Porphyrin and phthalocyanine glycodendritic ...

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Porphyrin and phthalocyanine glycodendritic conjugates: synthesis, photophysical and photochemical propertiesw Sandrina Silva,a Patrı´ cia M. R. Pereira,a Patrı´ cia Silva,b Filipe A. Almeida Paz,b Maria A. F. Faustino,a Jose´ A. S. Cavaleiroa and Joaõ P. C. Tome´*a Received 3rd December 2011, Accepted 30th January 2012 DOI: 10.1039/c2cc17561d Synthesis of water soluble porphyrin and phthalocyanine derivatives with, respectively, eight and sixteen galactose units has been carried out. The combined preliminary photophysical and photochemical features of the new products suggest that they might be promising photodynamic therapeutic agents. Knowledge about porphyrins (Pors) and phthalocyanines (Pcs), and their unique photophysical and photochemical properties boosted the development of synthetic strategies in order to increase the range of new molecules, for broad biological and technological potentialities.1,2 Their outstanding properties, such as red shifted Q-bands in the UV-vis spectra and Reactive Oxygen Species (ROS) generation, have highlighted their application in medical sciences as photosensitisers (PSs) in photodynamic therapy (PDT). This therapy requires a combination of a PS, oxygen and light, which under specific conditions generates highly cytotoxic ROS.3 In fact this emerging treatment is already used worldwide for a vast number of diseases, mainly age-related macular degeneration, certain solid tumours and infection maladies.3,4 In some medical cases, PDT has several advantages over conventional surgery, chemotherapy and radiotherapy.5 One of the appealing aspects of PDT is that the used PSs should be retained in tumours to a greater extent than in normal tissues. In this way, the efforts to find ideal PSs have occupied many research groups. Looking at the Por and Pc cores, such compounds have serious limitations concerning biocompatibility and biosolubility, requiring chemical formulations to be used in biomedical applications.6,7 In order to overcome these difficulties, those cores have been chemically modified by decoration with biocompatible motifs such as carbohydrates (CHs).8,9 These are attractive candidates for drug development because sugars are involved in many, if not most, complex human diseases including cancer, immune dysfunction, congenital disorders, and infectious diseases.10 Many research groups have prepared a

QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail: [email protected]; Fax: +351 234370084; Tel: +351 234370342 b CICECO, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal w Electronic supplementary information (ESI) available: Synthesis, photophysical and photochemical studies and spectral data. See DOI: 10.1039/c2cc17561d

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a large variety of new Por–CH conjugates assuming that the presence of the CH moiety could improve the membrane interaction of such conjugates across lectins (CHs-binding proteins up-expressed in cancer cells), increasing their tumourselectivity/uptake.11,12 The corresponding Pc–CH conjugates are, however, rather uncommon being thus of great interest as new targets.9,13–15 To study the optimal CH number/arrangement to be employed on the PS macrocycle periphery, a recent approach has been studied which incorporates glycodendritic units into Pors.16 These multi-branched materials are versatile components to incorporate several CH units in various arrangements, and enable adequate amphiphilic character which is useful for drug administration and transport through the organism (hydrophilicity), and across the cell membranes (lipophilicity).17 In the case of Pcs, as far as we know, there are no reports of Pcs decorated with glycodendritic units. To explore the potential influence of galactopyranose branches on the photophysical properties of these two photoactive families we have designed a synthetic procedure to construct a CH shell of eight and sixteen galactopyranoses around fluorinated Por and Pc, respectively. Herein we report the synthesis, characterization, electronic absorption and some photo-physical and -chemical properties of the new products and their interaction with human serum albumin (HSA). Indeed, knowledge of the interaction between HAS and these compounds proves to be essential for the understanding of their absorption, distribution, metabolism and excretion properties and also their potential use as drugs. The selected dendritic framework was 2,4,6-trichloro-1,3, 5-triazine (TCT) because of its well-known selective reactivity concerning substitution of the chlorine atoms at different temperatures. 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose was chosen as the CH moiety, in order to relate these new compounds with our previous studies, which have already shown the importance of this CH unit in the photophysical, chemical and biological results.8,9 The synthesis of dendritic unit 2 was carried out in two steps as depicted in Scheme 1. Di-nucleophilic substitution of TCT by the galactose moiety was carried out in dry toluene, in the presence of an excess of DIPEA, providing 1 in 92% yield. Reaction of di-galactotriazine 1 with 1,3-dimercaptopropane provided the dendrimeric unit 2 in 89% yield. This journal is

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Scheme 1 Synthesis of the glycodendritic unit 2.

Aiming at the synthesis of new PSs using this new unit, we employed commercially available platforms, such as 5,10,15, 20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (TPPF20) and hexadecafluorophthalocyaninatozinc(II) (ZnPcF16), which after reacting with four and eight equivalents of dendritic unit 2 led to the corresponding Por 3 and Pc 4 in 78% and 81% yields, respectively (Scheme 2). Hydrolysis of the isopropylidene protective groups with TFA/H2O (9 : 1) produced a/b mixtures of unprotected glycodendrimer–PS conjugates 3a (80%) and 4a (78%). All experimental conditions are provided in ESI.w Synthon 1 was easily identified by the resonances of CH units, and the double substitution was confirmed by two triazine carbon resonances at d 171.9 and 172.6 ppm in a ratio of 2 : 1, respectively. 1H and 13C NMR of 2 show, additionally, the resonance of the mercaptopropane chain. 1H, 19F and 13 C NMR spectra of Por 3 and Pc 4 clearly illustrate the CH conjugation and the symmetry of both structures. In the case of 3, looking at the ratio 1 : 1 between b-pyrrolic protons of Por and anomeric protons of CH, the tetra-substitution of the Por macrocycle is evident. Tetra- and octa-substitutions of TPPF20 and ZnPcF16 are further supported by 19F NMR data, showing the absence of the signals corresponding to the Por para and Pc beta fluorine atoms. While 3 shows two quartet signals at d 157.4 and 160.2 ppm, corresponding to the meta and ortho fluorine atoms, 4 has a single peak at d 131.5 ppm corresponding to the alpha fluorine atoms. Unprotection of the isopropylidene groups was confirmed by the complete disappearance of the corresponding 1H and 13C resonances. For both anomeric mixtures, 3a and 4a, 19F NMR spectra show now two multiplets between d 158.0 to 158.1 and 162.7 to 162.8 ppm corresponding to meta and ortho fluorine atoms of 3a, and a singlet at d 134.4 ppm for the alpha atoms of 4a. Besides the NMR data, all structures were further confirmed by HRMS mass spectrometry.

Fig. 1 Electronic absorption spectra in DMF ( PBS ( ): (a) Por 3a, (b) Pc 4a, both at 2 mM.

), DMSO (

) and

Electronic absorption spectra of 3a and 4a were measured in DMF, DMSO and PBS (phosphate buffered saline) and are depicted in Fig. 1. The very sharp Soret band of 3a and the Q-band of 4a in DMF and DMSO showed that these derivatives are highly soluble in these two solvents. The lower intensity and the broadening of these bands in PBS are related with a reasonable water solubility. To evaluate their aggregation behaviour in DMSO and PBS, UV-Vis studies at different concentrations were performed (see Fig. S1–S4 in the ESIw). Both compounds strictly follow the Beer–Lambert law in both solvents at the studied concentrations, suggesting no aggregation in PBS at concentrations below 19 mM for 3a and 9 mM for 4a. The steady-state fluorescence emission spectra of 3a and 4a were analysed in DMF and show two bands in the red spectral region (see Fig. S5, ESIw) characteristic for Pors and Pcs. The fluorescence quantum yields (FF) are summarised in Table 1, and they were determined in DMF, using mesotetraphenylporphyrin (TPP) as a standard (FF = 0.11 in DMF).18 Pc 4a showed the highest fluorescence quantum yield (FF = 0.13). To evaluate the PS potentialities of our compounds for PDT, their photostability, ability to generate singlet oxygen (1O2) and to interact with HSA carriers were determined. The photostability of the compounds is an important parameter because it is necessary to certify that they do not undergo extensive destruction when exposed to UV-Vis light and oxygen. Photobleaching studies Table 1 Photophysical data of 3a and 4a in DMF Compound

lmax/nm

log e

lEmission/nm

DStokes/nm

FF

3a 4a

655 722

5.24 5.18

658, 703 734, 805

4 12

0.09a 0.13a

a

Reference: TPP in DMF (FF = 0.11).18

Scheme 2 Synthesis of Por- and Pc-based on galactodendritic units.

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of 3a and 4a were performed by monitoring the decrease of the Soret and Q band intensities, respectively. PBS solutions of both compounds under white (400–800 nm) and red light (620–750 nm), at a fluence rate of 150 mW cm 2, showed high photostability over the investigated irradiation period (30 min; see Table S1 in the ESIw). Singlet oxygen production is fundamentally important for photoinduced processes in PDT. We have thus evaluated the ability of the glycodendritic conjugates to generate 1O2 because its production is significantly related to the photodynamic activity.3,19 Singlet oxygen generation was determined by a chemical method using 1,3-diphenylisobenzofuran (DPBF) as the 1O2 scavenger in DMF : H2O (9 : 1).4 Compounds 3a and 4a have shown similar ability to photooxidize DPBF when compared to the well known 1O2 reference compounds, TPP and ZnPc, respectively (see Fig. S6 and S7, ESIw). These results allowed us to envisage 3a and 4a as suitable PSs for PDT. In addition, the dendritic units do not appear to affect the singlet oxygen production in DMF : H2O (9 : 1). The quenching of the intrinsic tryptophan fluorescence of HSA was used as a tool to study the interaction of 3a and 4a with HSA. The maximum HSA emission wavelength is around 340 nm and the fluorescence intensity of HSA decreases with the addition of 3a or 4a (see Fig. S8 in the ESIw). Fluorescence quenching unequivocally demonstrates that there are PS binding sites close to the tryptophan residues of HSA. The binding constant (Ka) and the number of binding sites (n) were determined as described in the literature (see S1.7, eqn S2, ESIw).20 The values of Ka and n for 3a were 1.544 104 M 1 and 1.2, respectively. For 4a the values of Ka and n were 2.351 105 M 1 and 1.4, respectively. These results clearly indicate that there is only one binding site for 3a or 4a on HSA. A curious behaviour of Pc 4 was observed upon its precipitation from solution in DMSO as long needles, visible even by the naked eye (Fig. 2, left). Electron microscopy studies on a portion of these needles reveal a unique solid-state arrangement: Pc 4 self-assembles in a highly irregular fashion forming a spongelike material with uniform distribution of the diameter of the individual strands (globally, diameters are smaller than ca. 10 mm; see Fig. 2 and section SI 1.8, ESIw). Energy dispersive X-ray spectroscopy mapping studies (not shown) reveal that the heaviest elements of Pc 4 (namely, Zn, S and F) are uniformly distributed throughout the strands, thus confirming that these supramolecular aggregates are indeed formed by the molecular recognition of individual Pc units. Identical studies for the remnant DMSO solution still show the presence of similar fragments of these supramolecular arrangements, although exhibiting smaller sizes.

Fig. 2 Optical and SEM images of Pc 4 aggregates.

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In conclusion, we have synthesised and characterised a new galactodendrimeric unit, and prepared Por and Pc derivatives which incorporate a CH shell of eight and sixteen D-galactopyranose units, respectively. All new products were obtained in high yields. The presence of CH moieties in the periphery of Por and Pc PSs embodies hydrophilicity to the new compounds, which is important in connection with drug administration. In addition, considering the specific affinity of CHs for cancer cells and their strong influence on the bioavailability of the corresponding conjugates, good perspectives can be anticipated for this new generation of PSs based on PS–CH conjugates. Their interaction with HSA and high singlet oxygen production suggest high capacity for their use as PDT agents. Thanks are due to the University of Aveiro, FCT (Fundaçaõ para a Cieˆncia e Tecnologia, Portugal) and FEDER for funding the QOPNA, CICECO research units, the Portuguese National NMR Network and the project PTDC/QUI/65228/2006. S. Silva, P. Pereira and P. Silva also thank FCT for their post-doctoral, BI and PhD grants: SFRH/BPD/64812/2009, BI/UI55/5457/2011 and SFRH/BD/46601/2008, respectively.

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