Mendeleev Communications Mendeleev Commun., 2008, 18, 135–137 – 135 – © 2008 Mendeleev Communications. All rights reserved. Synthesis of chlorin–carbohydrate conjugates by ‘click chemistry’ Mikhail A. Grin,* a Ivan S. Lonin, a Alexey I. Makarov, a Anna A. Lakhina, a Filipp V. Toukach, b Vadim V. Kachala, b Anna V. Orlova b and Andrey F. Mironov a a M. V. Lomonosov Moscow State Academy of Fine Chemical Technology, 119571 Moscow, Russian Federation. Fax: +7 495 936 8901; e-mail: michael_grin@mail.ru b N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russian Federation DOI: 10.1016/j.mencom.2008.05.008 A synthesis of a conjugate of chlorin e 6 with β-D-lactose has been carried out by 1,3-dipolar cycloaddition of a sugar azide to a propargyl derivative of chlorin e 6 . Combinatory chemistry, which makes it possible to synthesise the libraries of new organic compounds for creating medicines with required therapeutic properties, has been rapidly developed. New approaches to the synthesis of biologically active com- pounds are being developed. It is mandatory for reactions of this kind that the starting compounds be readily accessible and easy to obtain, the reaction conditions be mild and the yields of target products be high. The [3 + 2] cycloaddition of azides to alkynes with a terminal triple bond suggested by Huisgen 1 is versatile and usable for constructing complex bioconjugates, immobilising biomolecules on various supports and attaching medicines to drug targeting systems. 2,3 The use of the above reac- tion in biological and pharmaceutical chemistry was encouraged by a considerable increase in the reaction rates owing to the use of Cu I -compounds as catalysts. 4 The insertion of mono- or disaccharides into a pigment molecule makes it possible to adjust the amphiphilicity of pharmaceuticals directly. Carbohydrate substituents not only increase the water solubility of porphyrins and chlorins but also ensure the vector delivery of pharmaceuticals into the cell by receptor-mediated endocytosis. 5 It is well known that cancer cells have high expression of galectins (e.g., Galectin-1), that is, proteins having a carbohydrate-recognising domain with high affinity to β-galactosides. 6 The photocytotoxicity of purpurin- imides conjugated with galactose and lactose is much higher than that of free pigments. 7 The photodynamic efficiency of porphyrin glycoconjugates with galactose and mannose residues due to enhanced expression of their surface receptors that have high affinity to the above sugars has been shown in in vitro experiments with a retinoblastoma cell line. 8 In this work, the ‘click chemistry’ method described above was used to synthesise glycoconjugates based on chlorin e 6 in order to obtain new amphiphile photosensitisers for the photo- dynamic therapy of cancer. Methylpheophorbide a 1 was used as a key compound in the synthesis; it was converted into a chlorin e 6 derivative with a terminal triple bond (Scheme 1). It is well known that the pentanone exocycle is opened on treatment with nucleophiles, including primary and secondary amines. In the latter case, corresponding amides are formed. 9 In this study, the reaction with propargylamine gave amide 2 in ~90% yield. The structure of compound 2 was confirmed by NMR experi- ments. † The signal of the ethynyl proton is a triplet at d 2.39, while the protons of the methylene group of the propargyl residue manifest themselves as a multiplet at d 4.50, giving correlation in the COSY spectrum with the adjacent NH proton of the amide group and with the proton at the triple bond. Ethynyl derivative 2 was condensed with 1-O-(2-azidoethyl)- β-D-lactose peracetate 4, which was obtained by the treatment of a chloroethoxy derivative of acetylated lactose with sodium azide using a procedure described previously. 10 The cycloaddition was carried out in the presence of a catalytic amount of copper(I) iodide. Within the first 10 min, the colour of the reaction mixture changed due to a hypso- † The solvents were purified using standard procedures. Pheophorbide a was obtained using a published procedure. 12 All of the reactions were carried out away from direct light in an argon atmosphere. Electronic spectra were recorded in chloroform using a Jasco-UV 7800 spectro- photometer. NMR experiments were carried out at 303 K on a Bruker DRX spectrometer (500 MHz) for solutions in CDCl 3 . Mass spectra were obtained by the MALDI method on a Bruker Ultraflex TOF/TOF mass spectrometer using dihydroxybenzene (DHB) as the substrate. High- resolution mass spectra were obtained with an LTQ FT hybrid mass spectrometer (Thermo, Germany). A Finnigan Ion Max Source in the positive ion mode was used as the ion source. The sample was electro- sputtered at 3.6 kV across the capillary emitter and 1 µl min –1 sample solution feed rate to the emitter needle. The sample was dissolved in an acetonitrile–water mixture (50:50). Accurate measurements of mole- cular ion masses were performed using an FTICR mass spectrometer. Fragmentation of ions and measurement of the mass spectra of fragments were carried out in a linear quadrupole ion trap. Mass spectra were processed and analysed using the Qual Browser software (Thermo, Germany). IR spectra of compounds in KBr pellets were recorded on a Bruker EQUINOX 55 spectrometer. Column chromatography was carried out on 40/60 silica gel (Merk). Preparative TLC was performed on silica gel 60 (Merck) using 20×20 plates with a layer thickness of 1 mm. Analytical TLC was carried out on Kieselgel 60 F 245 plates (Merck). 13'-(N-Propargylamide)chlorin e 6 methyl ester 2: yield 86%. 1 H NMR, d: 9.70 (s, H, 10-H), 9.63 (s, H, 10-H), 8.82 (s, H, 20-H), 8.07 (dd, H, 3 1 -H), 6.71 (t, H, 13 2 -NH), 6.35 (dd, H, E-3 2 -H), 6.14 (dd, H, Z-3 2 -H), 5.50 (d, H, 15-CH 2 ), 5.26 (d, H, 15-CH 2 ), 4.50 (m, 2H, 13 3 -CH 2 ), 4.46 (m, H, 18-H), 4.39 (d, H, 17-H), 3.83 (s, 3 H, 15 4 -COOMe), 3.78 (q, 2 H, 8 1 -CH 2 ), 3.62 (s, 3H, 17 5 -COOMe), 3.56 (s, 3H, 12 3 -Me), 3.49 (s, 3H, 2-Me), 3.30 (s, 3H, 7-Me), 2.56 (m, H, 17 2 -CH 2 ), 2.39 (t, H, 13 5 -H), 2.25 (m, H, 17 1 -CH 2 ), 2.15 (m, H, 17 2 -CH 2 ), 1.80 (m, H, 17 1 -CH 2 ), 1.73 (d, 3 H, 18-Me), 1.71 (t, 3 H, 8 2 -Me), –1.60 (H, NH), –1.83 (H, NH). MS (MALDI), m/z: 662 (M + ). UV-VIS [CH 2 Cl 2 , l max /nm (e/dm 3 mol –1 cm –1 )]: 391 (116500), 500 (11550), 528 (4300), 557 (2100), 608 (4200), 663 (33500). IR (KBr, n/cm –1 ): 3294 (C–H alkyne), 2120 (C–C alkyne), 1734 (C=O ester), 1659 (‘amide-I’), 1512 (‘amide-II’).