- Email: [email protected]
Novel water-soluble porphyrin-based receptors for saccharide recognition
Materials Science and Engineering C 18 Ž2001. 135–140 www.elsevier.comrlocatermsec
Novel water-soluble porphyrin-based receptors for saccharide recognition Oleksandr Rusin, Martin Hub, Vladimır ´ Kral ´) Department of Analytical Chemistry, Institute of Chemical Technology, Technicka´ 5, 166 28 Prague, Czech Republic
Abstract A binding study of porphyrin phosphonates Ž1,2., porphyrin–oligopeptide conjugates Ž3–5., porphyrin–fluorescein Ž6., and porphyrin–methyl red Ž7. conjugates to saccharides and alkyl pyranosides showed strong interaction in aqueous media and DMSO. The UV–Vis, fluorescence, 1 H-, 31 P-NMR, and IR spectroscopy were used to study the mechanisms of complexation. Participation of hydrogen bonds in the formation of porphyrin–saccharide complexes was demonstrated. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Porphyrin; Saccharide; Complexation
1. Introduction The phenomenon of saccharide recognition is one of the interesting topics of modern chemistry and biomedicine. The role of oligosaccharides in biological regulation has attracted a great deal of attention in recent years w1x. The modern concept of the role of oligosaccharides in the living cells includes not only energetic and structural functions, but also their participance in many recognition events. Carbohydrates, by their unique multilinkage monomers and branched structure, contain more information in a short sequence than any other biological oligomer. Therefore, on one hand, a high level of information potential is inherent in biological recognition systems comprised of complex carbohydrate ligands that are recognized for targeted activities and, on the other hand, in specific protein receptors such as lectins, enzymes, or antibodies. The information-carrying potential of oligosaccharides is greater than that of proteins and nucleic acids of equivalent molecular weight and their presence on cell surfaces and on many proteins suggests their importance that remained previously unrecognized w2x. Among all biological molecules, carbohydrates, in a short sequence, can potentially display the largest number of ligand structures to the binding sites of proteins in molecular recognition systems. Three-dimensional carbohydrate structures comprise a Ahigh level languageB biochemical code. In this view, ) Corresponding author. Tel.: q420-2-2435-4299; fax: q420-2-312828. E-mail address: [email protected] ŽV. Kral ´ ..
DNA can be looked upon as Amachine language,B coding for the lectins and the sets of transferases that assemble the sugars. Oligosaccharides are now known to mediate cell– cell recognition, infection of cells by pathogens, several aspects of immune response, distribution and reactivity of proteins within cells, and membrane transport. Synthetic carbohydrate receptors could be used as drugs, to target cell types, to transport saccharides or related pharmaceuticals across cell membranes, and in carbohydrate sensors. Principles of saccharide recognition by natural receptors are not well understood. To the chemists, the problem of the carbohydrate recognition represents a challenge to architectural and synthetic talents, and an opportunity to answer fundamental questions in an important area w2x. Besides, investigation of carbohydrate recognition can be very important for the biomedical and industrial applications. Thus, great efforts have been focused on the preparation of water-soluble porphyrins as potential ligands for the saccharides. A sufficient solubility is achieved by the introduction of water-solubilizing groups on the porphyrin periphery. Water-soluble porphyrins have been recently extensively studied, mainly due to their possible medicobiological applications w3,4x. Most of described derivatives possess positively charged groups. On the other hand, there are only a few examples of negatively charged porphyrins that are prepared mainly by the introduction of carboxylate or sulfonate groups into the porphyrin periphery. The use of phosphonate groups thus came as a new alternative toward known anionic groups. The P5O group is widespread in nature in numerous biomolecules. It plays
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 3 8 1 - 2
136
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
Fig. 1. Structures of receptors 1–7.
an important role in noncovalent bonding of proteins or other specific ligands to their substrates. This group is a strong hydrogen bond acceptor and macrocyclic phosphonates have been used successfully for the saccharide recognition w2x. Recently, porphyrin–oligopeptide conjugates have been intensively studied for their activity w5x, electro- and photochemical properties w6,7x, O 2 sensing w8x, and interactions with other molecules w9–12x. The interaction saccharide– polypeptide is essential in natural saccharide receptors such as lectins. This fact was employed for the design of a series of water-soluble porphyrin–peptide conjugates for the saccharide recognition.
We present complexation studies of 4-Ž2,8,12,18-tetraethyl-3,7,13,17-tetramethyl-1,5,9-Ž p-phosphonomethyl phenyl.-porphyrin-5-yl. benzylphosphonate Ž1,2. w13x and porphyrin–peptide conjugates Ž3–5. in comparison with porphyrin–fluorescein Ž6., and the porphyrin–methyl red Ž7. conjugates. All compounds are designed as artificial receptors for the saccharide recognition and can serve as recognition and signaling units of chemical sensors. 2. Experimental Fig. 1 shows the structures of hosts 1–7 possessing proton donorracceptor groups. The planar surface of the
Fig. 2. Typical spectral UV–Vis changes upon incremental addition of a-D-glucose to porphyrin phosphonate Ž2. in aqueous media.
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140 Table 1 Association constants for binding of saccharides with receptors in DMSO Ž1. and water Ž2–5, UV–Vis titration. a Association constant Ž K a ., 10 2 wMy1 x
Saccharide
1 Octyl a-Dglucopyranoside Octyl b-Dglucopyranoside Methyl a-Dglucopyranoside Methyl b-Dglucopyranoside p-Nitrophenyl galacto-b-pyranoside D-Galactose a-D-Glucose D-Fructose D-Ribose D-Trehalose a-D-Lactose b-D-Lactose Maltotriose
2
3
4
5
137
2.1. Preparations 2.1.1. Preparation of porphyrin phosphonates (1,2) The preparation of porphyrin phosphonates 1,2 was described elsewhere w13x.
51.11 48.90 11.50 10.90 60.00 135.00 190.00 233.00 60.00 194.00 213.00 210.10 200.20
1.20 0.22 0.51 1.26 2.74 2.09 1.48 3.15
0.68 1.40 2.78 1.00 4.53 4.08 1.48 4.85
0.35 0.29 0.63 0.11 0.67 0.97 0.33 1.00
a
The formation and UV–Vis estimation constants of sugar–receptor complexes. A 6.15=10y6 -M solution of macrocycle in DMSO or H 2 O containing 5% of MeOH Žvrv. was placed in a 1-cm2 quartz cuvette. A known amount of saccharide was added in increments Ž0–100 equivalents; the solution contained the same concentration of receptor as in cuvette.. The absorbance changes were measured at the absorption maxima Žroom temperature., and data were then evaluated with the aid of the least squares curve fitting. The K a was calculated for 1:1 complexes. The reproducibility of the K a values was "10% in triplicate runs.
porphyrin macrocycle favours stacking interactions in solution, which leads to the formation of aggregates w14x. Dilution of the 1–7 aggregate solutions results in disaggregation. Here we carried out spectral measurements at the porphyrin concentration 6.15 = 10y6 M where no deviation from Beer’s law was observed.
2.1.2. Preparation of porphyrin–peptide conjugates (3–5) A mixture of p-tetracarboxy phenyl porphyrin Ž0.1 g, 0.126 mM., oxalyl chloride Ž0.127 g, 0.63 mM., and dimethylformamide Ž0.01 ml. in dry CHCl 3 was stirred for 1 h, then the solvent was evaporated. The obtained acylchloride was redissolved in dry CHCl 3 . The protected peptides H 2 N-Lys-Ala-Ala, H 2 N-Lys-Ala-Asp, or H 2 NLys-Leu-Asp Ž0.63 mM. in dry CHCl 3 with excess of triethylamine were added dropwise. The reaction mixture was stirred for 48 h, the solvent was evaporated, and the conjugate was isolated by column chromatography on silica gel ŽCH 2 Cl 2rMeOH, 9:1. and deprotected. The yield was 70%. 2.1.3. Preparation of the porphyrin–fluorescein conjugate (6) Tetrakis Ž o-aminophenyl.porphyrin Ž a ,a ,a ,a-conformer. was prepared from the corresponding tetrakisŽnitrophenyl.porphyrin w15,16x. A solution of tetrakis Ž o-aminophenyl.porphyrin Ž1 mmol. and fluorescein isothiocyanate Ž5 mmol. in 2 ml of dry DMF was left in the dark at room temperature for 3 days w17x. The solution was diluted with methanol and diethyl ether to precipitate the fluorescein derivative. The conjugate was purified by silica gel chromatography ŽCH 2 Cl 2rMeOH, 9:1.. The yield was 80%. 2.1.4. Preparation of porphyrin–methyl red conjugate (7) A solution of methyl red in acetonitrile Ž15 mmol. with two drops of DMF was treated at room temperature by
Fig. 3. Typical UV–Vis spectral changes of 3–5 in aqueous media; lma x s 403 nm. Interaction of 3 with a-D-glucose.
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
138
squares curve fitting for 1:1 complexes. The mixture waterrmethanol Ž95:5. and water and DMSO were used as solvents, respectively.
3. Results and discussion 3.1. Porphyrin phosphonates (1,2)
Fig. 4. Fluorescence spectral changes of 6 in aqueous media; lma x s 514 nm. Interaction of 6 with a-D-glucose.
excess of the oxalyl chloride for 3 h. The solvent was evaporated, solid acyl chloride was redissolved in dry CH 2 Cl 2 and added dropwise to the solution of tetrakisŽ oaminophenyl.porphyrin in CH 2 Cl 2 Ž5 mmol. with excess of triethyl amine at 0 8C. The mixture was stirred 3 days at room temperature then filtered off, and evaporated. The product was purified by recrystallization from MeOH– Et 2 O. The yield was 75%. 2.2. Apparatus and procedures Absorption spectra were recorded with a ACary 400 ScanB spectrometer. Fluorescence spectra were recorded with a FluoroMax-2 spectrometer. 1 H- and 31 P-NMR spectra were measured using a 500-MHz ABrukerB spectrometer. IR spectra were obtained on FTIR spectrometer ANicolet 210.B K a values were determined by the least
Binding studies of the receptors were carried out in DMSO for 1 and in waterrmethanol Ž95:5. mixture for 2. Alkyl and aryl pyranosides were tested for the complexation with the water-insoluble receptor 1. Several monoand oligosaccharides were used for the complexation with the water-soluble receptor 2. The host 1 showed the Soret maximum in UV–Vis region at 403 nm in DMSO; the host 2 at 430 nm in aqueous media ŽFig. 2.. The measurements indicated preferable binding of 1 to the long or bulky side chain-substituted saccharides in comparison to O-methyl analogues ŽTable 1.. It indicates the presence of hydrophobic CH–p and p–p interactions. The water-soluble host 2 showed strong interaction with different saccharides ŽTable 1.. The receptor 2 also displayed stronger binding to a-D-glucose and D-fructose. The values of association constants calculated for di- and trisaccharides are slightly higher than for the majority of monosaccharides. Recently, a binding mode between macrocyclic phosphonates and saccharide species has been proposed w2x. The mechanism of interaction includes the formation of hydrogen bonds between the Õic-diol of the pyranose cycle and the two oxygen atoms of the corresponding phosphonate group. We suppose that a decisive role in porphyrin–saccharide complex formation between 1,2 and the vicinal diol segment of saccharide is played by
Fig. 5. Typical UV–Vis spectral changes of 7; lma x s 419 nm. Interaction of 7 with a-D-glucose-1-phosphate.
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
139
hydrogen bonds. The 1 H-, 31 P-NMR, and IR investigations clearly indicated an involvement of phosphonate and saccharide hydroxylic groups in the complex formation process. All spectroscopic methods indicate characteristic behavior of the signals of hydroxy and phosphonate groups under complexation, namely shift, splitting, and broadening, which clearly proves this involvement. 3.2. Porphyrin–peptide conjugates (3–5) We examined several peptide sequences attached to the porphyrin core as a recognition element for saccharide binding. Porphyrin–peptide conjugates 3–5 were tested in the abovementioned waterrmethanol system. The addition of saccharide to the macrocycle solution was accompanied with the change of Soret maximum intensity ŽFig. 3.. The maximum in UV–Vis region for 3 is at 403 nm, for 4 at 406 nm, and for 5 at 407 nm. All three receptors display higher specificity to di- and trisaccharides except for b-Dlactose ŽTable 1.. The interaction of 3–5 with saccharides was investigated also by 1 H-NMR spectroscopy in DMSOd 6 and CDCl 3 . This method allows to determine the shift of saccharide OH group proton signals caused by complexation. The association constants calculated for Asp-containing 5 are lower than that for 3 and 4. Nevertheless, 5 is able to extract a-D-lactose from aqueous media to CHCl 3 . This fact was confirmed by 1 H-NMR investigation where splitting and broadening of signals of amino, amido, and CH groups of peptide tails were monitored. The IR spectral data also clearly showed the involvement of saccharide OH– and carboxylate group of the receptor in the interaction process. 3.3. Porphyrin–fluorescein conjugate (6) Introduction of the fluorescein subunits on the porphyrin periphery via thiourea bridges allows to obtain a receptor with interesting binding properties. No changes were registered by UV–Vis spectroscopy on addition of saccharides to a solution of receptor 6 in aqueous media. However, significant changes were observed in its fluorescence spectra ŽFig. 4.. The fluorescence maximum of 6 is at 514 nm, which was increasing by gradual addition of saccharide species. The selectivity to certain saccharides is reflected in Fig. 6. The receptor 6 is able to distinguish certain saccharide species, namely D-galactose and D-glucose, D-fucose, and L-fucose. Oligosaccharides tend to bind stronger than monosaccharides. Moreover, selective binding of a-D-lactose was also observed.
Fig. 6. Interaction of receptors 6 and 7 with saccharides. Gal— D-galactose, Glu— a-D-glucose, Fru—fructose, Rib—ribose, Man—mannose, D-Fuc— D-fucose, L-Fuc— L-fucose, Tre—trehalose, a-Lac— a-D-lactose, b-Lac— b-D-lactose, Mtri—maltotriose, G-1-P— a-D-glucose-1-phosphate, G-6-P— b-D-glucose-6-phosphate, F-1,6-P— D-fructose-1,6-diphosphate, F-2,6-P— D-fructose-2,6-diphosphate, F-6-P— D-fructose-6phosphate, R-5-P—riboso-5-phosphate.
in UV–Vis and fluorescence spectra. All measurements were carried out in a 0.005-M phosphate buffer at pH 7.0. On the other hand, UV–Vis spectroscopy of 7 with phosphorylated saccharides showed expressed changes of the maxima at 419 nm ŽFig. 5.. The association constants to saccharides are presented in Fig. 6. The configuration of the glycoside hydroxyl and the position of the PO4 group at the C-6 probably influence binding of the substrates. The measurements indicate very low affinity of receptor 7 to b-glucose-6-phosphate and D-fructose-6-phosphate.
4. Conclusion 3.4. Porphyrin–methyl red conjugate (7) The specific environment of the porphyrin core of receptor 7 make this conjugate sensitive to the phosphorylated saccharide derivatives. Only weak changes were observed under the addition of the unmodified saccharides
Several water-soluble porphyrins were proposed as receptors for the recognition of saccharides. Anionic groups, as phosphonate, provide strong hydrogen donatingraccepting effect and are able to form effective bonds with saccharides in aqueous media. Porphyrin–peptide conju-
140
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
gates also form complexes with saccharides, where carboxylic and amido groups are important for binding. Water-soluble conjugates of porphyrin with fluorescein and methyl red are applicable for the fluorescence and UV–Vis tests of the saccharide species. Spectroscopic methods allow to establish the binding mode of the complex formation. All compounds can serve as suitable analytical tools for the saccharide recognition. Acknowledgements The project was supported by the grant of Ministry of Education of the Czech Republic, Research project CEZ: J19r98:223400008.
w5x w6x w7x w8x w9x w10x w11x w12x
References w1x R.U. Lemieux, Chem. Soc. Rev. 18 Ž1989. 347–374. w2x A.P. Davis, R.S. Wareham, Angew. Chem., Int. Ed. 38 Ž1999. 2978–2996. w3x R. Bonnett, Chem. Soc. Rev. 24 Ž1995. 19–33. w4x V. Kral, ´ O. Rusin, F.P. Schmidtchen, Org. Lett. 3 Ž6. Ž2001. 873–876; K. Lang, P. Anzenbacher Jr., P. Kapusta, P. Kubat, ´ V. Kral, ´ D.M. Wagnerova, ´ Photochem. Photobiol. 57 Ž2000. 51–59;
w13x w14x w15x w16x w17x
P. Kubat, ´ V. Kral, ´ K. Lang, P. Anzenbacher Jr., V. Kral, ´ B. Ehrenberg, J. Chem. Soc., Perkin Trans. 1 Ž2000. 933–941; A. Synytsya, V. Kral, ´ P. Pouckova, ´ K. Volka, App. Spectrosc. 55 Ž2. Ž2001. 175–180; O. Rusin, V. Kral, ´ Sensor Actuator B 3775 Ž2001. 1–5. P. Bhyrappa, J.K. Young, J.S. Moore, K.S. Suslick, J. Am. Chem. Soc. 118 Ž1999. 5708. R. Sadamoto, N. Tomioka, T. Aida, J. Am. Chem. Soc. Rev. 118 Ž1996. 3978. D.L. Jiang, T. Aida, J. Am. Chem. Soc. 120 Ž1998. 10895. S.A. Vinogradov, L.W. Lo, D.F. Wilson, Chem.-Eur. J. 5 Ž1999. 1338. Y. Tomoyose, D.-L. Jiang, R.-H. Jin, T. Aida, T. Yamashita, K. Horie, E. Yashima, Y. Okamoto, Macromolecules 29 Ž1996. 5236. N. Tomioka, D. Takasu, D. Takahashi, T. Aida, Angew. Chem. 110 Ž1998. 1611. N. Tomioka, D. Takasu, D. Takahashi, T. Aida, Angew. Chem., Int. Eng. 37 Ž1998. 1331. M. Numata, A. Ikeda, C. Fukuhara, S. Shinkai, Tetrahedron Lett. 40 Ž1999. 6945. V. Kral, ´ O. Rusin, J. Charvatova, ´ ´ P. Anzenbacher Jr., Tetrahedron Lett. 41 Ž2000. 10147. D. Dolphin ŽEd.., The Porphyrins, vol. 5, Academic Press, New York, 1978, p. 303. A. Bettelheim, B.A. White, S.A. Raybuck, R.W. Murray, Inorg. Chem. 26 Ž1987. 1009. J.P. Collman, R.R. Gagne, C.A. Reed, T.R. Halbert, G. Land, W.T. Robinson, J. Am. Chem. Soc. 97 Ž1975. 1427. V. Bakthavachalam, N. Baindur, B.K. Madras, J.L. Neumeyer, J. Med. Chem. 34 Ž1991. 3235.
Novel water-soluble porphyrin-based receptors for saccharide recognition Oleksandr Rusin, Martin Hub, Vladimır ´ Kral ´) Department of Analytical Chemistry, Institute of Chemical Technology, Technicka´ 5, 166 28 Prague, Czech Republic
Abstract A binding study of porphyrin phosphonates Ž1,2., porphyrin–oligopeptide conjugates Ž3–5., porphyrin–fluorescein Ž6., and porphyrin–methyl red Ž7. conjugates to saccharides and alkyl pyranosides showed strong interaction in aqueous media and DMSO. The UV–Vis, fluorescence, 1 H-, 31 P-NMR, and IR spectroscopy were used to study the mechanisms of complexation. Participation of hydrogen bonds in the formation of porphyrin–saccharide complexes was demonstrated. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Porphyrin; Saccharide; Complexation
1. Introduction The phenomenon of saccharide recognition is one of the interesting topics of modern chemistry and biomedicine. The role of oligosaccharides in biological regulation has attracted a great deal of attention in recent years w1x. The modern concept of the role of oligosaccharides in the living cells includes not only energetic and structural functions, but also their participance in many recognition events. Carbohydrates, by their unique multilinkage monomers and branched structure, contain more information in a short sequence than any other biological oligomer. Therefore, on one hand, a high level of information potential is inherent in biological recognition systems comprised of complex carbohydrate ligands that are recognized for targeted activities and, on the other hand, in specific protein receptors such as lectins, enzymes, or antibodies. The information-carrying potential of oligosaccharides is greater than that of proteins and nucleic acids of equivalent molecular weight and their presence on cell surfaces and on many proteins suggests their importance that remained previously unrecognized w2x. Among all biological molecules, carbohydrates, in a short sequence, can potentially display the largest number of ligand structures to the binding sites of proteins in molecular recognition systems. Three-dimensional carbohydrate structures comprise a Ahigh level languageB biochemical code. In this view, ) Corresponding author. Tel.: q420-2-2435-4299; fax: q420-2-312828. E-mail address: [email protected] ŽV. Kral ´ ..
DNA can be looked upon as Amachine language,B coding for the lectins and the sets of transferases that assemble the sugars. Oligosaccharides are now known to mediate cell– cell recognition, infection of cells by pathogens, several aspects of immune response, distribution and reactivity of proteins within cells, and membrane transport. Synthetic carbohydrate receptors could be used as drugs, to target cell types, to transport saccharides or related pharmaceuticals across cell membranes, and in carbohydrate sensors. Principles of saccharide recognition by natural receptors are not well understood. To the chemists, the problem of the carbohydrate recognition represents a challenge to architectural and synthetic talents, and an opportunity to answer fundamental questions in an important area w2x. Besides, investigation of carbohydrate recognition can be very important for the biomedical and industrial applications. Thus, great efforts have been focused on the preparation of water-soluble porphyrins as potential ligands for the saccharides. A sufficient solubility is achieved by the introduction of water-solubilizing groups on the porphyrin periphery. Water-soluble porphyrins have been recently extensively studied, mainly due to their possible medicobiological applications w3,4x. Most of described derivatives possess positively charged groups. On the other hand, there are only a few examples of negatively charged porphyrins that are prepared mainly by the introduction of carboxylate or sulfonate groups into the porphyrin periphery. The use of phosphonate groups thus came as a new alternative toward known anionic groups. The P5O group is widespread in nature in numerous biomolecules. It plays
0928-4931r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 Ž 0 1 . 0 0 3 8 1 - 2
136
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
Fig. 1. Structures of receptors 1–7.
an important role in noncovalent bonding of proteins or other specific ligands to their substrates. This group is a strong hydrogen bond acceptor and macrocyclic phosphonates have been used successfully for the saccharide recognition w2x. Recently, porphyrin–oligopeptide conjugates have been intensively studied for their activity w5x, electro- and photochemical properties w6,7x, O 2 sensing w8x, and interactions with other molecules w9–12x. The interaction saccharide– polypeptide is essential in natural saccharide receptors such as lectins. This fact was employed for the design of a series of water-soluble porphyrin–peptide conjugates for the saccharide recognition.
We present complexation studies of 4-Ž2,8,12,18-tetraethyl-3,7,13,17-tetramethyl-1,5,9-Ž p-phosphonomethyl phenyl.-porphyrin-5-yl. benzylphosphonate Ž1,2. w13x and porphyrin–peptide conjugates Ž3–5. in comparison with porphyrin–fluorescein Ž6., and the porphyrin–methyl red Ž7. conjugates. All compounds are designed as artificial receptors for the saccharide recognition and can serve as recognition and signaling units of chemical sensors. 2. Experimental Fig. 1 shows the structures of hosts 1–7 possessing proton donorracceptor groups. The planar surface of the
Fig. 2. Typical spectral UV–Vis changes upon incremental addition of a-D-glucose to porphyrin phosphonate Ž2. in aqueous media.
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140 Table 1 Association constants for binding of saccharides with receptors in DMSO Ž1. and water Ž2–5, UV–Vis titration. a Association constant Ž K a ., 10 2 wMy1 x
Saccharide
1 Octyl a-Dglucopyranoside Octyl b-Dglucopyranoside Methyl a-Dglucopyranoside Methyl b-Dglucopyranoside p-Nitrophenyl galacto-b-pyranoside D-Galactose a-D-Glucose D-Fructose D-Ribose D-Trehalose a-D-Lactose b-D-Lactose Maltotriose
2
3
4
5
137
2.1. Preparations 2.1.1. Preparation of porphyrin phosphonates (1,2) The preparation of porphyrin phosphonates 1,2 was described elsewhere w13x.
51.11 48.90 11.50 10.90 60.00 135.00 190.00 233.00 60.00 194.00 213.00 210.10 200.20
1.20 0.22 0.51 1.26 2.74 2.09 1.48 3.15
0.68 1.40 2.78 1.00 4.53 4.08 1.48 4.85
0.35 0.29 0.63 0.11 0.67 0.97 0.33 1.00
a
The formation and UV–Vis estimation constants of sugar–receptor complexes. A 6.15=10y6 -M solution of macrocycle in DMSO or H 2 O containing 5% of MeOH Žvrv. was placed in a 1-cm2 quartz cuvette. A known amount of saccharide was added in increments Ž0–100 equivalents; the solution contained the same concentration of receptor as in cuvette.. The absorbance changes were measured at the absorption maxima Žroom temperature., and data were then evaluated with the aid of the least squares curve fitting. The K a was calculated for 1:1 complexes. The reproducibility of the K a values was "10% in triplicate runs.
porphyrin macrocycle favours stacking interactions in solution, which leads to the formation of aggregates w14x. Dilution of the 1–7 aggregate solutions results in disaggregation. Here we carried out spectral measurements at the porphyrin concentration 6.15 = 10y6 M where no deviation from Beer’s law was observed.
2.1.2. Preparation of porphyrin–peptide conjugates (3–5) A mixture of p-tetracarboxy phenyl porphyrin Ž0.1 g, 0.126 mM., oxalyl chloride Ž0.127 g, 0.63 mM., and dimethylformamide Ž0.01 ml. in dry CHCl 3 was stirred for 1 h, then the solvent was evaporated. The obtained acylchloride was redissolved in dry CHCl 3 . The protected peptides H 2 N-Lys-Ala-Ala, H 2 N-Lys-Ala-Asp, or H 2 NLys-Leu-Asp Ž0.63 mM. in dry CHCl 3 with excess of triethylamine were added dropwise. The reaction mixture was stirred for 48 h, the solvent was evaporated, and the conjugate was isolated by column chromatography on silica gel ŽCH 2 Cl 2rMeOH, 9:1. and deprotected. The yield was 70%. 2.1.3. Preparation of the porphyrin–fluorescein conjugate (6) Tetrakis Ž o-aminophenyl.porphyrin Ž a ,a ,a ,a-conformer. was prepared from the corresponding tetrakisŽnitrophenyl.porphyrin w15,16x. A solution of tetrakis Ž o-aminophenyl.porphyrin Ž1 mmol. and fluorescein isothiocyanate Ž5 mmol. in 2 ml of dry DMF was left in the dark at room temperature for 3 days w17x. The solution was diluted with methanol and diethyl ether to precipitate the fluorescein derivative. The conjugate was purified by silica gel chromatography ŽCH 2 Cl 2rMeOH, 9:1.. The yield was 80%. 2.1.4. Preparation of porphyrin–methyl red conjugate (7) A solution of methyl red in acetonitrile Ž15 mmol. with two drops of DMF was treated at room temperature by
Fig. 3. Typical UV–Vis spectral changes of 3–5 in aqueous media; lma x s 403 nm. Interaction of 3 with a-D-glucose.
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
138
squares curve fitting for 1:1 complexes. The mixture waterrmethanol Ž95:5. and water and DMSO were used as solvents, respectively.
3. Results and discussion 3.1. Porphyrin phosphonates (1,2)
Fig. 4. Fluorescence spectral changes of 6 in aqueous media; lma x s 514 nm. Interaction of 6 with a-D-glucose.
excess of the oxalyl chloride for 3 h. The solvent was evaporated, solid acyl chloride was redissolved in dry CH 2 Cl 2 and added dropwise to the solution of tetrakisŽ oaminophenyl.porphyrin in CH 2 Cl 2 Ž5 mmol. with excess of triethyl amine at 0 8C. The mixture was stirred 3 days at room temperature then filtered off, and evaporated. The product was purified by recrystallization from MeOH– Et 2 O. The yield was 75%. 2.2. Apparatus and procedures Absorption spectra were recorded with a ACary 400 ScanB spectrometer. Fluorescence spectra were recorded with a FluoroMax-2 spectrometer. 1 H- and 31 P-NMR spectra were measured using a 500-MHz ABrukerB spectrometer. IR spectra were obtained on FTIR spectrometer ANicolet 210.B K a values were determined by the least
Binding studies of the receptors were carried out in DMSO for 1 and in waterrmethanol Ž95:5. mixture for 2. Alkyl and aryl pyranosides were tested for the complexation with the water-insoluble receptor 1. Several monoand oligosaccharides were used for the complexation with the water-soluble receptor 2. The host 1 showed the Soret maximum in UV–Vis region at 403 nm in DMSO; the host 2 at 430 nm in aqueous media ŽFig. 2.. The measurements indicated preferable binding of 1 to the long or bulky side chain-substituted saccharides in comparison to O-methyl analogues ŽTable 1.. It indicates the presence of hydrophobic CH–p and p–p interactions. The water-soluble host 2 showed strong interaction with different saccharides ŽTable 1.. The receptor 2 also displayed stronger binding to a-D-glucose and D-fructose. The values of association constants calculated for di- and trisaccharides are slightly higher than for the majority of monosaccharides. Recently, a binding mode between macrocyclic phosphonates and saccharide species has been proposed w2x. The mechanism of interaction includes the formation of hydrogen bonds between the Õic-diol of the pyranose cycle and the two oxygen atoms of the corresponding phosphonate group. We suppose that a decisive role in porphyrin–saccharide complex formation between 1,2 and the vicinal diol segment of saccharide is played by
Fig. 5. Typical UV–Vis spectral changes of 7; lma x s 419 nm. Interaction of 7 with a-D-glucose-1-phosphate.
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
139
hydrogen bonds. The 1 H-, 31 P-NMR, and IR investigations clearly indicated an involvement of phosphonate and saccharide hydroxylic groups in the complex formation process. All spectroscopic methods indicate characteristic behavior of the signals of hydroxy and phosphonate groups under complexation, namely shift, splitting, and broadening, which clearly proves this involvement. 3.2. Porphyrin–peptide conjugates (3–5) We examined several peptide sequences attached to the porphyrin core as a recognition element for saccharide binding. Porphyrin–peptide conjugates 3–5 were tested in the abovementioned waterrmethanol system. The addition of saccharide to the macrocycle solution was accompanied with the change of Soret maximum intensity ŽFig. 3.. The maximum in UV–Vis region for 3 is at 403 nm, for 4 at 406 nm, and for 5 at 407 nm. All three receptors display higher specificity to di- and trisaccharides except for b-Dlactose ŽTable 1.. The interaction of 3–5 with saccharides was investigated also by 1 H-NMR spectroscopy in DMSOd 6 and CDCl 3 . This method allows to determine the shift of saccharide OH group proton signals caused by complexation. The association constants calculated for Asp-containing 5 are lower than that for 3 and 4. Nevertheless, 5 is able to extract a-D-lactose from aqueous media to CHCl 3 . This fact was confirmed by 1 H-NMR investigation where splitting and broadening of signals of amino, amido, and CH groups of peptide tails were monitored. The IR spectral data also clearly showed the involvement of saccharide OH– and carboxylate group of the receptor in the interaction process. 3.3. Porphyrin–fluorescein conjugate (6) Introduction of the fluorescein subunits on the porphyrin periphery via thiourea bridges allows to obtain a receptor with interesting binding properties. No changes were registered by UV–Vis spectroscopy on addition of saccharides to a solution of receptor 6 in aqueous media. However, significant changes were observed in its fluorescence spectra ŽFig. 4.. The fluorescence maximum of 6 is at 514 nm, which was increasing by gradual addition of saccharide species. The selectivity to certain saccharides is reflected in Fig. 6. The receptor 6 is able to distinguish certain saccharide species, namely D-galactose and D-glucose, D-fucose, and L-fucose. Oligosaccharides tend to bind stronger than monosaccharides. Moreover, selective binding of a-D-lactose was also observed.
Fig. 6. Interaction of receptors 6 and 7 with saccharides. Gal— D-galactose, Glu— a-D-glucose, Fru—fructose, Rib—ribose, Man—mannose, D-Fuc— D-fucose, L-Fuc— L-fucose, Tre—trehalose, a-Lac— a-D-lactose, b-Lac— b-D-lactose, Mtri—maltotriose, G-1-P— a-D-glucose-1-phosphate, G-6-P— b-D-glucose-6-phosphate, F-1,6-P— D-fructose-1,6-diphosphate, F-2,6-P— D-fructose-2,6-diphosphate, F-6-P— D-fructose-6phosphate, R-5-P—riboso-5-phosphate.
in UV–Vis and fluorescence spectra. All measurements were carried out in a 0.005-M phosphate buffer at pH 7.0. On the other hand, UV–Vis spectroscopy of 7 with phosphorylated saccharides showed expressed changes of the maxima at 419 nm ŽFig. 5.. The association constants to saccharides are presented in Fig. 6. The configuration of the glycoside hydroxyl and the position of the PO4 group at the C-6 probably influence binding of the substrates. The measurements indicate very low affinity of receptor 7 to b-glucose-6-phosphate and D-fructose-6-phosphate.
4. Conclusion 3.4. Porphyrin–methyl red conjugate (7) The specific environment of the porphyrin core of receptor 7 make this conjugate sensitive to the phosphorylated saccharide derivatives. Only weak changes were observed under the addition of the unmodified saccharides
Several water-soluble porphyrins were proposed as receptors for the recognition of saccharides. Anionic groups, as phosphonate, provide strong hydrogen donatingraccepting effect and are able to form effective bonds with saccharides in aqueous media. Porphyrin–peptide conju-
140
O. Rusin et al.r Materials Science and Engineering C 18 (2001) 135–140
gates also form complexes with saccharides, where carboxylic and amido groups are important for binding. Water-soluble conjugates of porphyrin with fluorescein and methyl red are applicable for the fluorescence and UV–Vis tests of the saccharide species. Spectroscopic methods allow to establish the binding mode of the complex formation. All compounds can serve as suitable analytical tools for the saccharide recognition. Acknowledgements The project was supported by the grant of Ministry of Education of the Czech Republic, Research project CEZ: J19r98:223400008.
w5x w6x w7x w8x w9x w10x w11x w12x
References w1x R.U. Lemieux, Chem. Soc. Rev. 18 Ž1989. 347–374. w2x A.P. Davis, R.S. Wareham, Angew. Chem., Int. Ed. 38 Ž1999. 2978–2996. w3x R. Bonnett, Chem. Soc. Rev. 24 Ž1995. 19–33. w4x V. Kral, ´ O. Rusin, F.P. Schmidtchen, Org. Lett. 3 Ž6. Ž2001. 873–876; K. Lang, P. Anzenbacher Jr., P. Kapusta, P. Kubat, ´ V. Kral, ´ D.M. Wagnerova, ´ Photochem. Photobiol. 57 Ž2000. 51–59;
w13x w14x w15x w16x w17x
P. Kubat, ´ V. Kral, ´ K. Lang, P. Anzenbacher Jr., V. Kral, ´ B. Ehrenberg, J. Chem. Soc., Perkin Trans. 1 Ž2000. 933–941; A. Synytsya, V. Kral, ´ P. Pouckova, ´ K. Volka, App. Spectrosc. 55 Ž2. Ž2001. 175–180; O. Rusin, V. Kral, ´ Sensor Actuator B 3775 Ž2001. 1–5. P. Bhyrappa, J.K. Young, J.S. Moore, K.S. Suslick, J. Am. Chem. Soc. 118 Ž1999. 5708. R. Sadamoto, N. Tomioka, T. Aida, J. Am. Chem. Soc. Rev. 118 Ž1996. 3978. D.L. Jiang, T. Aida, J. Am. Chem. Soc. 120 Ž1998. 10895. S.A. Vinogradov, L.W. Lo, D.F. Wilson, Chem.-Eur. J. 5 Ž1999. 1338. Y. Tomoyose, D.-L. Jiang, R.-H. Jin, T. Aida, T. Yamashita, K. Horie, E. Yashima, Y. Okamoto, Macromolecules 29 Ž1996. 5236. N. Tomioka, D. Takasu, D. Takahashi, T. Aida, Angew. Chem. 110 Ž1998. 1611. N. Tomioka, D. Takasu, D. Takahashi, T. Aida, Angew. Chem., Int. Eng. 37 Ž1998. 1331. M. Numata, A. Ikeda, C. Fukuhara, S. Shinkai, Tetrahedron Lett. 40 Ž1999. 6945. V. Kral, ´ O. Rusin, J. Charvatova, ´ ´ P. Anzenbacher Jr., Tetrahedron Lett. 41 Ž2000. 10147. D. Dolphin ŽEd.., The Porphyrins, vol. 5, Academic Press, New York, 1978, p. 303. A. Bettelheim, B.A. White, S.A. Raybuck, R.W. Murray, Inorg. Chem. 26 Ž1987. 1009. J.P. Collman, R.R. Gagne, C.A. Reed, T.R. Halbert, G. Land, W.T. Robinson, J. Am. Chem. Soc. 97 Ž1975. 1427. V. Bakthavachalam, N. Baindur, B.K. Madras, J.L. Neumeyer, J. Med. Chem. 34 Ž1991. 3235.