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Iron(III) complexing ability of carbohydrate derivatives
JOURNAL OF
Inorganic Biochemistry Journal of Inorganic Biochemistry 98 (2004) 1002–1008 www.elsevier.com/locate/jinorgbio
Iron(III) complexing ability of carbohydrate derivatives E. Ferrari, M. Saladini
*
Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy Received 17 October 2003; received in revised form 6 February 2004; accepted 17 February 2004 Available online 18 March 2004
Abstract A solution study on the coordinative ability of galactaric acid (GalAH2 ), D -glucosamine (GlcN) and D -glucosaminic acid (GlcNAH) toward Fe3þ ion is reported. UV spectroscopic study provides useful information to identify complex species formation and their stability constants are determined by means of potentiometric measurements. GalAH2 behaves as chelating ligand through carboxylic oxygen and a-hydroxylic oxygen in the protonated or dissociated form depending on pH value. Two complex species [Fe2 GalA(OH)4 ] and Na[FeGalAH2 ] 2H2 O are also isolated in the solid state and characterised through IR spectroscopy. GlcNAH also binds the Fe3þ ion through carboxylic and hydroxylic groups, while NH2 group is probably involved in metal coordiantion up to pH 4. GlcN demonstrates low ligating ability at acidic pH and does not prevent metal hydroxyde precipitation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Galactaric acid;
D -glucosaminic
acid;
D -glucosamine;
Fe3þ complexes; Potentiometric study
1. Introduction Iron(III) complexes with sugar type ligands (sugars, sugar acids, other carbohydrates) are of vital importance in human and veterinary iron therapy [1,2]. The stability of iron(III) complexes must be high enough to prevent the precipitation of metal hydrolysis products damaging the living tissue, however a stability too high hinders the utilization of iron by the living organism. Fe3þ ion may coordinate to the oxygen donor atom of carbohydrates but it is generally acknowledged that D -aldoses have low tendency to bind iron(III) [3]. Nevertheless, a polysaccharide-iron complex named ‘‘Nifrex’’ has been synthesized from FeCl3 and glucose and is currently used for the treatment of iron deficiency anaemia [4]. Introduction of an anchoring group into a sugar molecule as primary coordination site may promote the coordination and deprotonation of the alcoholic hydroxylic groups of the carbohydrate moiety. Potentiometric and spectroscopic studies showed that amino sugars are effective ligands toward metal ions *
Corresponding author. Tel.: +39-059-2055040; fax: +39-059273543. E-mail address: [email protected] (M. Saladini). 0162-0134/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.02.017
such as Cu(II), Ni(II), and Co(II) [5]. Sugar acids, containing both carboxylic and hydroxylic groups, are expected to chelate metal ions via various different binding modes, depending on the metal itself and the reaction conditions. Sugar acids iron complexes are potential pharmaceuticals since their stability is high enough to prevent metal ion hydrolysis in biological systems; in particular D -gluconic acid is the more effective chelating agent. Among sugar acids, galactaric acid exhibits properties of great interest in biosynthesis and in the pharmaceutical industry [6]. Previous study on M(II)galactaric acid systems [7,8] described the chelating ability of such sugar with the formation of polymeric species, whose stability is also maintained at basic pH thanks to the deprotonation of the co-ordinated alchoholic hydroxylic group. Now, we report a potentiometric and spectroscopic study on iron(III) complex systems with different sugars type ligand such as: galactaric acid (GalAH2 ), D -glucosamine (GlcN) and D -glucosaminic acid (GlcNAH) (Scheme 1) in order to compare the calculated stability constants of the complex species formed at physiological pH with the aim to test their possible use in the iron up taking therapy.
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
1003
varied by adding small amounts of concentrated NaOH and HNO3 ). All Fe(III)–L systems were investigated in the 1:1, 1:2 and 1:4 molar ratios in the 240–400 nm spectral range. The infrared spectra of the solid complexes in KBr pellets were obtained by means of a Perkin–Elmer FTIR 1600, in the 4000–400 cm1 spectral range. 2.3. Preparation of the solid complexes
Scheme 1.
2. Experimental 2.1. Potentiometric measurements The concentration of the aqueous solution of galactaric acid, D -glucosamine hydrocloride and D -glucosaminic acid (1.0 103 M) was determined potentiometrically. FeCl3 solution (5.0 103 M) was standardized by means of SPECTROFLAME D ICP plasma spectrometer; sample contained 1% of HNO3 (BDH-Aristar). Potentiometric measurements were performed in aqueous solution at 25 0.1 °C, using a fully automated ORION 960 Autochemistry System and following the general procedures previously reported [8]. All experiments were carried out in a nitrogen atmosphere at ionic strength 0.05 M (adjusted with solid NaNO3 ). The stability constants (bpqr ), which are defined by Eqs. (1) and (2) pFe þ qL þ rH ¡ Fep Lq Hr p
q
bprs ¼ ½Fep Lq Hr =½Fe ½L ½H
ð1Þ r
ð2Þ
where L is the ligand in the completely dissociated form and H is proton, were refined by least-squares calculation, using computer program HYPERQUAD [9] taking into account the presence of the species [Fe(OH)]2þ ; [Fe(OH)2 ]þ ; [Fe2 (OH)2 ]4þ [10]. The starting solution for each titration was prepared by addition of known volumes of Fe3þ solution and the appropriate ligand solution in 1:1, 1:2, and 1:4 metal to ligand molar ratios. Known volumes of HNO3 were added to have acidic pH. Aqueous NaOH (0.01 M) was used as titrant. At least 10 measurements were performed for each system with 40 data points in each titration in the pH range 2–9. 2.2. Spectrophotometric measurements Electronic spectra were recorded with a Perkin–Elmer Lamda B20 spectrophotometer (T ¼ 25 °C; pH being
[Fe2 GalA(OH)4 ] 2 mmol of Fe(NO3 )3 were dissolved in 20 ml of water and slowly added with 1 mmol of GalAH2 suspended in 20 ml of water; the pH diminishes until 1.2 and suddenly a yellow powder precipitates. Found: C, 18.7; H, 3.2; Fe, 29.8. Yield: 80%. Calculated for C6 Fe2 H12 O12 : C, 18.6; H, 3.1; Fe, 28.8. Na[FeGalAH2 ] 2H2 O 2 mmol of GalAH2 were dissolved in 50 ml of water by adding NaOH until pH 11; by adding 20 ml of a water solution of Fe(NO3 )3 (2 mmol) pH drops to 6.5. pH was adjusted to 11 with NaOH, then the solution slowly evaporated. By addition of EtOH, a brown powder precipitated. Found: C, 21.9; H, 3.3; Fe, 17.9; Na, 8.1. Yield: 60%. Calculated for C6 FeH10 NaO10 : C, 22.4; H, 3.1; Fe, 17.4; Na, 7.2. Fe% and Na% contents were determined by means of SPECTROFLAME D ICP plasma spectrometer.
3. Results and discussion 3.1. Galactaric acid In the pH-metric titration curves of Fe3þ /GalAH2 system in all molar ratios investigated a drop in pH with respect to the ‘‘free’’ acid is observed, suggesting that the formation of complex species takes place before the complete dissociation of the carboxylic groups. Increasing pH one equivalent point is observed according to the equation mNaOH ¼ 2mL þ 2mFe3þ where L stands for GalAH2 . In order to verify the complexing process at low pH, a titration of Fe(NO3 )3 0.001 M solution with a solution of GalAH2 0.1 M was performed. As shown in Fig. 1, a diminishing in pH is observed until the M/L 1:1 molar ratio is reached, suggesting the formation of a carboxylate complex around pH 2.6. Table 1 reports the log b value of the complex species, while Fig. 2(a) shows the species distribution curves calculated on the basis of the protonation and association constants reported in Table 1; Scheme 2 suggests the complexes coordination mode. At acidic pH, the first complex species formed is the polymeric [FeGalA(OH)] (I) where two galactaric acids coordinate the Fe3þ ion through the carboxylic group and the a-hydroxylic group giving rise to five membered
1004
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008 100
[FeLH-2]-
% of species
80
[FeL(OH)]
60
40 Fe3+
20 [Fe2L(OH)4]
0 2
3
4
5
6
(a) Fig. 1. Variation of pH by adding GalAH2 to 50 cm3 of aqueous solution: j in the presence of 0.05 mmol of Fe3þ ; d in the absence of Fe3þ .
8
9
100
[FeL2]+ 80
[Fe(LH-1)]% of species
chelate rings; the coordination sphere is completed by a water molecule and a OH ion. In order to confirm the M/L 1:1 ratio of the species, a spectrophotometric titration of a Fe3þ solution with GalAH2 is performed. Fig. 3(b) reports a plot of absorbance vs. mmol of GalAH2 added at k ¼ 360 nm and states the formation of a complex species with an 1:1 molar ratio as the absorbance increases until the 1:1 ratio is obtained. The formation of polynuclear species may not be excluded, on the contrary a polymeric arrangement (Scheme 2) is suggested on the basis of the crystal structure of [CuGalA(bpy)]n nH2 O [8]. The spectrophotometric titration of the Fe/GalAH2 1:4 system at acidic pH (Fig. 3(a)) shows the presence of an absorption maximum at k ¼ 300 nm typical of hydroxylated Fe3þ ion. Therefore, the presence of a complex species with a coordinated OH ion is confirmed. On increasing pH, a new band appears at k ¼ 340 nm in accordance with the formation of a new complex species
7
pH
60
Fe3+ [FeHL]3+ [Fe(HL)2]3+
40
20
[FeL]2+ 0 1
2
(b)
3
4
5
6
7
8
9
pH
Fig. 2. Species distribution curves of Fe3þ /GalAH2 (a) and Fe3þ / GlcHNA (b) in the 1:4 molar ratio; [Fe3þ ] ¼ 2.5 104 M; (a) L stands for Galactaric acid in the dissociated form, (b) HL stands for neutral glucosaminic acid with carboxylic group in the dissociated form and aminic group in the protonated one.
Table 1 log b of protonation constants of the ligands and of the formation of complex species at 25 °C (I ¼ 0:1 M NaNO3 ); estimated standard deviation 0.05 log b011 log b012 log b111 log b112 log b214 log b111 log b122 log b110 log b120 log b122
GalAH GalAH2 [FeGalA(OH)] [FeGalAH2 ] [Fe2 GalA(OH)4 ]
3.05 7.09 2.99 )2.12 )0.62
GlcHNA GlcHNAHþ
[FeGlcHNA]3þ [Fe(GlcHNA)2 ]3þ [Fe(GlcHNA) (OH)]2þ [FeGlcNA]2þ [Fe(GlcNA)2 ]þ [Fe(GlcNAH1 )2 ]
9.05 12.28
13.52 26.31 11.45 21.02 7.32
GlcHN
7.66
[FeGlcHN(OH)2 ]2þ
3.21
[FeGlcHN]4þ
9.02
[FeGlcHN(OH)]3þ
8.01
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
1005
Scheme 2.
which is completely formed at pH 5, no more increasing in absorbance is present up to this pH value. By plotting absorbance vs. pH at k ¼ 340 nm (Fig. 4(a)), a titolative trend is observed with two equivalent points corresponding to the formation of species I and III. As we can see from the species distribution curves (Fig. 2(a)), the prevailing species at physiological pH is complex III in which galactaric acid coordinates Fe3þ through carboxylic oxygen and deprotonated a-hydroxylic group. A polymeric structure also for this complex is proposed. The stability constant of complex I (log b ¼ 2:99) is higher compared to those found with other sugar acids (lactobionic acid log b ¼ 2:03; gluconic acid log b ¼ 2:43) [11], this difference is probably due to electroneutrality of our complex species. Previous studies on Fe3þ /galacturonic acid [12] showed the formation, at acidic pH, of FeL3 species in which galacturonic acid bounds the metal ion through carboxylic oxygen. The ability of Fe3þ ion to promote hydroxylic deprotonation in sugars was previously observed with fructose [13], but the measured log b value was )2.8. The formation of [FeLH2 ] species was observed also with lactobionic (log b ¼ 3:89) and gluconic (log b ¼ 3:23)
[11] with stability constants lower than that of complex III (log b ¼ 2:12) despite the ionicity of this complex. This study confirms the greater stability in aqueous media of Fe3þ /sugar acid complexes with respect to simple carbohydrates as was reported by Nagy and Gyurcsik [2] although the existence of polymeric species was not ascertained. Complex II remains a minor species but, thanks to its low solubility, it is separated in the solid state at low pH (pH 3) in the presence of an excess of Fe3þ ion. At pH>7 the solid species obtained is complex II and the elemental analysis suggests the Na[FeGalAH2 ] 2H2 O formula. The IR spectra of these complexes show a general shift of the OH stretching vibration toward higher frequencies due to the general weakening of the strong sugar hydrogen bonding system upon metal coordination [14,15] (mOH ¼ 3287 cm1 in GalAH2 ; 3395 cm1 in [Fe2 GalA(OH)4 ]; 3437 cm1 in Na[FeGalAH2 ] 2H2 O). The same trend is observed in the bending vibration of the COH and CCH groups as a consequence of sugar metalation [16]. In the IR spectra of the complexes is also missing the carbonylic stretching observed at 1719 cm1 in the free ligand and two strong absorption bands
1006
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008 0.40
0.35
0.30
A
0.25
0.20
0.15
0.10
0.05 1
2
3
4
5
6
7
8
9
10
pH
(a) 0.50 0.45 0.40
A
0.35 0.30 0.25
Fig. 3. (a) Spectrophotometric titration of the Fe3þ /GalAH2 system in the 1:4 molar ratio; [Fe3þ ] ¼ 2.5 104 M, pH varying from 1.06 to 3.09. (b) Plot of absorbance vs. mmol of GalAH2 added to a solution containing 1 mmol of Fe3þ ; [Fe3þ ] ¼ 2.5 104 M, k ¼ 340 nm.
0.20 0.15
appear at 1629–1579, 1386 in [Fe2 GalA(OH)4 ] and 1629, 1378 in Na[FeGalAH2 ] 2H2 O due to the antisymmetric and symmetric stretching vibrations of the OCO groups in the sugar anion. The separation of the two OCO components (243 and 252 cm1 ) is indicative of a bidentate mode of carboxylic group [17] in agreement with the crystal structure of sodium and potassium galactarate where the carboxylic group is found to act as bridging bidentate [18]. 3.2. Glucosaminic acid The potentiometric titration of the Fe/GlcNA system shows two equivalent points according to equations mNaOH ¼ 2mFe3þ mNaOH ¼ 4mFe3þ The stability constants of the complex species are reported in Table 1, while the species distribution curves are shown in Fig. 2(b). At low pH the prevailing species are [Fe(GlcHNA)]3þ and [Fe(GlcHNA)2 ]3þ where the ligand is in the COO /NHþ 2 form and the aminic group
0.10
2
(b)
3
4
5
6
7
8
9
10
pH
Fig. 4. Plot of absorbance vs. pH of the Fe3þ /GalAH2 (a) and Fe3þ / GlcHNA (b) systems in the 1:4 molar ratio; [Fe3þ ] ¼ 2.5 104 M, k ¼ 340 nm.
is not involved in metal coordination as was found in Fe/Glycine system [10] and in the crystal structure of [FeII (HAla)2 ] complex [19]. Nevertheless, the stability constant of [Fe(GlcHNA)]3þ (log b ¼ 13:52) is much higher than the one of [FeHGly]3þ (log b ¼ 10:36) [10] suggesting a possible involvement of an hydroxylic group in metal coordination as observed in Cu/Glucosaminic acid system [20,21]. Increasing pH the release of a proton by the complex species is compatible with both reactions: F3þ þ H2 O þ GlcHNA ¡ ½FeðOHÞGlcHNA Fe3þ þ GlcHNA ¡ ½FeGlcNA2þ þ Hþ
2þ
þ Hþ
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
In the first case the ligand is coordinated through carboxyl oxygen like in [Fe(OH)HGly] [10], in the second case the protolytic dissociation of [FeGlcHNA]3þ species may be followed by closing up the five-membered glycine like ring as in [FeAla] complex [22]. In equilibrium calculations the two complexes cannot be discriminated but the evaluated stability constant is higher than those of [FeGly] and [FeAla] (11.45 vs. 8.57 and 8.96, respectively), suggesting also for this complex the involvement of hydroxylic group in metal coordination as shown in Scheme 3. The formation of a new complex species is confirmed by the analysis of the absorbance of the Fe/GlcNA system on varying pH (Fig. 4(b)) which shows increasing until pH 5 where the complex species is completely formed. Over pH 7 a new change in absorbance suggests the formation of another species which is identified as [Fe(GlcNAH1 )2 ] . In this species, we suppose the dissociation of the coordinated hydroxylic oxygen in agreement to what is observed in Cu2þ /Glucosaminic acid system [19,20]. 3.3. Glucosamine The potentiometric titration of the Fe3þ /GlcN system is hindered by the precipitation of Fe-hydroxyde at pH 5; the treatment of data below this pH value allowed to evaluate the stability constants reported in Table 1. In all the complex species, the amino nitrogen is in the protonated NHþ 3 form and the ligand coordinates the metal ion through hydroxylic groups. Previous studies on Fe3þ /sugars systems suggested the formation of complex species in which the sugar molecule (fructose [13], mannitol [23]) chelates the metal ion through deprotonated hydroxylic groups in very acidic conditions. In our case, by progressive addition of GlcN to a solution of Fe3þ no variation in pH is observed, therefore excluding any ionization process in the ligand upon metal coordination.
1007
Increasing pH the formation of mixed hydroxo species is reached but their stability is not sufficient to avoid metal hydroxyde precipitation. Crystallographic studies on solid complexes with carbohydrates isolated in alkaline solution showed octahedral coordination with Fe3þ ion surrounded by alcoholic groups, hydroxylic oxygens and water molecules [2]. In our case, the precipitation of Fe-hydroxyde excludes the formation of this kind of complexes.
4. Conclusions In order to establish the effective Fe3þ chelating ability of these ligands, we may consider the prevailing species in physiological conditions. But since they have different stoichiometry, a simple comparison of the stability constants is not useful, a more proper parameter is represented by pFe3þ , defined as )log [Fe3þ ] [24]. This value is usually calculated with [L] ¼ 105 M and [Fe3þ ] ¼ 106 M and, unlike the stability constants takes into account the effects of ligand protonation and dentiticy as well as differences in metal–ligand stoichiometry. The pFe3þ vs. pH plot provides a useful method to compare the ability of chelators to bind Iron(III) at different pH; it shows that a high value of log K is not necessarily characterized by a high value of pFe3þ . In Fig. 5, pFe3þ vs. pH is reported for our ligands and is compared to OH ligating ability. The only ligand which may compete with OH is Galactaric acid, thus we may propose to use this molecule in the oral iron up taking therapy.
20
18
16
pFe3+
14
12
10
8
6 3
4
5
6
7
8
9
pH
Scheme 3.
Fig. 5. pFe3þ vs. pH for: GalA ––––; GlcNA –––––; GlcN . . . . . . . . .; OH ––––––. [Fe3þ ] ¼ 106 M and [L] ¼ 105 M.
1008
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
Acknowledgements We are grateful to the Ministero dellÕUniversita e della Ricerca Scientifica e Tecnologica of Italy for financial support.
References [1] A. Bezkorovany, Biochemistry of Nonheme Iron, Plenum press, London, 1980. [2] B. Gyurcsik, L. Nagy, Coord. Chem. Rev. 203 (2000) 81–149. [3] M.M. H€ am€ al€ aimen, H. L€ onnberg, Carbohydr. Res. 215 (1991) 357–360. [4] D.M. Whitfield, S. Stjkovski, B. Sarkar, Coord. Chem. Rev. 122 (1993) 171–225. [5] J. Urbanska, H. Kozlowski, J. Coord. Chem. 21 (1990) 175–182. [6] Life Science Patents. Available from. [7] M. Saladini, E. Ferrari, L. Menabue, J. Inorg. Biochem. 92 (2002) 121–127. [8] M. Saladini, M. Candini, D. Iacopino, L. Menabue, Inorg. Chim. Acta 292 (1999) 189–197. [9] P. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 1739–1753.
[10] G.M. Escandar, A.C. Olivieri, M. Gonzales-Sierra, L.F. Sala, J. Chem. Soc., Dalton Trans. (1994) 1189–1192. [11] S. Deiana, C. Gessa, V. Solinas, P. Pin, R. Seeber, Anal. Chim. Acta 226 (1989) 315–322. [12] K. Araki, S. Shiraishi, Bull. Chem. Soc. Jpn 59 (1986) 3661–3662. [13] H.A. Tajmir-Riahi, J. Inorg. Biochem. 24 (1986) 127–136. [14] H.A. Tajmir-Riahi, Carbohydr. Res. 125 (1984) 13–20. [15] M. Hineno, Carbohydr. Res. 56 (1977) 219–227. [16] P. Djurdjevic, Trans. Met. Chem. 15 (1990) 345–350. [17] L.P. Battaglia, A. Bonamartini Corradi, G. Marcotrigiano, L. Menabue, G.C. Pellacani, J. Am. Chem. Soc. 102 (1980) 1663– 1669. [18] T. Taga, T. Shimada, N. Nimura, Acta Crystallogr. C 50 (1994) 1076–1079. [19] I. Lindqvist, R. Rosenstein, Acta Chem. Scand. 14 (1960) 1228– 1229. [20] J. van Haveren, H. van Bekkum, J.A. Peters, Inorg. Chim. Acta 205 (1993) 1–7. [21] T. Gajda, B. Gyurcsik, T. Jakusch, K. Burger, B. Henry, J.J. Delpuech, Inorg. Chim. Acta 130 (1998) 275–276. [22] P. Djurdjevic, R. Jelie, Trans. Met. Chem. 18 (1993) 457–461. [23] L. Nagy, H. Ohtaki, T. Yamaguchi, M. Nomura, Inorg. Chim. Acta 159 (1989) 201–207. [24] R.C. Hider, Z.D. Liu, S. Piyamongkol, Transf. Sci. 23 (2000) 201– 209.
Inorganic Biochemistry Journal of Inorganic Biochemistry 98 (2004) 1002–1008 www.elsevier.com/locate/jinorgbio
Iron(III) complexing ability of carbohydrate derivatives E. Ferrari, M. Saladini
*
Department of Chemistry, University of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy Received 17 October 2003; received in revised form 6 February 2004; accepted 17 February 2004 Available online 18 March 2004
Abstract A solution study on the coordinative ability of galactaric acid (GalAH2 ), D -glucosamine (GlcN) and D -glucosaminic acid (GlcNAH) toward Fe3þ ion is reported. UV spectroscopic study provides useful information to identify complex species formation and their stability constants are determined by means of potentiometric measurements. GalAH2 behaves as chelating ligand through carboxylic oxygen and a-hydroxylic oxygen in the protonated or dissociated form depending on pH value. Two complex species [Fe2 GalA(OH)4 ] and Na[FeGalAH2 ] 2H2 O are also isolated in the solid state and characterised through IR spectroscopy. GlcNAH also binds the Fe3þ ion through carboxylic and hydroxylic groups, while NH2 group is probably involved in metal coordiantion up to pH 4. GlcN demonstrates low ligating ability at acidic pH and does not prevent metal hydroxyde precipitation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Galactaric acid;
D -glucosaminic
acid;
D -glucosamine;
Fe3þ complexes; Potentiometric study
1. Introduction Iron(III) complexes with sugar type ligands (sugars, sugar acids, other carbohydrates) are of vital importance in human and veterinary iron therapy [1,2]. The stability of iron(III) complexes must be high enough to prevent the precipitation of metal hydrolysis products damaging the living tissue, however a stability too high hinders the utilization of iron by the living organism. Fe3þ ion may coordinate to the oxygen donor atom of carbohydrates but it is generally acknowledged that D -aldoses have low tendency to bind iron(III) [3]. Nevertheless, a polysaccharide-iron complex named ‘‘Nifrex’’ has been synthesized from FeCl3 and glucose and is currently used for the treatment of iron deficiency anaemia [4]. Introduction of an anchoring group into a sugar molecule as primary coordination site may promote the coordination and deprotonation of the alcoholic hydroxylic groups of the carbohydrate moiety. Potentiometric and spectroscopic studies showed that amino sugars are effective ligands toward metal ions *
Corresponding author. Tel.: +39-059-2055040; fax: +39-059273543. E-mail address: [email protected] (M. Saladini). 0162-0134/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2004.02.017
such as Cu(II), Ni(II), and Co(II) [5]. Sugar acids, containing both carboxylic and hydroxylic groups, are expected to chelate metal ions via various different binding modes, depending on the metal itself and the reaction conditions. Sugar acids iron complexes are potential pharmaceuticals since their stability is high enough to prevent metal ion hydrolysis in biological systems; in particular D -gluconic acid is the more effective chelating agent. Among sugar acids, galactaric acid exhibits properties of great interest in biosynthesis and in the pharmaceutical industry [6]. Previous study on M(II)galactaric acid systems [7,8] described the chelating ability of such sugar with the formation of polymeric species, whose stability is also maintained at basic pH thanks to the deprotonation of the co-ordinated alchoholic hydroxylic group. Now, we report a potentiometric and spectroscopic study on iron(III) complex systems with different sugars type ligand such as: galactaric acid (GalAH2 ), D -glucosamine (GlcN) and D -glucosaminic acid (GlcNAH) (Scheme 1) in order to compare the calculated stability constants of the complex species formed at physiological pH with the aim to test their possible use in the iron up taking therapy.
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
1003
varied by adding small amounts of concentrated NaOH and HNO3 ). All Fe(III)–L systems were investigated in the 1:1, 1:2 and 1:4 molar ratios in the 240–400 nm spectral range. The infrared spectra of the solid complexes in KBr pellets were obtained by means of a Perkin–Elmer FTIR 1600, in the 4000–400 cm1 spectral range. 2.3. Preparation of the solid complexes
Scheme 1.
2. Experimental 2.1. Potentiometric measurements The concentration of the aqueous solution of galactaric acid, D -glucosamine hydrocloride and D -glucosaminic acid (1.0 103 M) was determined potentiometrically. FeCl3 solution (5.0 103 M) was standardized by means of SPECTROFLAME D ICP plasma spectrometer; sample contained 1% of HNO3 (BDH-Aristar). Potentiometric measurements were performed in aqueous solution at 25 0.1 °C, using a fully automated ORION 960 Autochemistry System and following the general procedures previously reported [8]. All experiments were carried out in a nitrogen atmosphere at ionic strength 0.05 M (adjusted with solid NaNO3 ). The stability constants (bpqr ), which are defined by Eqs. (1) and (2) pFe þ qL þ rH ¡ Fep Lq Hr p
q
bprs ¼ ½Fep Lq Hr =½Fe ½L ½H
ð1Þ r
ð2Þ
where L is the ligand in the completely dissociated form and H is proton, were refined by least-squares calculation, using computer program HYPERQUAD [9] taking into account the presence of the species [Fe(OH)]2þ ; [Fe(OH)2 ]þ ; [Fe2 (OH)2 ]4þ [10]. The starting solution for each titration was prepared by addition of known volumes of Fe3þ solution and the appropriate ligand solution in 1:1, 1:2, and 1:4 metal to ligand molar ratios. Known volumes of HNO3 were added to have acidic pH. Aqueous NaOH (0.01 M) was used as titrant. At least 10 measurements were performed for each system with 40 data points in each titration in the pH range 2–9. 2.2. Spectrophotometric measurements Electronic spectra were recorded with a Perkin–Elmer Lamda B20 spectrophotometer (T ¼ 25 °C; pH being
[Fe2 GalA(OH)4 ] 2 mmol of Fe(NO3 )3 were dissolved in 20 ml of water and slowly added with 1 mmol of GalAH2 suspended in 20 ml of water; the pH diminishes until 1.2 and suddenly a yellow powder precipitates. Found: C, 18.7; H, 3.2; Fe, 29.8. Yield: 80%. Calculated for C6 Fe2 H12 O12 : C, 18.6; H, 3.1; Fe, 28.8. Na[FeGalAH2 ] 2H2 O 2 mmol of GalAH2 were dissolved in 50 ml of water by adding NaOH until pH 11; by adding 20 ml of a water solution of Fe(NO3 )3 (2 mmol) pH drops to 6.5. pH was adjusted to 11 with NaOH, then the solution slowly evaporated. By addition of EtOH, a brown powder precipitated. Found: C, 21.9; H, 3.3; Fe, 17.9; Na, 8.1. Yield: 60%. Calculated for C6 FeH10 NaO10 : C, 22.4; H, 3.1; Fe, 17.4; Na, 7.2. Fe% and Na% contents were determined by means of SPECTROFLAME D ICP plasma spectrometer.
3. Results and discussion 3.1. Galactaric acid In the pH-metric titration curves of Fe3þ /GalAH2 system in all molar ratios investigated a drop in pH with respect to the ‘‘free’’ acid is observed, suggesting that the formation of complex species takes place before the complete dissociation of the carboxylic groups. Increasing pH one equivalent point is observed according to the equation mNaOH ¼ 2mL þ 2mFe3þ where L stands for GalAH2 . In order to verify the complexing process at low pH, a titration of Fe(NO3 )3 0.001 M solution with a solution of GalAH2 0.1 M was performed. As shown in Fig. 1, a diminishing in pH is observed until the M/L 1:1 molar ratio is reached, suggesting the formation of a carboxylate complex around pH 2.6. Table 1 reports the log b value of the complex species, while Fig. 2(a) shows the species distribution curves calculated on the basis of the protonation and association constants reported in Table 1; Scheme 2 suggests the complexes coordination mode. At acidic pH, the first complex species formed is the polymeric [FeGalA(OH)] (I) where two galactaric acids coordinate the Fe3þ ion through the carboxylic group and the a-hydroxylic group giving rise to five membered
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[FeLH-2]-
% of species
80
[FeL(OH)]
60
40 Fe3+
20 [Fe2L(OH)4]
0 2
3
4
5
6
(a) Fig. 1. Variation of pH by adding GalAH2 to 50 cm3 of aqueous solution: j in the presence of 0.05 mmol of Fe3þ ; d in the absence of Fe3þ .
8
9
100
[FeL2]+ 80
[Fe(LH-1)]% of species
chelate rings; the coordination sphere is completed by a water molecule and a OH ion. In order to confirm the M/L 1:1 ratio of the species, a spectrophotometric titration of a Fe3þ solution with GalAH2 is performed. Fig. 3(b) reports a plot of absorbance vs. mmol of GalAH2 added at k ¼ 360 nm and states the formation of a complex species with an 1:1 molar ratio as the absorbance increases until the 1:1 ratio is obtained. The formation of polynuclear species may not be excluded, on the contrary a polymeric arrangement (Scheme 2) is suggested on the basis of the crystal structure of [CuGalA(bpy)]n nH2 O [8]. The spectrophotometric titration of the Fe/GalAH2 1:4 system at acidic pH (Fig. 3(a)) shows the presence of an absorption maximum at k ¼ 300 nm typical of hydroxylated Fe3þ ion. Therefore, the presence of a complex species with a coordinated OH ion is confirmed. On increasing pH, a new band appears at k ¼ 340 nm in accordance with the formation of a new complex species
7
pH
60
Fe3+ [FeHL]3+ [Fe(HL)2]3+
40
20
[FeL]2+ 0 1
2
(b)
3
4
5
6
7
8
9
pH
Fig. 2. Species distribution curves of Fe3þ /GalAH2 (a) and Fe3þ / GlcHNA (b) in the 1:4 molar ratio; [Fe3þ ] ¼ 2.5 104 M; (a) L stands for Galactaric acid in the dissociated form, (b) HL stands for neutral glucosaminic acid with carboxylic group in the dissociated form and aminic group in the protonated one.
Table 1 log b of protonation constants of the ligands and of the formation of complex species at 25 °C (I ¼ 0:1 M NaNO3 ); estimated standard deviation 0.05 log b011 log b012 log b111 log b112 log b214 log b111 log b122 log b110 log b120 log b122
GalAH GalAH2 [FeGalA(OH)] [FeGalAH2 ] [Fe2 GalA(OH)4 ]
3.05 7.09 2.99 )2.12 )0.62
GlcHNA GlcHNAHþ
[FeGlcHNA]3þ [Fe(GlcHNA)2 ]3þ [Fe(GlcHNA) (OH)]2þ [FeGlcNA]2þ [Fe(GlcNA)2 ]þ [Fe(GlcNAH1 )2 ]
9.05 12.28
13.52 26.31 11.45 21.02 7.32
GlcHN
7.66
[FeGlcHN(OH)2 ]2þ
3.21
[FeGlcHN]4þ
9.02
[FeGlcHN(OH)]3þ
8.01
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
1005
Scheme 2.
which is completely formed at pH 5, no more increasing in absorbance is present up to this pH value. By plotting absorbance vs. pH at k ¼ 340 nm (Fig. 4(a)), a titolative trend is observed with two equivalent points corresponding to the formation of species I and III. As we can see from the species distribution curves (Fig. 2(a)), the prevailing species at physiological pH is complex III in which galactaric acid coordinates Fe3þ through carboxylic oxygen and deprotonated a-hydroxylic group. A polymeric structure also for this complex is proposed. The stability constant of complex I (log b ¼ 2:99) is higher compared to those found with other sugar acids (lactobionic acid log b ¼ 2:03; gluconic acid log b ¼ 2:43) [11], this difference is probably due to electroneutrality of our complex species. Previous studies on Fe3þ /galacturonic acid [12] showed the formation, at acidic pH, of FeL3 species in which galacturonic acid bounds the metal ion through carboxylic oxygen. The ability of Fe3þ ion to promote hydroxylic deprotonation in sugars was previously observed with fructose [13], but the measured log b value was )2.8. The formation of [FeLH2 ] species was observed also with lactobionic (log b ¼ 3:89) and gluconic (log b ¼ 3:23)
[11] with stability constants lower than that of complex III (log b ¼ 2:12) despite the ionicity of this complex. This study confirms the greater stability in aqueous media of Fe3þ /sugar acid complexes with respect to simple carbohydrates as was reported by Nagy and Gyurcsik [2] although the existence of polymeric species was not ascertained. Complex II remains a minor species but, thanks to its low solubility, it is separated in the solid state at low pH (pH 3) in the presence of an excess of Fe3þ ion. At pH>7 the solid species obtained is complex II and the elemental analysis suggests the Na[FeGalAH2 ] 2H2 O formula. The IR spectra of these complexes show a general shift of the OH stretching vibration toward higher frequencies due to the general weakening of the strong sugar hydrogen bonding system upon metal coordination [14,15] (mOH ¼ 3287 cm1 in GalAH2 ; 3395 cm1 in [Fe2 GalA(OH)4 ]; 3437 cm1 in Na[FeGalAH2 ] 2H2 O). The same trend is observed in the bending vibration of the COH and CCH groups as a consequence of sugar metalation [16]. In the IR spectra of the complexes is also missing the carbonylic stretching observed at 1719 cm1 in the free ligand and two strong absorption bands
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E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008 0.40
0.35
0.30
A
0.25
0.20
0.15
0.10
0.05 1
2
3
4
5
6
7
8
9
10
pH
(a) 0.50 0.45 0.40
A
0.35 0.30 0.25
Fig. 3. (a) Spectrophotometric titration of the Fe3þ /GalAH2 system in the 1:4 molar ratio; [Fe3þ ] ¼ 2.5 104 M, pH varying from 1.06 to 3.09. (b) Plot of absorbance vs. mmol of GalAH2 added to a solution containing 1 mmol of Fe3þ ; [Fe3þ ] ¼ 2.5 104 M, k ¼ 340 nm.
0.20 0.15
appear at 1629–1579, 1386 in [Fe2 GalA(OH)4 ] and 1629, 1378 in Na[FeGalAH2 ] 2H2 O due to the antisymmetric and symmetric stretching vibrations of the OCO groups in the sugar anion. The separation of the two OCO components (243 and 252 cm1 ) is indicative of a bidentate mode of carboxylic group [17] in agreement with the crystal structure of sodium and potassium galactarate where the carboxylic group is found to act as bridging bidentate [18]. 3.2. Glucosaminic acid The potentiometric titration of the Fe/GlcNA system shows two equivalent points according to equations mNaOH ¼ 2mFe3þ mNaOH ¼ 4mFe3þ The stability constants of the complex species are reported in Table 1, while the species distribution curves are shown in Fig. 2(b). At low pH the prevailing species are [Fe(GlcHNA)]3þ and [Fe(GlcHNA)2 ]3þ where the ligand is in the COO /NHþ 2 form and the aminic group
0.10
2
(b)
3
4
5
6
7
8
9
10
pH
Fig. 4. Plot of absorbance vs. pH of the Fe3þ /GalAH2 (a) and Fe3þ / GlcHNA (b) systems in the 1:4 molar ratio; [Fe3þ ] ¼ 2.5 104 M, k ¼ 340 nm.
is not involved in metal coordination as was found in Fe/Glycine system [10] and in the crystal structure of [FeII (HAla)2 ] complex [19]. Nevertheless, the stability constant of [Fe(GlcHNA)]3þ (log b ¼ 13:52) is much higher than the one of [FeHGly]3þ (log b ¼ 10:36) [10] suggesting a possible involvement of an hydroxylic group in metal coordination as observed in Cu/Glucosaminic acid system [20,21]. Increasing pH the release of a proton by the complex species is compatible with both reactions: F3þ þ H2 O þ GlcHNA ¡ ½FeðOHÞGlcHNA Fe3þ þ GlcHNA ¡ ½FeGlcNA2þ þ Hþ
2þ
þ Hþ
E. Ferrari, M. Saladini / Journal of Inorganic Biochemistry 98 (2004) 1002–1008
In the first case the ligand is coordinated through carboxyl oxygen like in [Fe(OH)HGly] [10], in the second case the protolytic dissociation of [FeGlcHNA]3þ species may be followed by closing up the five-membered glycine like ring as in [FeAla] complex [22]. In equilibrium calculations the two complexes cannot be discriminated but the evaluated stability constant is higher than those of [FeGly] and [FeAla] (11.45 vs. 8.57 and 8.96, respectively), suggesting also for this complex the involvement of hydroxylic group in metal coordination as shown in Scheme 3. The formation of a new complex species is confirmed by the analysis of the absorbance of the Fe/GlcNA system on varying pH (Fig. 4(b)) which shows increasing until pH 5 where the complex species is completely formed. Over pH 7 a new change in absorbance suggests the formation of another species which is identified as [Fe(GlcNAH1 )2 ] . In this species, we suppose the dissociation of the coordinated hydroxylic oxygen in agreement to what is observed in Cu2þ /Glucosaminic acid system [19,20]. 3.3. Glucosamine The potentiometric titration of the Fe3þ /GlcN system is hindered by the precipitation of Fe-hydroxyde at pH 5; the treatment of data below this pH value allowed to evaluate the stability constants reported in Table 1. In all the complex species, the amino nitrogen is in the protonated NHþ 3 form and the ligand coordinates the metal ion through hydroxylic groups. Previous studies on Fe3þ /sugars systems suggested the formation of complex species in which the sugar molecule (fructose [13], mannitol [23]) chelates the metal ion through deprotonated hydroxylic groups in very acidic conditions. In our case, by progressive addition of GlcN to a solution of Fe3þ no variation in pH is observed, therefore excluding any ionization process in the ligand upon metal coordination.
1007
Increasing pH the formation of mixed hydroxo species is reached but their stability is not sufficient to avoid metal hydroxyde precipitation. Crystallographic studies on solid complexes with carbohydrates isolated in alkaline solution showed octahedral coordination with Fe3þ ion surrounded by alcoholic groups, hydroxylic oxygens and water molecules [2]. In our case, the precipitation of Fe-hydroxyde excludes the formation of this kind of complexes.
4. Conclusions In order to establish the effective Fe3þ chelating ability of these ligands, we may consider the prevailing species in physiological conditions. But since they have different stoichiometry, a simple comparison of the stability constants is not useful, a more proper parameter is represented by pFe3þ , defined as )log [Fe3þ ] [24]. This value is usually calculated with [L] ¼ 105 M and [Fe3þ ] ¼ 106 M and, unlike the stability constants takes into account the effects of ligand protonation and dentiticy as well as differences in metal–ligand stoichiometry. The pFe3þ vs. pH plot provides a useful method to compare the ability of chelators to bind Iron(III) at different pH; it shows that a high value of log K is not necessarily characterized by a high value of pFe3þ . In Fig. 5, pFe3þ vs. pH is reported for our ligands and is compared to OH ligating ability. The only ligand which may compete with OH is Galactaric acid, thus we may propose to use this molecule in the oral iron up taking therapy.
20
18
16
pFe3+
14
12
10
8
6 3
4
5
6
7
8
9
pH
Scheme 3.
Fig. 5. pFe3þ vs. pH for: GalA ––––; GlcNA –––––; GlcN . . . . . . . . .; OH ––––––. [Fe3þ ] ¼ 106 M and [L] ¼ 105 M.
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Acknowledgements We are grateful to the Ministero dellÕUniversita e della Ricerca Scientifica e Tecnologica of Italy for financial support.
References [1] A. Bezkorovany, Biochemistry of Nonheme Iron, Plenum press, London, 1980. [2] B. Gyurcsik, L. Nagy, Coord. Chem. Rev. 203 (2000) 81–149. [3] M.M. H€ am€ al€ aimen, H. L€ onnberg, Carbohydr. Res. 215 (1991) 357–360. [4] D.M. Whitfield, S. Stjkovski, B. Sarkar, Coord. Chem. Rev. 122 (1993) 171–225. [5] J. Urbanska, H. Kozlowski, J. Coord. Chem. 21 (1990) 175–182. [6] Life Science Patents. Available from
[10] G.M. Escandar, A.C. Olivieri, M. Gonzales-Sierra, L.F. Sala, J. Chem. Soc., Dalton Trans. (1994) 1189–1192. [11] S. Deiana, C. Gessa, V. Solinas, P. Pin, R. Seeber, Anal. Chim. Acta 226 (1989) 315–322. [12] K. Araki, S. Shiraishi, Bull. Chem. Soc. Jpn 59 (1986) 3661–3662. [13] H.A. Tajmir-Riahi, J. Inorg. Biochem. 24 (1986) 127–136. [14] H.A. Tajmir-Riahi, Carbohydr. Res. 125 (1984) 13–20. [15] M. Hineno, Carbohydr. Res. 56 (1977) 219–227. [16] P. Djurdjevic, Trans. Met. Chem. 15 (1990) 345–350. [17] L.P. Battaglia, A. Bonamartini Corradi, G. Marcotrigiano, L. Menabue, G.C. Pellacani, J. Am. Chem. Soc. 102 (1980) 1663– 1669. [18] T. Taga, T. Shimada, N. Nimura, Acta Crystallogr. C 50 (1994) 1076–1079. [19] I. Lindqvist, R. Rosenstein, Acta Chem. Scand. 14 (1960) 1228– 1229. [20] J. van Haveren, H. van Bekkum, J.A. Peters, Inorg. Chim. Acta 205 (1993) 1–7. [21] T. Gajda, B. Gyurcsik, T. Jakusch, K. Burger, B. Henry, J.J. Delpuech, Inorg. Chim. Acta 130 (1998) 275–276. [22] P. Djurdjevic, R. Jelie, Trans. Met. Chem. 18 (1993) 457–461. [23] L. Nagy, H. Ohtaki, T. Yamaguchi, M. Nomura, Inorg. Chim. Acta 159 (1989) 201–207. [24] R.C. Hider, Z.D. Liu, S. Piyamongkol, Transf. Sci. 23 (2000) 201– 209.