm)
6.0
5.5
5.0
4.5
4.0
3.5
Figure S5. 1H NMR spectrum of porphyrin dimer 5 (MeOD / 5% pyridine-d5, 500 MHz).
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Conjugated porphyrin dimer 5
Observed data
Theoretical [M-3H]3-
Figure S6. ESI-TOF of the porphyrin dimer 5 m/z ESI-TOF 770.48
3-
(C116H103N8O28S4Zn2 , [M]3-, requires 770.47).
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Conjugated porphyrin dimer 3 (P2C2-NMeI)
550
450
350
250
150
50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
-50
Time, min
Figure S7. HPLC chromatograph of the porphyrin dimer 3 monitored 440 nm (Soret band, blue line)
and 770 nm (Q band, red line).
Table S3. Solvent gradient used for HPLC purification of the porphyrin dimer 3 (Method C)
Time/min MeOH
DMF
40 %
80 %
80 %
40 %
40 %
1g/L NH4OAc
40 %
0
6
20 %
6.7 %
6.7 %
20 %
20 %
13.3 %
13.3 %
40 %
13
13.01
15
40 %
Linear ramping between solvent mixtures
1H_000001r.esp
water
pyridine
pyridine
0.4
0.3
0.2
0.1
0
pyridine
dichloromethane
10.0
9.5
9.0
8.5
8.0
7.5
7.0 6.5
Chemical Shift (ppm)
6.0
5.5
5.0
4.5
4.0
3.5
3.0
Figure S8. 1H NMR spectrum of porphyrin dimer 3 (MeOD / 5% pyridine-d5, 400 MHz).
- 7 -

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Conjugated porphyrin dimer 3
Figure S9. ESI-TOF of the porphyrin dimer 3 m/z MALDI-TOF 1978.5
(C112H106N10O16Zn2, [M-2I]+, requires 1978.6, 100%)
Figure S10. ESI-TOF of the porphyrin dimer 3 m/z MALDI-TOF 1978.5
(C112H106N10O16Zn2, [M-2I]+, requires 1978.6, 100%).
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Conjugated porphyrin dimer 6 (P2C2-CO2NH4)
650
550
450
350
250
150
50
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
-50
Time, min
Figure S11. HPLC chromatograph of the porphyrin dimer 6 monitored 440 nm (Soret band, blue line)
and 680 nm (Q band, red line).
Table S4. Solvent gradient used for HPLC purification of the porphyrin dimer 6 (Method C)
Time/min MeOH
DMF
40 %
80 %
80 %
40 %
40 %
1g/L NH4OAc
40 %
0
6
20 %
6.7 %
6.7 %
20 %
20 %
13.3 %
13.3 %
40 %
13
13.01
15
40 %
Linear ramping between solvent mixtures
1H_000000fid.esp
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
pyridine
pyridine
water
pyridine
10.0
9.5
9.0
8.5
8.0
7.5
7.0 6.5
Chemical Shift (ppm)
6.0
5.5
5.0
4.5
4.0
3.5
3.0
Figure S12. 1H NMR spectrum of porphyrin dimer 6 (DMSO / 5% pyridine-d5, 400 MHz).
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Conjugated porphyrin dimer 6
Figure S13. ESI-TOF of the porphyrin dimer 6 m/z MALDI-TOF 2122.6
(C116H102N8O24Zn2, M+, requires 2122.6, 100%)
Figure S14. ESI-TOF of the porphyrin dimer 6 m/z MALDI-TOF 2122.6
(C116H102N8O24Zn2, M+, requires 2122.6, 100%)
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Conjugated porphyrin dimer 4 (P2-NMe3OAc)
300
250
200
150
100
50
DAD-CH1 440 - 790 nm
MB-NMe3-pure-Bob-MB-Suc-H2O-THF-AcOH-SP-3.met027.dat
Retention Time
DAD-CH4 695 - 790 nm
MB-NMe3-pure-Bob-MB-Suc-H2O-THF-AcOH-SP-3.met027.dat
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Minutes
Figure S15. HPLC chromatogram of the porphyrin dimer 4 monitored by at two different
wavelengths, 440 nm (Soret band, blue line) and 695 nm (Q band, red line).
Chromatographic condition: T = 40oC, flow = 4 mL / min, gradient solvent system: 1% acetic
acid in water / THF, Table S5. Porphyrin dimer 4 was dissolved in THF and 30 µL was
injected.
Table S5. Solvent gradient used for HPLC purification of the porphyrin dimer 4: HPLC method D
time / min
1% aqueous
acetic acid
60 %
THF
0
3
6
40 %
50 %
70 %
80 %
80 %
40 %
40 %
50 %
30 %
20 %
20 %
60 %
60 %
9
12
13
15
HDO
DMSO
4H
4H 4H 4H 8H
12H 4H
8H
26H 8 8 8 8H 12H
6H
10.0
7.5
5.0
2.5
0.0
ppm (t1)
Figure S16. 1H NMR spectrum of porphyrin dimer 4 (DMSO-d6, 400MHz).
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Conjugated porphyrin dimer 4
Figure S17. MALDI-TOF of the porphyrin dimer 4 . MALDI TOF m/z 1006.9
(C114H118N10O16Zn2, [M-2OAc]2+, requires 1007.4).
Matrix: 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB)
MALDI-TOF m/z 1007.4 [M-2OAc]2+, calculated for C114H118N10O16Zn2 1007.4
MALDI-TOF m/z 1998.7 [M-2OAc-Me]+, calculated for C113H115N10O16Zn2 1998.7
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Conjugated porphyrin dimer 7 (P2-Suc)
DAD-CH1 440 - 790 nm
MB-suc-pure-Bob-MB-Suc-H2O-THF-AcOH-SP-3.met022.dat
Retention Time
DAD-CH4 695 - 790 nm
MB-suc-pure-Bob-MB-Suc-H2O-THF-AcOH-SP-3.met022.dat
400
300
200
100
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Minutes
Figure S18. HPLC chromatograph of the porphyrin dimer 7 monitored at two different wavelengths,
440 nm (Soret band, blue line) and 695 nm (Q band, red line).
Chromatographic condition: T = 40oC, flow = 4 mL / min, gradient solvent system: 1% acetic
acid in water / THF, Table S6. The porphyrin dimer 7 was dissolved in THF and 30 µL was
injected each time.
Table S6. Solvent gradient used for HPLC purification of porphyrin dimer 7: HPLC method D
time / min
1% aqueous
acetic acid
60 %
THF
0
3
6
40 %
50 %
70 %
80 %
80 %
40 %
40 %
50 %
30 %
20 %
20 %
60 %
60 %
9
12
13
15
HDO
DMSO
THF
2H
2H
4H
4H 8H
8H 12H 4H
8H
8H 24H 8H 12H 8H
12.5
10.0
7.5
5.0
2.5
0.0
ppm (f1)
Figure S19. 1H NMR spectrum of porphyrin dimer 7 (DMSO-d6, 400MHz).
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Conjugated porphyrin dimer 7
Figure S20. MALDI-TOF of the porphyrin dimer 7. m/z MALDI-TOF 2128.7 (C116H112N10O22Zn2,
M+, requires 2128.96).
Matrix: 2-[(2E)-3-(4-tert-Butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB)
MALDI-TOF m/z 2128.7 [M]+, calculated for C116H112N10O22Zn2 2128.9643
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HPLC method E (linear gradient)
time / min
1% aqueous
acetic acid
60 %
THF
0
40 %
80 %
40 %
40 %
20
22
24
20 %
60 %
60 %
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