Derivatizable phthalocyanine with single carboxyl group: Synthesis and purification

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/229242077 Derivatizable phthalocyanine with single carboxyl group: Synthesis and purification Article in Inorganic Chemistry Communications · March 2006 DOI: 10.1016/j.inoche.2005.12.002 CITATIONS READS 47 121 5 authors, including: Jincan Chen Mingdong Huang 24 PUBLICATIONS 357 CITATIONS 177 PUBLICATIONS 3,410 CITATIONS Chinese Academy of Sciences SEE PROFILE Fuzhou University SEE PROFILE All content following this page was uploaded by Mingdong Huang on 04 February 2014. The user has requested enhancement of the downloaded file. Inorganic Chemistry Communications 9 (2006) 313–315 www.elsevier.com/locate/inoche Derivatizable phthalocyanine with single carboxyl group: Synthesis and purification Jincan Chen a, Naisheng Chen b, Jinling Huang b, Jundong Wang a b,* , Mingdong Huang a,* State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, 155 Yang Qiao Xi Lu, Fuzhou, Fujian 350002, China b Institute of Functional Materials, College of Chemistry and Chemical Engineering, Fuzhou University, Fujian 350002, China Received 19 July 2005; accepted 2 December 2005 Available online 18 January 2006 Abstract Phthalocyanine zinc (ZnPc) is an important class of photosensitizers. Derivatization of phthalocyanine ring with chemical functional groups provides anchor points for covalent attachment of biological targeting agents, thus increasing the selectivity of the photosensitizers. ZnPc with symmetric and multiple functional groups, e.g., tetracarboxyphthalocyanine zinc, is easy to be prepared but is unfavorable for linking targeting agents due to many reasons. On the other hand, single-substituted ZnPc, is more challenging to synthesize and, especially, to purify. Here we report the synthesis of an unsymmetrical phthalocyanine, 2-carboxyphthalocyanine zinc 1, with a single carboxylic group on phthalocyanine ring, and the design of its chromatographic purification. Ó 2005 Elsevier B.V. All rights reserved. Keywords: Unsymmetrical phthalocyanine; Carboxyl phthalocyanine; Photodynamic therapy; Biological targeting agents In photodynamic therapy [1], infrared light with specific wavelength is shined onto tissue/tumor pre-administrated with photosensitizing agent. In the presence of physiological oxygen, the light-absorbing photosensitizer produces reactive oxygen species (ROS) and/or free radicals in its immediate locale, rendering biological toxicity. Phthalocyanine zincs, and several other metallophthalocyines, have been successfully used as a photosensitizing agents in photodynamic therapy at both cellular and animal levels [2–6]. Conjugation of sensitizer with biological targeting agents, e.g., monoclonal antibodies or lipoproteins, increases the concentration of sensitizer on targeted tissue over normal tissue, providing additional level of selectivity and possibility to reduce effective sensitizer concentration, and thus phototoxicity to normal tissue [7,8]. Phthalocyanine zinc (ZnPc) with multiple and symmetrical functionalities in the phthalocyanine ring, e.g., tetracarboxyphthalocyanine zinc, is easy to be prepared * Corresponding author. Tel.: +86 591 83795550 (J. Wang); Tel.: +86 591 83704996; fax: +86 591 83714946 (M. Huang). E-mail address: mhuang@fjirsm.ac.cn (M. Huang). 1387-7003/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.inoche.2005.12.002 but is often unfavorable for linking targeting agents. For example, once conjugated to antibodies, tetracarboxyphthalocyanine zinc will become insoluble polymer. Thus, conjugation of ZnPc with targeting agents prefers a single functionality in the phthalocyanine ring. The synthesis of such unsymmetrical phthalocyanine is typically through the expansion of subphthalocyanine that is composed of three isoindole units [9,10], or through statistical condensation. Purification is always the key to obtain intended products for all these synthetic methods. Here we report a two step synthesis of an unsymmetrical phthalocyanine, 2-carboxyphthalocyanine zinc 1, with a single carboxylic group on phthalocyanine ring. We also reported the development of a simple column-based purification scheme of 1. Condensation of trimellitic anhydride or phthalic anhydride in the presence of urea and catalyst at high temperature will lead to formation of tetra-formamidophathalocyanine or phthalocyanine, respectively. Using a mixture of trimellitic anhydride and phthalic anhydride at a ratio of 1:7 for condensation at 170 °C and in the presence of urea, zinc acetate, and ammonium molybdate, we have made a mixture of 2-formamidophthalocyanine 314 J. Chen et al. / Inorganic Chemistry Communications 9 (2006) 313–315 zinc 2 and phthalocyanine zinc (Scheme 1). The mixture was then hydrolyzed under alkaline condition, yielding 2carboxyphthalocyanine zinc 1 (Scheme 2). The final product of 1 was then purified from the hydrolysis mixture. Its purity was confirmed based on a major elution band on an analytical C18 HPLC column (Fig. 1b). At our molar ratio of starting materials (1:7) for condensation, only trace amount presumably polycarboxy substituted phthalocyanine zinc was found (small peaks eluted ahead of 1 in Fig. 1b). Compound 1 was further characterized by UV/VIS, IR, proton NMR, and mass spectra. Large amount of phthalocyanine zinc byproduct exists in this synthetic scheme. This phthalocyanine zinc in our reaction mixture was soluble in DMF (Fig. 1), presumably due to axial coordination to zinc ion. Hence, the separation of desired product from reaction mixture was necessary for the success of the preparation of 1. We performed extensive analysis of reaction mixture by analytical HPLC (see Supplementary data) to guide our design of the purification scheme. We chose not to carry out purification after condensation reaction (Scheme 1) but after hydrolysis reaction (Scheme 2), even though this seems added another level of complexity (i.e., incomplete hydrolysis) to the separation. This is because the HPLC analysis indicated that compound 2 from condensation reaction was quite similar to phthalocyanine zinc (Fig. 1a) on a C18 HPLC column, and thus its separation from phthalocyanine zinc will be difficult. On the other hand, compound 1 is quite distinct from both 2 and phthalocyanine zinc on a HPLC column (Fig. 1b), which indicates larger polarity of compound 1. Indeed, we were able to work out a convenient protocol Fig. 1. Analytical HPLC monitored at 670 nm of (a) the mixture after condensation, indicating the presence of 2-formamido-phthalocyanine zinc 2 and phthalocyanine zinc at about 1:4 ratio; and (b) 2-carboxyphthalocyanine zinc 1 purified twice over a silica gel column. See Supplementary Data for detailed HPLC conditions. Basically, a C18 column was gradient eluted from water to DMF (5–100%) at a flow rate of 1 ml/min. to separate 1 from both 2 and phthalocyanine zinc on a silica gel column eluted with a mixed elution solvent (DMF:acetone at 3:1 ratio). Compound 2 and phthalocyanine zinc migrated as a leading band and compound 1 was retained as a well-separated second band due to its larger polarity shown on HPLC column (Fig. 1b). The existence of carboxylic group on phthalocyanine ring was confirmed by IR spectrum (1719 cm 1, COOH stretching, and 1655 cm 1, C@O stretching). On ESI Mass spectrum, molecular ion of 1 appears as major peak (Fig. 2. obs: 621.8, expected: 621.92). Upon second ionization (Fig. 2 inset), this ion lost a COOH group and fragmented into phthalocyanine zinc (obs: 576.4, expected: 576.90). A strong mass peak at 1242.9 corresponded to the dimer of 2-carboxyphthalocyanine zinc, which was further fragmented into a peak with m/z of 1119.1, presumably due to the lost of two zinc atoms. Trace amount of trimer of compound 1 (1864.5) was also detected on mass spectrum. The existence of the mono-substitution of carboxy group on phthalocyanine ring is also confirmed by proton Scheme 1. Condensation. Scheme 2. Hydrolysis. Fig. 2. ESI Mass spectrum of purified 2-carboxyphthalocyanine zinc. Molecular ion appears at 621.8 (expected: 621.92). Upon second ionization (inset), this ion lost a COOH group and fragmented into a peak at 576.4, corresponding to phthalocyanine zinc (576.9). 315 J. Chen et al. / Inorganic Chemistry Communications 9 (2006) 313–315 Table 1 Solubility of compound 1 in common organic solvent Fig. 3. Proton NMR of 2-carboxyphthalocyanine zinc in deuterated DMSO and the tentative assignment of proton chemical shifts. Solvent Solubility (g/L) N,N-Dimethylformamide Tetrahydrofuran Ethanol Acetone 11.25 3.60 4.65 5.55 In summary, we have synthesized a new unsymmetrical phthalocyanine, 2-carboxyphthalocyanine zinc 1, and designed a convenient column based purification scheme for the product. The carboxylic acid functional group on phthalocyanine allows site specific covalent attachment of biological markers to enhance the target specificity of phthalocyanine zinc as photosensitizer in photodynamic therapy. Acknowledgements We thank Prof. Yu Zheng for helpful discussion on the Infrared spectrum of phthalocyanines, and financial support from a Fujian NSF grant (C0320003) and from Chinese Academy of Sciences (Bairen grant to MH). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.inoche.2005. 12.002. Fig. 4. UV–VIS spectra of 2-carboxyphthalocyanine zinc 1 (dotted line) and phthalocyanine zinc (solid line), 5 lM in DMF. NMR (Fig. 3). Chemical shifts of protons on the phthalocyanine ring are assigned as follows: d 9.710(a), 9.264(b), 8.656(c), 8.204(d), 7.728(c). Carboxy proton was not observed due to its exchange with solvent. UV–VIS spectrum (Fig. 4) of compound 1 in DMF shows a slightly broadened Q band, presumably due to the aggregation of compound 1. Its major absorption wavelength (675.3 nm) is red-shifted compared to that of phthalocyanine zinc (669.99 nm). Compound 1 has good solubility in polar organic solvent except water (Table 1), but is not soluble in other less polar or non polar solvents, including dichloromethane, ethyl acetate, n-hexane, carbon tetrachloride. View publication stats References [1] D.E. Dolmans, D. Fukumura, R.K. Jain, Nat. Rev. Cancer. 3 (2003) 380–387. [2] I. Rosenthal, Photochem. Photobiol. 53 (1991) 859–870. [3] N. Brasseur, H. Ali, R. Langlois, J.E. van Lier, Photochem. Photobiol. 47 (1988) 705–711. [4] S.J. Stern, S. Thomsen, S. Small, S. Jacques, Arch. Otolaryngol. Head Neck Surg. 116 (1990) 1259–1266. [5] C. Ometto, C. Fabris, C. Milanesi, G. Jori, M.J. Cook, D.A. Russell, Br. J. Cancer 74 (1996) 1891–1899. [6] D.J. Ball, S. Mayhew, S.R. Wood, J. Griffiths, D.I. Vernon, S.B. Brown, Photochem. Photobiol. 69 (1999) 390–396. [7] E. Reddi, J. Photoch. Photobiol. B. 37 (1997) 189–195. [8] Y.N. Konan, R. Gurny, E. Allemann, J. Photoch. Photobiol. B. 66 (2002) 89–106. [9] N. Kobayashi, R. Kondo, S. Nakajima, T. Osa, J. Am. Chem. Soc. 112 (1990) 9640–9641. [10] C. Claessens, T. Torres, Chem. Eur. J. 6 (2000) 2168–2172.