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Computational analysis of native and modified oligofructosides
Journal of Molecular Structure (Theochem) 710 (2004) 61–64 www.elsevier.com/locate/theochem
Computational analysis of native and modified oligofructosides Svetlana Gontaa,*, M. Utinansb, O. Neilandsb, I. Vı¯naa a Institute of Microbiology and Biotechnology, Latvian University, Kronvalda Boulevard 4, Riga, LV-1586, Latvia Faculty of Material Science and Applied Chemistry, Riga Technical University, Azenes street 14, Riga, LV-1048, Latvia
b
Received 23 April 2004; revised 14 July 2004; accepted 30 July 2004 Available online 12 October 2004
Abstract This paper presents a quantum chemical calculation of native (2–7 fructoside residues) and chemically modified (2–4 fructoside residues) levan molecule models. A levan modification was carried out by oxidation and the following reduction or hyrdazonation of the fructoside rings. The conformational particularity and reaction ability was studied for the native and for the modified levan molecules. q 2004 Elsevier B.V. All rights reserved. Keywords: Levan; Computational analysis; Structure–activity relationship; 3-Dimensional structure
1. Introduction The use of immunomodulating agents provides distinct advantages over conventional therapies. Most important, the enhancement of the host immune system’s innate ability to combat bacterial infection obviates the problem associated with the antibiotic resistance. Recently, certain polysaccharides of microbial origin have been described that act as potent immunomodulators with the specific activity for both T cells and antigen-presenting cells such as monocytes and macrophages [1]. While the activity of some of these polymers has been known for over 30 years, the lack of defined structural and mechanistic information has limited efforts to study their potential for clinical use. Relatively few polysaccharides have been examined in detail where both the structurefunction and mechanism of action have been performed. Recent investigations have led to a more detailed understanding of the structural aspects of polysaccharides that influence the host immune responses [1]. The immunomodulating activity was shown for such microbial origin polysaccharides as b-[1/3]-glucans, mannan [1] and levan [2–5]. Levan is a biologically active polyfructoside produced by various bacteria. It is a neutral * Corresponding author. Tel.: C371-7034-889; fax: C371-7034-885. E-mail address: [email protected] (S. Gonta). 0166-1280/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2004.07.029
globular macromolecule with a highly branched structure, b-[2/6] linkages in the main chain and b-[2/1] linkages in the branching point [6]. Levan stimulates both, leukopoiesis and antibody production by splenocytes, activates the formation of reactive oxygen intermediates by phagocytes, and promotes the tumoricidal activity of phagocytes. The antitumor action of levan is implemented via the immune system of the organism. Levan prolongs the life-span of tumor-bearing mice. Levan has also radioprotective properties [5]. The C3–C4 region of the levan b-fructoside ring might be one of the essential elements of its immunological activity [7]. However, there is not much information as yet about the levan immunomodulating mechanisms. Previously it was shown that chemical modification of levan produced by Zymomonas mobilis enhanced its immunomodulating activity [8]. To elucidate an effect of levan chemical modifications on its conformational particularity and reaction ability a conformational analysis of native and modified model molecules of levan has been done by using AM1 simulations based on the ‘HyperChem’ programm package.
2. Method The semiempirical quantum chemical programme AM1 from the ‘HyperChem’ programm package [9] was used for
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Fig. 1. The structure of levan and its modification form: I—levan, fragment of structure; II—oxidized form; III—reduced form.
a theoretical modification and analysis of native and modified fructosides with 2–7 fructosyl residues and b-[2/6] links in the chain. The conformational analysis of oligofructoside was done according to the molecular mechanics programm MMC. The possible molecule conformation was calculated by using ‘HyperChem’ programm package utilities. The torsion angles between fructoside fragments have been varied. Electronic system of fructoside model molecules was studied after their geometry optimisation with AM1 method.
[10]. As the periodate oxidation altered the configuration of C3 and C4 region we suggested that this region could be connected with the biological activity of levan. 3.2. An electronic structure model of native and modified trifructosides
3.1. An effect of chemical modifications of C3–C4 region on its structure
Calculations of unmodified trifructoside with AM1 method showed, –O–H group hydrogen atom to have a positive charge (C0.21 to C0.23 e), but oxygen atoms in the fructoside cycle and in the oxygen bridge between fructoside cycles to have a negative charge (K0.26 to K0.31 e). Structures were stabilised with relatively strong hydrogen bonds between these atoms. The length of O–H/O bonds ˚ , which resulted in strengthenfluctuated from 2.17 to 2.54 A ing of hydrogen bonds and explained the difficulties in
Chemical modification of fructoside I by its oxidation with potassium periodate II and following reduction with sodium borohydride III (Fig. 1) destroyed the fructoside ring: in line I–II–III (Fig. 2). The distance between C3 and ˚ ) and led to C4 atoms was increased (1.55 /2.80 /3.00 A an increase in C5–O–C2 angle (111.3/120.1/122.48). It was earlier shown that levan molecules treated with sodium borohydride possessed a higher immunostimulating activity
Fig. 2. The computational model of native (I), oxidized (II) and reducing (III) 3-fructoside.
3. Results and discussion
S. Gonta et al. / Journal of Molecular Structure (Theochem) 710 (2004) 61–64
conformational changes in these structures. The same effect was observed for all calculated heptafructozide V structures. The same character of the hydrogen bond formation was observed for trifructozide II oxidised in the first cycle. Besides, in the same cycle there was observed the next type of the charge localisation on carbonyl groups: from CZC0.15 to 0.17 e, and for OZK0.25 to 0.26 e. These data allowed to resume that nucleophilic reagents, such as amines or hydrazines, could have an advantage in reacting with carbonyl group C in the condensation reactions. The difference in the distribution of hydroxylgroups charges in reduced trifructosyl forms III from other polysaccharide hydroxylgroups was insignificant. A small negative charge in the CaN region (fragment) (K0.19 to K0.20 e) localised mainly on the C atom was registered in trifructoside dihidrazone derivatives IV. The changes in the electron density on the atoms in residual (others) parts of the molecule were insignificant in comparing with unmodified trifructoside. Upon comparing the trifructoside HOMO and LUMO energy (Fig. 3) it was shown, that after oxidation of one fructoside cycle the difference between HOMO and LUMO decreased by approximately 2.1 eV. This could be explained by the formation of a double bound (CaO) after the oxidation of the carboxyl group. The reduced form of trifructoside III HOMO and LUMO did not differ essentially in energy from the unmodified trifructoside frontier orbitals. In a hydrazine modified trifructoside molecule IV the difference between HOMO and LUMO was even less than in II case and less by w2.6 eV in case of native trifructoside I. In a hydrazine modified trifructoside molecule small absorption in a visible part of the spectrum was observed. The distribution of the electron charge density in a native trifructoside molecule I in the area of oxygen atoms in a cycle, hydroxyl groups, and on the oxygen bridge between
63
Fig. 4. Models of 7-fructoside (V).
fructoside fragments was studied. Both, in native I and reduced trifructozide III HOMO and LUMO were strongly delocalized. In oxidized trifructoside II HOMO was partially localized in –O–C–CaO fragment (grey in Fig. 1), but LUMO in CaO fragment. With hydrazine modified trifructoside both, HOMO and LUMO were localized in CaN–N fragment. Upon comparing II with trifructoside III it was observed that the electron density in CaO and CaN group carbon atom varied strongly. If in CaO case the carbon atom carried w0.16 e positive charge, then in CaN case the carbon atom carried w0.2 e negative charge. 3.3. A quantum chemical calculation of the spatial structure of hepta-b-D-fructoside chain The conformer geometry of oligofructosides that consisted of 3–7 fructosyl residues was optimized by AM1 method. According to the obtained results (Fig. 4) an increase in the fructosyl residue number led to spiralisation of the levan chain and that a full spiral clockwise consists of 5–6 residues. Table 1 Heptafructoside energy optimized on AM1 method (first 16)
Fig. 3. The HOMO and LUMO energy of native and modified trifructoside.
Number
Energy (kcal/mol)
Energy difference (kcal/mol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
K1709.92 K1707.28 K1704.54 K1704.46 K1703.50 K1703.27 K1702.76 K1701.09 K1700.85 K1699.97 K1699.90 K1699.67 K1699.59 K1699.21 K1699.08 K1698.79
– K2.64 K5.38 K5.46 K6.42 K6.65 K7.16 K8.83 K9.07 K9.95 K10.02 K10.25 K10.33 K10.71 K10.84 K11.13
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Various configurations of heptafructoside were investigated by variating of torsion angles. The MMC method did not give a sufficient accuracy and thus it was substituted by AM1 method. Using AM1 method 63 configurations with energy varying from K170,992 to K167,090 kcal/mol (difference is 39 kcal/mol) were obtained (Table 1). Obviously, a downturn of the energy of a stable conformation was connected not only with optimum torsion angles, but also with possible hydrogen binding formations. It was assumed that in the first 10 optimized conformations (Table 1) hydrogen bindings could be formed in 2–4 points ˚ and with with the internuclear distances from 2.08 to 2.3 A the difference of charges on atoms about G0.5 e in average.
4. Conclusions The computational analysis of oligofructosides as the levan fragment showed that the periodate oxidation increased the distance between C3 and C4 atoms and the angle C5–O–C2 in the fructosyl ring and as a result led to an increase in the fructoside cycle spatial structure. We assume that such increase of the dimensional structure of the oligofructoside chain located on the surface of a levan molecule can retard or prevent the approach of oxidant to the interior of the molecule. This theoretical considerations are in agreement with earlier obtained experimental data on the low oxidation degree of high molecular levan (17–23%)
during the periodate oxidation [8]. Possible hydrogen binding formations in 2–4 points in optimized 7-fructoside models can stabilize carbohydrate chains and prevent levan oxidation, as well.
References [1] A.O. Tzianabos, Clin. Microbiol. Rev. 13 (2000) 523. [2] V. Liepa, G. Zakenfelds, E. Volpe, Zˇ. Koronova, R. Lapsa, M. Laivenieks, M. Bek¸ers, I. Pospishil, Proc. Latvian Acad. Sci. Part B 5 (1993) 59. [3] I. Vina, A. Karsakevich, M. Toma, M. Bekers, Eighth European Congress on Biotechnology, 70th Event of the European Federation of Biotechnology (Book of Abstracts), Hungarian Biochemical Society, Budapest, 1997 p.105. [4] I. Vina, A. Karsakevich, M. Bekers, S. Gonta, R. Linde, A. Zhilevica, R. Treimane, H. Grante, Book of Abstracts, XVth EFMC International Symposium on Medicinal Chemistry, Edinburh, September, 1998 p.171. [5] V. Liepa, et al., Proc. Latvian Acad. Sci. Part B 5 (1993) 59. [6] T. Tanaka, S. Oi, T. Yamamoto, J. Biochem. 87 (1980) 297. [7] C.P.J. Glaudemans, Advances in Carbohydrate Chemistry vol. 31 Academic Press, New York, 1975 p. 311. [8] S. Gonta, A. Karsakevich, I. Vı¯na, Proc. Latvian Acad. Sci. Part B 53 (1999) 20. [9] M.J.S. Dewar, E.G. Zoebisch, E.F. Heally, J.J.P. Stewart, J. Am. Chem. Soc. 107 (1985) 3902. [10] I. Vina, A. Zilevica, R. Treimane, A. Karsakevich, S. Gonta, Book of Abstract, Fourth International Fructan Symposium, Arolla, Switzerland, August 10–20, 2000 p. 8.13.
Computational analysis of native and modified oligofructosides Svetlana Gontaa,*, M. Utinansb, O. Neilandsb, I. Vı¯naa a Institute of Microbiology and Biotechnology, Latvian University, Kronvalda Boulevard 4, Riga, LV-1586, Latvia Faculty of Material Science and Applied Chemistry, Riga Technical University, Azenes street 14, Riga, LV-1048, Latvia
b
Received 23 April 2004; revised 14 July 2004; accepted 30 July 2004 Available online 12 October 2004
Abstract This paper presents a quantum chemical calculation of native (2–7 fructoside residues) and chemically modified (2–4 fructoside residues) levan molecule models. A levan modification was carried out by oxidation and the following reduction or hyrdazonation of the fructoside rings. The conformational particularity and reaction ability was studied for the native and for the modified levan molecules. q 2004 Elsevier B.V. All rights reserved. Keywords: Levan; Computational analysis; Structure–activity relationship; 3-Dimensional structure
1. Introduction The use of immunomodulating agents provides distinct advantages over conventional therapies. Most important, the enhancement of the host immune system’s innate ability to combat bacterial infection obviates the problem associated with the antibiotic resistance. Recently, certain polysaccharides of microbial origin have been described that act as potent immunomodulators with the specific activity for both T cells and antigen-presenting cells such as monocytes and macrophages [1]. While the activity of some of these polymers has been known for over 30 years, the lack of defined structural and mechanistic information has limited efforts to study their potential for clinical use. Relatively few polysaccharides have been examined in detail where both the structurefunction and mechanism of action have been performed. Recent investigations have led to a more detailed understanding of the structural aspects of polysaccharides that influence the host immune responses [1]. The immunomodulating activity was shown for such microbial origin polysaccharides as b-[1/3]-glucans, mannan [1] and levan [2–5]. Levan is a biologically active polyfructoside produced by various bacteria. It is a neutral * Corresponding author. Tel.: C371-7034-889; fax: C371-7034-885. E-mail address: [email protected] (S. Gonta). 0166-1280/$ - see front matter q 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.theochem.2004.07.029
globular macromolecule with a highly branched structure, b-[2/6] linkages in the main chain and b-[2/1] linkages in the branching point [6]. Levan stimulates both, leukopoiesis and antibody production by splenocytes, activates the formation of reactive oxygen intermediates by phagocytes, and promotes the tumoricidal activity of phagocytes. The antitumor action of levan is implemented via the immune system of the organism. Levan prolongs the life-span of tumor-bearing mice. Levan has also radioprotective properties [5]. The C3–C4 region of the levan b-fructoside ring might be one of the essential elements of its immunological activity [7]. However, there is not much information as yet about the levan immunomodulating mechanisms. Previously it was shown that chemical modification of levan produced by Zymomonas mobilis enhanced its immunomodulating activity [8]. To elucidate an effect of levan chemical modifications on its conformational particularity and reaction ability a conformational analysis of native and modified model molecules of levan has been done by using AM1 simulations based on the ‘HyperChem’ programm package.
2. Method The semiempirical quantum chemical programme AM1 from the ‘HyperChem’ programm package [9] was used for
62
S. Gonta et al. / Journal of Molecular Structure (Theochem) 710 (2004) 61–64
Fig. 1. The structure of levan and its modification form: I—levan, fragment of structure; II—oxidized form; III—reduced form.
a theoretical modification and analysis of native and modified fructosides with 2–7 fructosyl residues and b-[2/6] links in the chain. The conformational analysis of oligofructoside was done according to the molecular mechanics programm MMC. The possible molecule conformation was calculated by using ‘HyperChem’ programm package utilities. The torsion angles between fructoside fragments have been varied. Electronic system of fructoside model molecules was studied after their geometry optimisation with AM1 method.
[10]. As the periodate oxidation altered the configuration of C3 and C4 region we suggested that this region could be connected with the biological activity of levan. 3.2. An electronic structure model of native and modified trifructosides
3.1. An effect of chemical modifications of C3–C4 region on its structure
Calculations of unmodified trifructoside with AM1 method showed, –O–H group hydrogen atom to have a positive charge (C0.21 to C0.23 e), but oxygen atoms in the fructoside cycle and in the oxygen bridge between fructoside cycles to have a negative charge (K0.26 to K0.31 e). Structures were stabilised with relatively strong hydrogen bonds between these atoms. The length of O–H/O bonds ˚ , which resulted in strengthenfluctuated from 2.17 to 2.54 A ing of hydrogen bonds and explained the difficulties in
Chemical modification of fructoside I by its oxidation with potassium periodate II and following reduction with sodium borohydride III (Fig. 1) destroyed the fructoside ring: in line I–II–III (Fig. 2). The distance between C3 and ˚ ) and led to C4 atoms was increased (1.55 /2.80 /3.00 A an increase in C5–O–C2 angle (111.3/120.1/122.48). It was earlier shown that levan molecules treated with sodium borohydride possessed a higher immunostimulating activity
Fig. 2. The computational model of native (I), oxidized (II) and reducing (III) 3-fructoside.
3. Results and discussion
S. Gonta et al. / Journal of Molecular Structure (Theochem) 710 (2004) 61–64
conformational changes in these structures. The same effect was observed for all calculated heptafructozide V structures. The same character of the hydrogen bond formation was observed for trifructozide II oxidised in the first cycle. Besides, in the same cycle there was observed the next type of the charge localisation on carbonyl groups: from CZC0.15 to 0.17 e, and for OZK0.25 to 0.26 e. These data allowed to resume that nucleophilic reagents, such as amines or hydrazines, could have an advantage in reacting with carbonyl group C in the condensation reactions. The difference in the distribution of hydroxylgroups charges in reduced trifructosyl forms III from other polysaccharide hydroxylgroups was insignificant. A small negative charge in the CaN region (fragment) (K0.19 to K0.20 e) localised mainly on the C atom was registered in trifructoside dihidrazone derivatives IV. The changes in the electron density on the atoms in residual (others) parts of the molecule were insignificant in comparing with unmodified trifructoside. Upon comparing the trifructoside HOMO and LUMO energy (Fig. 3) it was shown, that after oxidation of one fructoside cycle the difference between HOMO and LUMO decreased by approximately 2.1 eV. This could be explained by the formation of a double bound (CaO) after the oxidation of the carboxyl group. The reduced form of trifructoside III HOMO and LUMO did not differ essentially in energy from the unmodified trifructoside frontier orbitals. In a hydrazine modified trifructoside molecule IV the difference between HOMO and LUMO was even less than in II case and less by w2.6 eV in case of native trifructoside I. In a hydrazine modified trifructoside molecule small absorption in a visible part of the spectrum was observed. The distribution of the electron charge density in a native trifructoside molecule I in the area of oxygen atoms in a cycle, hydroxyl groups, and on the oxygen bridge between
63
Fig. 4. Models of 7-fructoside (V).
fructoside fragments was studied. Both, in native I and reduced trifructozide III HOMO and LUMO were strongly delocalized. In oxidized trifructoside II HOMO was partially localized in –O–C–CaO fragment (grey in Fig. 1), but LUMO in CaO fragment. With hydrazine modified trifructoside both, HOMO and LUMO were localized in CaN–N fragment. Upon comparing II with trifructoside III it was observed that the electron density in CaO and CaN group carbon atom varied strongly. If in CaO case the carbon atom carried w0.16 e positive charge, then in CaN case the carbon atom carried w0.2 e negative charge. 3.3. A quantum chemical calculation of the spatial structure of hepta-b-D-fructoside chain The conformer geometry of oligofructosides that consisted of 3–7 fructosyl residues was optimized by AM1 method. According to the obtained results (Fig. 4) an increase in the fructosyl residue number led to spiralisation of the levan chain and that a full spiral clockwise consists of 5–6 residues. Table 1 Heptafructoside energy optimized on AM1 method (first 16)
Fig. 3. The HOMO and LUMO energy of native and modified trifructoside.
Number
Energy (kcal/mol)
Energy difference (kcal/mol)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
K1709.92 K1707.28 K1704.54 K1704.46 K1703.50 K1703.27 K1702.76 K1701.09 K1700.85 K1699.97 K1699.90 K1699.67 K1699.59 K1699.21 K1699.08 K1698.79
– K2.64 K5.38 K5.46 K6.42 K6.65 K7.16 K8.83 K9.07 K9.95 K10.02 K10.25 K10.33 K10.71 K10.84 K11.13
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Various configurations of heptafructoside were investigated by variating of torsion angles. The MMC method did not give a sufficient accuracy and thus it was substituted by AM1 method. Using AM1 method 63 configurations with energy varying from K170,992 to K167,090 kcal/mol (difference is 39 kcal/mol) were obtained (Table 1). Obviously, a downturn of the energy of a stable conformation was connected not only with optimum torsion angles, but also with possible hydrogen binding formations. It was assumed that in the first 10 optimized conformations (Table 1) hydrogen bindings could be formed in 2–4 points ˚ and with with the internuclear distances from 2.08 to 2.3 A the difference of charges on atoms about G0.5 e in average.
4. Conclusions The computational analysis of oligofructosides as the levan fragment showed that the periodate oxidation increased the distance between C3 and C4 atoms and the angle C5–O–C2 in the fructosyl ring and as a result led to an increase in the fructoside cycle spatial structure. We assume that such increase of the dimensional structure of the oligofructoside chain located on the surface of a levan molecule can retard or prevent the approach of oxidant to the interior of the molecule. This theoretical considerations are in agreement with earlier obtained experimental data on the low oxidation degree of high molecular levan (17–23%)
during the periodate oxidation [8]. Possible hydrogen binding formations in 2–4 points in optimized 7-fructoside models can stabilize carbohydrate chains and prevent levan oxidation, as well.
References [1] A.O. Tzianabos, Clin. Microbiol. Rev. 13 (2000) 523. [2] V. Liepa, G. Zakenfelds, E. Volpe, Zˇ. Koronova, R. Lapsa, M. Laivenieks, M. Bek¸ers, I. Pospishil, Proc. Latvian Acad. Sci. Part B 5 (1993) 59. [3] I. Vina, A. Karsakevich, M. Toma, M. Bekers, Eighth European Congress on Biotechnology, 70th Event of the European Federation of Biotechnology (Book of Abstracts), Hungarian Biochemical Society, Budapest, 1997 p.105. [4] I. Vina, A. Karsakevich, M. Bekers, S. Gonta, R. Linde, A. Zhilevica, R. Treimane, H. Grante, Book of Abstracts, XVth EFMC International Symposium on Medicinal Chemistry, Edinburh, September, 1998 p.171. [5] V. Liepa, et al., Proc. Latvian Acad. Sci. Part B 5 (1993) 59. [6] T. Tanaka, S. Oi, T. Yamamoto, J. Biochem. 87 (1980) 297. [7] C.P.J. Glaudemans, Advances in Carbohydrate Chemistry vol. 31 Academic Press, New York, 1975 p. 311. [8] S. Gonta, A. Karsakevich, I. Vı¯na, Proc. Latvian Acad. Sci. Part B 53 (1999) 20. [9] M.J.S. Dewar, E.G. Zoebisch, E.F. Heally, J.J.P. Stewart, J. Am. Chem. Soc. 107 (1985) 3902. [10] I. Vina, A. Zilevica, R. Treimane, A. Karsakevich, S. Gonta, Book of Abstract, Fourth International Fructan Symposium, Arolla, Switzerland, August 10–20, 2000 p. 8.13.