Tetrahedron Letters xxx (2012) xxx–xxx
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Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Pentafluorophenylcorrole–D-galactose conjugates Teresa A. F. Cardote a, Joana F. B. Barata a,⇑, M. Amparo F. Faustino a,⇑, Annegret Preuß c, M. Graça P. M. S. Neves a, José A. S. Cavaleiro a, Catarina I. V. Ramos b, M. Graça O. Santana-Marques b, Beate Röder c a
Organic Chemistry Laboratory, QOPNA Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal Mass Spectrometry Laboratory, QOPNA Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal c Institut für Physik, Photobiophysik, Humboldt-Universität zu Berlin, Newtonstrasse 15, 12489 Berlin, Germany b
a r t i c l e
i n f o
Article history: Received 20 June 2012 Revised 5 September 2012 Accepted 11 September 2012 Available online xxxx Keywords: Corroles Glycocorroles PDT
a b s t r a c t New corrole–D-galactose conjugates were synthesized from the reaction of 5,10,15-tris(pentafluorophenyl)corrole and 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose. The monosubstituted isomers were fully characterized by NMR spectroscopy and mass spectrometry. The latter studies can be used to differentiate the two conjugate isomers. The photophysical properties and the photodynamic effect on Jurkat cells were evaluated for the main conjugate isolated. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction The design of new molecules with anti-cancer properties is a hot and always urgent topic. The development of molecular platforms for improved cancer-targeting efficacy is the key point of cancer therapies, including photodynamic therapy (PDT).1 In PDT, light, oxygen and a photosensitizing drug are combined to produce a selective therapeutic effect. Amongst the various types of photosensitizers used in PDT, porphyrin derivatives are the most extensively studied and are the ones approved so far for clinical use.2 The design of molecules which can target specific cells can be reached by incorporating some biological subunits like sugars on the photosensitizer core to control water solubility, biodistribution, pharmacokinetics and affinity or selectivity for cancer cells.3 Porphyrins with carbohydrate moieties have been described as efficient photosensitizers to be used in PDT.4 That is mainly due to the specific affinity of several carbohydrates for cancer cells and molecular recognition receptors in the surface.5 In this way the conjugation of carbohydrates to corroles should increase the hydrophilic character and will promote a higher tumour specificity. Corroles are tetrapyrrolic macrocycles that share close similarities with porphyrins and other related macrocycles. Nowadays corroles can be functionalized by several approaches allowing great flexibility in the synthesis of molecules with unique physical
⇑ Corresponding authors. Tel.: +351 234 370 710; fax: +351 234 370 084. E-mail addresses:
[email protected] (J.F.B. Barata),
[email protected] (M.A.F. Faustino).
and chemical properties.6,7 Although corroles have been investigated in several areas such as catalysis and as sensors,8 the therapeutic potential of corroles has only recently been disclosed.9 They present interesting and unique properties, which confer them the applicability as photosensitizers in PDT. Therefore we have decided to covalently link a sugar moiety to 5,10,15-tris(pentafluorophenyl)corrole in order to study the influence of the carbohydrate moiety on the photophysical properties of corroles and also to evaluate their potential to be used as photosensitizers for PDT. The photodynamic effect of the main galactoconjugate was evaluated on Jurkat cell suspension. D-Galactose was selected because the required derivative (1,2:3,4-di-O-isopropylidene-a-D-galactopyranose) is commercially available and due to the promising biological results obtained with porphyrin-D-galactose conjugates.10,11 In addition, there are several studies indicating that many cancer cells express high sugar receptors such as galectin with preferential affinity for galactose residues.12 The results obtained also prompted us to investigate the potential of ESI-MS/MS to differentiate galactocorrole isomers, based on the analysis of specific fragmentation. Results and discussion The starting corrole 1 was synthesized by condensation of pyrrole with pentafluorobenzaldehyde following Gryko’s procedure.13 The synthetic strategy to prepare the new corrole–galactose conjugates 2a and 2b involved the nucleophilic substitution14 of a para-fluorine atom of 5,10,15-tris(pentafluorophenyl)corrole 1
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2012.09.038
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T. A. F. Cardote et al. / Tetrahedron Letters xxx (2012) xxx–xxx
Scheme 1.
by the commercially available 1,2:3,4-di-O-isopropylidene-a-Dgalactopyranose (Scheme 1). Based on the conditions previously established for the analogue porphyrin,15 the coupling reaction16 was performed in a pearshaped flask using a (1:1) corrole:sugar ratio, in dry toluene and in the presence of NaH (17 equiv) (entry 1, Table 1). After 18 h at 80 °C (no new product was detected at lower temperatures) and under a nitrogen atmosphere, the TLC analysis revealed the formation of two new purple-coloured compounds, with Rf values smaller than that of the starting corrole. After the workup and purification, the mass spectra of the two-formed compounds showed equal base peaks with m/z values at 1036 corresponding to the molecular ion M+, of the expected mono-conjugates 2a17 and 2b.18 Attempts to improve the outcome of the coupling reaction of 1 with 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose were performed, by changing the base and the number of equivalents of sugar (Table 1). In the presence of K2CO3 and using the same ratio of corrole:sugar (1:1) (entry 3, Table 1) the mono-conjugates 2a and 2b were obtained in 22% and 10% yields, respectively, after three days of reaction at 120 °C. When the ratio of corrole:sugar was changed to 1:2 (entry 2, Table 1), under the same reaction conditions (K2CO3 and 120 °C) and after three days we were able to isolate the mono-substituted corrole 2a in 34% yield. Under these conditions, a third fraction was also collected, in 21% yield. It was identified as a mixture of bis-substituted galactoconjugates based on the mass spectrum (peak base at m/z 1276 M+) and its 1H and 19F NMR spectra.19 We were not able to separate these two isomers, even using preparative TLC with different mixtures of eluents. Under the previous conditions, but using NaH as the base (entry 4, Table 1), no improvement was detected regarding the formation of 2a (28% yield) and 2b (13% yield). The preferential formation of 2a versus 2b can be justified by the two possibilities of substitution that lead to the former against just one possibility of substitution to achieve compound 2b. The structures of glycocorroles were confirmed by NMR and mass spectrometry. The 1H NMR spectra of corroles 2a and 2b
are very similar and present three distinctive regions. At lower field the doublets between 9.11 and 8.56 ppm belong to the resonances of the b-pyrrolic protons of the corrole macrocycle. It should be noticed that in 2a there are two doublets at 9.11 and 9.09 corresponding to the resonances of H-2 and H-18, and this is the evidence for the asymmetry of this compound due to the introduction of the galactose unit at the pentafluorophenyl ring at C-5 of the macrocycle. A second region can be observed between 5.67 and 4.38 ppm due to the CH of the carbohydrate moiety. The resonances of the anomeric protons of compounds 2a and 2b appear as doublets at 5.67 and 5.68 ppm, respectively. The resonances of the isopropylidene groups appear in the third region between 3.48–1.26 ppm. The 19F NMR spectra of 2a and 2b (Fig. 1) confirmed the substitution of the para-fluorine atoms by the sugar units. In the 19F NMR spectrum of 2b the resonances of the two equivalent para-fluorine atoms F-41, 43 appear as one triplet at 175.86 ppm. The resonances of the ortho- and meta-fluorine atoms in the non-substituted pentafluorophenyl groups appear as two multiplets between 161.17 and 161.25 and between 184.95 and 185.10 ppm, respectively. The asymmetric profile of 2a can be observed in the 19F NMR spectrum. The resonances of the para-fluorine atoms F-42 and F-43 appear as two signals at 175.91 and 176.44. In this case the resonances of the fluorine atoms in the ortho and meta positions of the non-substituted pentafluorophenyl groups are non equivalents and appear as a set of double doublets and a set of multiplets. The glycocorroles were also characterized by ESI mass spectrometry, in positive mode. The ESI mass spectra showed the corresponding protonated molecules, [M+H]+, at m/z 1037 for corrole isomers 2a, 2b and at m/z 1277 for the mixture of the bis-substituted corroles. The fragmentation of each [M+H]+ ion was induced by collision with argon, and the product ions observed in the corresponding ESI-MS/MS spectra were analysed in order to differentiate corroles 2a and 2b. At low energy collisions, similar fragmentation pathways were observed for compounds 2a and 2b, but with different relative percentages (Fig. 2).
Table 1 Reaction conditions tested for the nucleophilic substitution Entries
1 2 3 4
Ratio (eq.) Sugar
Corrole
1 1 1 1
2 2 1 1
Base
T (°C)
Time
Yield (%) 2a
2b
Bis-substituted
NaH K2CO3 K2CO3 NaH
80 120 120 120
18 h 3 days 6 days 6 days
27 34 22 28
14 16 10 13
21 4
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Figure 1.
19
3
F NMR spectra of corroles (a) 2a and (b) 2b, in CDCl3.
Figure 2. ESI-MS/MS spectra of [M+H]+ ion for isomers (a) 2a and (b) 2b; ⁄ precursor ion.
The ESI-MS/MS spectra of the galactocorrole isomers 2a and 2b obtained present two main fragment ions at m/z 795 and at m/z 775. The ion at m/z 795 is formed by the loss of the protected galactopyranoside substituent, formed by cleavage of the C–O bond,
with migration of one hydrogen to the oxygen of the ether bond (Gal - H) (Scheme 2). The m/z 775 ion can be justified by a joint loss of the same moiety and a HF molecule. These are the major ions corresponding to the base peak of the ESI-MS/MS spectra of both isomers. The different relative abundances of these ions, for example for the one at m/z 795 is 99.9% for 2a and 63.9% for 2b, can be used to differentiate these two isomers. A characteristic ion, with m/z 793 is also observed in both 2a and 2b spectra. This ion bearing a quinone ring is formed by loss of a galactopyranoside substituent plus one hydrogen (Gal + H) (Scheme 2). Also, differences in the relative abundances of this ion at m/z 793 can be observed by comparing both isomers showing higher relative abundance for 2a. Although some of these fragmentations were already described for other porphyrinoid compounds,20,21 this study reveals that these isomers can be characterized and differentiated by ESI-MS/MS. In PDT, the generation of singlet oxygen (1O2) in the target tissue by energy transfer from the first excited triplet state of the photosensitizer to molecular oxygen (3O2), is considered a key feature for an efficient photodynamic effect and the main mechanism responsible for cell death.1 So, considering the potential application of new conjugates corrole–sugar in medicine, namely in PDT, and in order to evaluate how the sugar moiety affects the corrole photophysical properties, we determined the fluorescence quantum yield (fl) and the ability of corroles 1 and 2a (the more abundant compound) to generate singlet oxygen in the same solvent, dimethylformamide (DMF). The methods used - steady state absorption and fluorescence, and time resolved singlet oxygen luminescence detection - have been described before.22–24 The excitation wavelength for both steady state fluorescence and singlet oxygen luminescence detection was at 532 nm with an OD of the galactocorroles of 0.05.
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NHHN
C6F5
C6F5
N HN F F
F
C6F5
NH HN
- (Gal + H) F
O
NHHN
C6F5
F
F
F
m/z 793
NHHN
C6F5 - (CH3)2CO F
F
H
H
O
O
H
O
F O
HO
H O
F
F
m/z 1037
OO
C6F5
NH HN
H
H
m/z 979
O
O
H O
- (Gal - H)
F
NHHN
C6F5
F
NH HN
F
F
F
F
F
OH
F - HF
C6F5
F
NH HN
F
m/z 795
F
NHHN F
F
F
F
F
OH
F
m/z 775
Scheme 2.
Figure 3. Normalized UV–Vis and normalized fluorescence (kexc = 532 nm, OD = 0.05) spectra of corroles 1 and 2a in DMF.
The UV–Vis spectra of the glycocorrole derivatives (Fig. 3) show, as expected, spectroscopic features analogous to those of the starting corrole. The linking of the carbohydrate units to the pentafluorophenyl rings does not change the macrocycle conjugation in order to affect the absorption properties of the new derivatives. The steady-state fluorescence spectra of both compounds were measured also in DMF solutions under normal conditions (open air) and the outcome showed that both compounds have a similar behaviour (Fig. 3).25 As shown in Table 2, for non-conjugated corrole 1 and the sugar-conjugate 2a the spectral position (around 626 nm) and the extinction coefficients at S1,0 S0,0 transition are similar. The fluorescence emission spectra of corroles 1 and 2a in DMF are characterized by a strong emission band with a maximum
Figure 4. Intracellular uptake of corroles 1 and 2a at concentration of 10 lM by Jurkat cells after different periods of incubation time.
intensity around 626 nm and the fluorescence quantum yield (fl) of both derivatives (Table 2) is similar. Considering the potential application of these compounds as photosensitizers for PDT, their ability to generate singlet oxygen was evaluated and both show to have a singlet oxygen quantum yield below that of the tetraphenylporphyrin (TPP) used as reference that shows a singlet oxygen quantum yield of 0.65.26 However, from the results we can conclude that both molecules are able to generate singlet oxygen although the singlet oxygen quantum yield of 2a (0.36) was reduced by a factor of 1.5 when compared with the one obtained for the starting corrole 1 (0.54).
Table 2 Photophysical parameters of corroles 1 and 2a in DMF Samples 1 2a
kmax/nm (Soret band)
e (mol1
kmax/nm (S0,0 ? S1,0)
e (mol1
cm1)
cm1)
Fluorescence peak (nm)
Fluorescence quantum yield (fl)
Singlet oxygen quantum yield (d)
435.5 434.5
22962 23015
616.0 616.5
16234 16220
625.0 626.5
0.27 0.29
0.54 0.36
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Figure 5. Photodynamic effect of corroles 1 and 2a in Jurkat cells after 1 min of irradiation with white light (from a LED array with a irradiance of 120 mW/cm2), at the concentration of 10 lM.
Preliminary studies concerning the intracellular uptake and phototoxic effects of corroles 1 and 2a at the concentration of 10 lM were determined using Jurkat cells (human acute T cell leukaemia) at different periods of incubation (1 h, 5 h and 24 h). The cell number per mL was assessed by a haemocytometer chamber (Improved Neubauer) and after corrole extraction from the cells with DMSO, the fluorescence intensity of cell extracts was measured in the fluorescence setup. The amount of corrole in the cells was quantified by comparison to standard curves and was calculated considering the cell number after each incubation time. The accumulation of corrole photosensitizers in Jurkat cells was found to be moderately dependent on the chemical structure as well as on the incubation time. The presence of the sugar unit seems to facilitate the uptake of 2a when compared with 1, principally after short periods of incubations (Fig. 4). The trend of reduced uptake at longer incubation times can be explained either by a decreasing uptake whilst cell division takes place or by an active transport of the photosensitizer molecules out of the cells by cytoplasmic membrane located carriers. The cell death mechanisms (apoptosis and or necrosis) and irradiation conditions were assessed according to previously established conditions.27 Samples of the cell suspension incubated without any photosensitizer were used as control. The toxicity of our tested corroles was studied by incubation of the Jurkat cells with corroles 1 and 2a (10 lM) during periods of 1 h, 5 h and 24 h in the absence of light. Both corroles did not show any dark toxicity and the results obtained at the studied concentration were comparable to the controls (data not shown). In phototoxicity tests a special setup was used for the cell irradiation. The cells were placed in a 96-well culture plate (200 lL per well) and then positioned on a plane and irradiated with white light from a LED array with an irradiance of 120 mJ/ cm2 during 1 min. After irradiation the cells were incubated in darkness for further 2 h. The phototoxic effect of the studied corroles was evaluated considering the two cell death pathways, apoptosis and necrosis. Apoptosis was estimated through the cell morphological change observation whilst necrosis mechanisms were evaluated by observing the loss of plasma membrane integrity and the ability of cells to exclude dyes like trypan blue. The results obtained after a pre-established incubation period in the presence of the studied compounds followed by irradiation (Fig. 5) show that both corroles gave almost the same total cell killing and there was an increase of about 10% of the apoptotic and necrotic processes when the period of incubation was 24 h. The glycosylation of the corrole core is responsible by an increase in Jurkat cell uptake. The low photodynamic effect of
conjugate 2a is probably related with the less efficient singlet oxygen production when compared with the starting corrole 1. The intracellular uptake experiment provides information about the amount of photosensitizers attached to the Jurkat cells, but no information about the intracellular localization of the photosensitizer molecules. Thus further investigations using confocal laser scanning microscopy methods are necessary due to the fact that for an efficient photosensitization the intracellular localization of the photosensitizer plays a major role.27 Therefore, the low phototoxicity of the investigated corroles in this study should not be over interpreted. Since the intracellular localization of the photosensitizers can be affected by the usage of nanocarriers taken up via endocytosis, further in vitro experiments are necessary to screen the potential of the pentafluorophenylcorrole–D-galactose conjugates as efficient photosensitizers. New glycocorroles have been synthesized and fully characterized. The ESI-MS/MS spectra of both isomers demonstrated several differences in the fragmentation pattern, useful to their differentiation. The photophysical properties revealed that glycocorrole 2a is a good fluorophore and is able to generate singlet oxygen. These studies open the way to further investigation on glycocorroles to be tested in PDT or fluorescence diagnosis. Acknowledgments Thanks are due to the University of Aveiro, ‘Fundação para a Ciência e a Tecnologia’ (FCT) and POCI 2010 (FEDER) for funding the Organic Chemistry Research Unit (Project PEst-C/QUI/UI0062/ 2011). J. F. B. Barata thanks FCT for the grant SFRH/BPD/63237/ 2009. MAFF thanks DAAD for funding her stay at HumboldtUniversitat zu Berlin. The authors also thank the FCT-DAAD Transnational cooperation program for financial assistance. References and notes 1. Bonnett, R. Chemical Aspects of Photodynamic Therapy; Gordon and Breach Science Publishers: London, 2000. 2. Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. Chem. Soc. Rev. 2011, 40, 340–362. 3. Soares, A. R. M.; Neves, M. G. P. M. S.; Tomé, A. C.; Cruz, M. C. I; Zamarrón, A.; Carrasco, E.; González, S.; Cavaleiro, J. A. S.; Torres, T.; Guldi, D. M.; Juarranz, A. Chem. Res. Toxicol. 2012, 25, 940–951. 4. Cavaleiro, J. A. S.; Tomé, J. P. C.; Faustino, M. A. F. Synthesis of Glycoporphyrins In Topics in Heterocyclic Chemistry, Heterocycles from Carbohydrate Precursors; Ashry, E. S. H. El., Ed.; Springer: Berlin/Heidelberg, 2007; Vol. 7, pp 179–248. 5. Peng, J.; Wang, K.; Tan, W.; He, X.; He, C.; Wu, P.; Liu, F. Talanta 2007, 71, 833– 840. 6. Aviv-Harel, I.; Gross, Z. Chem. Eur. J. 2009, 15, 8382–8394. 7. Barata, J. F. B.; Neves, M. G. P. M. S.; Tomé, A. C.; Cavaleiro, J. A. S. J. Porphyrins Phthalocyanines 2009, 13, 415–418.
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8. Santos, C. I. M.; Oliveira, E.; Barata, J. F. B.; Faustino, M. A. F.; Cavaleiro, J. A. S.; Neves, M. G. P. M. S.; Lodeiro, C. J. Mater. Chem. 2012, 22, 13811–13819. 9. (a) Lim, P.; Mahammed, A.; Okun, Z.; Saltsman, I.; Gross, Z.; Gray, H. B.; Termini, J. Chem. Res. Toxicol. 2012, 25, 400–409; (b) Agadjanian, H.; Weaver, J. J.; Mahammed, A.; Rentsendorj, A.; Bass, S.; Kim, J.; Dmochowski, I. J.; Margalit, R.; Gray, H. B.; Gross, Z.; Medina-Kauwe, L. K. Pharm. Res. 2006, 23, 367–377; (c) Agadjanian, H.; Ma, J.; Rentsendorj, A.; Valluripalli, V.; Hwang, J. Y.; Mahammed, A.; Farkas, D. L.; Gray, H. B.; Gross, Z.; Medina-Kauwe, L. K. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 6105–6110; (d) Haber, A.; Aviram, M.; Gross, Z. Inorg. Chem. 2012, 51, 28–30. 10. Tomé, J. P. C.; Silva, E. M. P.; Pereira, A. M. V. M.; Alonso, C. M. A.; Faustino, M. A. F.; Neves, M. G. P. M. S.; Tomé, A. C.; Cavaleiro, J. A. S.; Tavares, S. A. P.; Duarte, R. R.; Caeiro, M. F.; Valdeira, M. L. Bioorg. Med. Chem. 2007, 15, 4705–4713. 11. Tomé, J. P. C.; Neves, M. G. P. M. S.; Tomé, A. C.; Cavaleiro, J. A. S.; Mendonça, A. F.; Pegado, I. N.; Duarte, R.; Valdeira, M. L. Bioorg. Med. Chem. 2005, 13, 3878–3888. 12. (a) Zheng, G.; Graham, A.; Shibata, M.; Missert, J. R.; Oseroff, A. R.; Dougherty, T. J.; Pandey, R. K. J. Org. Chem. 2001, 66, 8709–8716; (b) Vedachalam, S.; Choi, B.; Pasunooti, K. K.; Ching, K. M.; Lee, K.; Yoon, H. S.; Liu, X. W. Med. Chem. Commun. 2011, 2, 371–377. 13. Gryko, D. T.; Koszarna, B. Org. Biomol. Chem. 2003, 1, 350–357. 14. Gross, Z.; Galili, N.; Saltsman, I. Angew. Chem., Int. Ed. 1999, 38, 1427–1429. 15. Costa, J. I. T.; Tomé, A. C.; Neves, M. G. P. M. S.; Cavaleiro, J. A. S. J. Porphyrins Phthalocyanines 2011, 15, 1116–1133. 16. General procedure: To a solution of 1,2:3,4-di-O-isopropylidene-a-Dgalactopyranose (1 equiv, 3.25 mg) and base in dried toluene, corrole 1 (2 equiv, 20 mg) was added. The reaction was carried out at 80–120 °C, under N2 atmosphere. After 18 h to 6 days, the reaction was finished. The crude mixture was neutralized with a saturated solution of citric acid. It was recovered to organic phase with chloroform and washed, at least two times, with distilled water. The reaction mixture was chromatographed using chloroform as eluent. 17. Compound 2a: 1H NMR (CDCl3, 300,13 MHz): d 9.11 (1H, d, J 4.3 Hz, H-2 or H18), 9.09 (1H, d, J 4.3 Hz, H-2 or H-18), 8.84 (1H,d, J 4.8, H-b), 8.75 (1H,d, J 4.6, H-b), 8.61–8.56 (4H, m), 5.67 (1H, d, J 5.0 Hz, Gal-H1), 4.77–4.72 (2H, m, Gal-H, Gal-CH2), 4.50 (1H, dd, J 1.9 Hz, J 7.8 Hz, Gal-H), 4.44 (1H, dd, J 2.5 Hz, J 5.0 Hz, Gal-H), 4.40–4.36 (1H, m, Gal-H), 1.65, 1.56, 1.43, 1.26 (12H, 4s, Gal-CH3). 19F NMR (CDCl3, 282 MHz): d 160.63 to 160.71 and 161.17 to 161.23 (4F, m, F-22,62,23,63), 163.72 to 163.78 (2F, m, F-21,61), 175.91 (1F, m, F-43), 176.44 (1F, t, J 21.2 Hz, F-42), 179.54 to 179.61 (2F, m, F-31,51), 185.04 to 185.11 and 185.40 to 185.58 (4F, m, F-32,52,33,53) UV–Vis em CHCl3 kmax (log e): 407 (4.60), 561 (3.73), 606 (3.33), MALDI-TOF: m/z 1036 M+. 18. Compound 2b: 1H NMR (CDCl3, 300,13 MHz): d 9.11 (2H, d, J 4.2 Hz, H-2,18), 8.75 (2H,d, J 4.7, H-b), 8.66 (2H,d, J 4.7, H-b), 8.56 (2H, d, J 4.2 Hz, H-3,17), 5.68
19.
20.
21.
22. 23. 24. 25.
26. 27.
(1H, d, J 5.0 Hz, Gal-H1), 4.77–4.73 (2H, m, Gal-H, Gal-CH2), 4.52–4.38 (4H, m, Gal-H), 3.49 (6H,1s, Gal (C(CH3)2) 1.65, 1.56, 1.43, 1.26 (12H, 4s, Gal-CH3). 19F NMR (CDCl3, 282 MHz): d 161.17 to 161.25 (4F, m, F-21,61,23,63), 163.26 (2F, dd, J1 24.0 J2 8.5 Hz, F-22,62), 175.86 (2F, t, J 19.4 Hz, F-41,43), 180.03 (2F, dd, J1 24.0 J2 8.5 Hz, F-32,52), 184.95 to 185.10 (4F, m, F-31,51,33,53) UV–Vis em CHCl3 kmax (log e): 407 (4.83), 562 (3.95), 603 (3.51), MALDI-TOF: m/z 1036 M+. Mixture of bis-substituted: 1H NMR (CDCl3, 300,13 MHz) d: 9.10–9.07 (m, 4H, H2,18), 8.81 (d, 4H, J 4.8 Hz, H-b), 8.72 (d, 1H, J 4.7 Hz, H-b), 8.64 (d, 2H, J 4.7 Hz, H-b), 8.60–8.53 (m, 6H, H-b), 5.68-5.65 (m, 4H, Gal-H1), 4.77–4.72 (m, 12H, Gal-H, Gal-CH2), 4.52–4.51 (m, 4H, Gal-H), 4.45–4.43 (m, 4H, Gal-H), 4.39–4.36 (m, 4H, Gal-H), 1.65, 1.55, 1.42 (3s, 48H, Gal-CH3). 19F NMR (CDCl3, 282 MHz) d:160.62 to 161.24 (m, 2F, F-ortho), 161.15 to 161.22 (m, 2F, F-ortho), 163.18 to 163.25 (m, 3F, F-ortho), 163.66 to 163.73 (m, 5F, F-ortho), 176.11 to 176.13 (m, 1F, F-para), 176.56 to 176.71 (m, 1F, F-para), 179.64 to 179.72 (m, 5F, F-meta), 180.06 to 180.17 (m, 3F, F-meta), 185.16 to 185.22 (m, 2F, F-meta), 185.51 to 185.69 (m, 2F, F-meta). ESIMS (fragment, relative abundance): 1219 [Mix(CH3)2CO, 29.8], 1199 [Mix(CH3)2COHF, 5.2], 1161 {Mix2 [(CH3)2CO], 19.0}, 1035 (MixGalH, 74.9), 1033 [Mix(Gal+H), 54.8], 1015 [Mix(GalH)+HF, 29.7], 995 [(MixGalH)2F, 11.3], 977 [Mix(GalH)(CH3)2CO, 74.5], 957 [Mix(GalH)(CH3)2COHF, 48.4], 793 [Mix2(GalH), 89.8], 791 [Mix(GalH)(Gal+H), 92.5], 773 [Mix2 (GalH)HF, 100], 753 [Mix2 (GalH)2 HF, 74.0], 733 [Mix2 (GalH)3 HF, 80.9]. MALDI-TOF: m/z 1276 M+. Izquierdo, R. A.; Barros, C. M.; Santana-Marques, M. G.; Ferrer Correia, A. J.; Silva, A. M. G.; Tomé, A. C.; Silva, A.; Neves, M. G. P. M. S.; Cavaleiro, J. A. S. Rapid Commun. Mass Spectrom. 2004, 18, 2601–2611. Domingues, M. R. M.; Domingues, P.; Reis, A.; Ferrer Correia, A. J.; Tomé, J. P. C.; Tomé, A. C.; Neves, M. G. P. M. S.; Cavaleiro, J. A. S. J. Mass Spectrom. 2004, 39, 158–167. Ermilov, E.; Menting, R.; Lau, J.; Leng, X.; Röder, B.; Ng, D. Phys. Chem. Chem. Phys. 2011, 13, 17633. Tannert, S.; Ermilov, E. A.; Vogel, J. O.; Choi, M. T. M.; Ng, D.; Röder, B. J. Phys. Chem. B 2007, 111, 8053. Hackbarth, S.; Schlothauer, J.; Preuß, A.; Röder, B. Proc. SPIE 2009, 7380, 738045–738047. Kuciauskas, D.; Lin, S.; Seely, G. R.; Moore, A. L.; Moore, T. A.; Gust, D.; Drovetskaya, T.; Reed, C. A.; Boyd, P. D. W. J. Phys. Chem. 1996, 100, 15926– 15932. A.A., Krasnovsky, Jr. Proc. Royal Soc. Edinburgh 1994, 102B, 219–235. Chen, K.; Preuß, A.; Hackbarth, S.; Wacker, M.; Langer, K.; Röder, B. J. Photochem. Photobiol. B: Biol. 2009, 96(1), 66–74.
Please cite this article in press as: Cardote, T. A. F.; et al. Tetrahedron Lett. (2012), http://dx.doi.org/10.1016/j.tetlet.2012.09.038
