Metal Binding to Heparin Monosaccharides: D=Glucosamine=6=Sulphate, D-Glucuronic Acid, and L-Iduronic Acid Dennis M. Whitfield and Bibudhendra Sarkar Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
ABSTRACT In order to ascertain which residues in heparin may be responsible for its metal bindin capacities we have investigated metal binding to some of its component monosaccharides by ‘H and $3C NMR. The diamagnetic Zn ion and the paramagnetic Ni ion were used as probes. 4-Methylumbelliferyl-2deoxy-2acetamido-6-O-sulpho-D-glucosamine was used as a model for 0-sulphates. Only weak interactions with the sulphate group were found. The 4C, ring conformation of sodium methyl-fl-D-glucopyranosiduronate was not perturbed by binding to its carboxylate and little evidence exists for chelation. By contrast, the ring conformation of the sodium methyl-ar-l-idopyranosiduronate is affected by the addition of Zn > Pb > Cd > Ca * K ions. The sodium salt is suggested to be an equilibrium mixture of the ‘So and ‘C, ring conformations. Cation binding to the carboxylate group shifts this equilibrium towards the ‘C, conformation and suggests additional binding to 05 or, less likely, 04. This effect appears to be electrostatic in nature, as excess Na and protonation produce similar shifts. Lead complexation is different from the other ions and suggests some covalent character. The control of the ring conformation of iduronic acid by metal ions may have biological implications for the action of heparin and heparin-like compounds.
INTRODUCTION D-glucosamine, D-glucuronic acid, and L-iduronic acid, the C-5 epimer of Dsuch glucuronic acid, are common constituents of mammalian mucopolysaccharides as heparin [l]. These monosaccharides are frequently sulphated. These polymers have been implicated as potential metal binding sites for both toxic and essential metals [2, 31. In spite of this, little work has been published on metal binding to small oligomers including the monosaccharides [4, 51. The anionic sulphate and carboxylate groups are the most probable binding sites but suitably oriented sugar oxygens can also coordinate [6]. Indeed, the carboxylate Address reprint requests to: Dr. Bibudhendra Sarkar, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. Journal of Inorganic Biochemistry, 41, 157-170 (1991) 0 1991 Elsevier Science Publishing Co,, Inc., 655 Avenue of the Americas, NY, NY 10010
157 0162-0134/91/$3.50
158
D. M, Whitfeld and B. Sarkar
(2-l FIGURE
1.
Structures of il). (2), and (3)
group of uranic acids has been shown to be a binding site for metals by Kohn [7] and others [8-lo]. Thus it seemed plausible that the uranic acids in heparin, including L-iduronic acid, could bind metals. The binding of metals to O-sulphate groups of carbohydrates has been scarcely studied [ 1 I]. NMR experiments [ 121 and semi-empirical calculations [ 13] by a number of roups [ 141 have clearly demonstrated that L-iduronic acid can adopt at least the and ‘C, ring conformations depending on ita substituents. We have synthesized the a,@-methyl glycosides of L-iduronic ~c:iJ and shown that the ring conformation of the a-anOmcr ( 1) is sensitive to pH, whereas the $-anomer is essentially in the ‘C, ring conformation [ 15. 161. In this c0mmanication WC rcpoi-t the investigation on the conformation of the pyran ring oi ~-1 -idurmx acrd in the presence of added metal ions like Tn. ‘ui. Cd. Ph, anti C;: EXPERIMENTAL Chemicals 4-Methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulpho-D-glucosamine (I) was from Toronto Research Chemicals (Toronto, Ontario). Methyl-P-D-glucopyranosiduronic acid (2) and methyl-cr-L-idopyranosiduronic acid (3) were synthesized as described previously 1151. For structures and numbering see Figure i The 1 and at this point no further additions were made. The stability constant determination was made using the sodium salt of (3) and Zn acetate. This salt did not promote precipitation and
160
D. M. Whitfield and B. Sarkar
allowed for direct determination of the Zn/sugar mole ratio by integration of the acetate signal and the sugar resonances, with interpulse delays > 10s to allow for complete relaxation. The complete list of experiments is tabulated in Table 1. Equilibrium constants were determined by curve fitting the NMR data using a basic program NMRTITM which iterates for the equilibrium constant and the chemical shift of the 1: 1 complex. Corrections for Zn binding to the acetate counter ion were made using the basic program ALPHA. Both programs were run on a 386-20 MHz PC. Molecular
Modeling MM2 calculations were performed on a 386-20 MHz PC using the software package CHEMCAD (Austin, Texas). Coupling constants were calculated from the MM2 H-H torsion angles using Equation 8E of Haasnoot et al. [ 181. The coordinates for these structures were then entered into the program CCM using the program KC both of which were run on a MicroVAXII computer at the Carbohydrate Research Centre (University of Toronto) ]19]. This program uses a full distance matrix approach to calculate NOE. Only NOE ratios are presented since the requisite correlation times for absolute NOE are unknown. The structures were printed using the program PLUTO.
RESULTS
AND DISCUSSION
4-Methylumbelliferyl-2-deoxy-2-acetamid~6-O-sulpho-D-~uco~mine
(I)
Since the 0-sulphate group of carbohydrates is completely ionized above pH 2 it could be anticipated that electrostatic binding to metal cations would take place. Thus we chose to study binding to the readily available monosaccharide 4-methylumbelliferyl-2-deoxy-2-acetamido-6-0-sulpho-D-glucosamine ( I). As in previous studies [20], we have chosen to use glycosides in order to avoid complications arising from mutarotation of reducing sugars. For structures and numbering see Figure 1. A number of groups have reported metal binding to polymeric sulphated carbohydrates (211 including heparin [22]. Since the extent of binding is proportional to the charge density in most cases, this binding has been ascribed to polyelectrolyte effects [23]. There is little evidence for site binding to sulphate in solution although some crystal structures like potassium 6-0-sulpho-B-D-glucopyranose do indeed show coordination 1241. The free amino D-glucosamine has been shown to bind Cu. Ni. and Co [25] but the 2-acetamido-2-deoxy-D-glucosamine derivative did not 1261. Thus the 0-sulphate group in (1) is a potential binding site. Figure 2 shows the ‘H chemical shifts produced by the addition of Zn acetate to (I). It is readily apparent that only small shifts are observed. The largest shifts are observed for the umbelliferyl aglycone which suggests that the lactone group successfully competes with the sulphate group as a Zn binding site. Figure 3 shows the results of T, measurements of (I) with increasing Ni sulphate concentrations. Because the H6 and HS resonances are slightly more efficiently relaxed than the HI resonance by the addition of Ni, it is tempting to suggest an interaction with the sulphate. However, as w-ith the Zn induced shifts. the lactonr also appears to be a binding site Thus, at least for {I), there appears to be only weak or nonexistent interactions with the sulphate group.
METAL BINDING TO HEPARIN MONOSACCHARIDES
-20
!
161
1 tit
~(2
~3
~4
HS
148
H8’
A0
A6
AS
A3
Resonances FIGURE 2. ‘H NMR chemical shifts for K (I) plus 1.3 equivalent for numbering).
Methyl-/3-D-Glucopyranosiduronic
Zn (OAc),
(see Fig. 1
Acid
Nickel has been shown by ESR to bind to the carboxylates of polygalacturonic acids [27] but no complex was found by potentiometry [28] with the reducing Dglucuronate. This same group reports values for 1: 1 complexes of 42 M- ’ for Pb and 13 M-’ for Cd with D-glucuronate and values of 100 M-’ for Pb and 14 M-’ for Cd with D-galacturonate. The corresponding values for Ca binding to (1: 1) D-glucuronate are 32 and for D-galacturonate 64 M- ’ [29]. These results concur with those of Kohn [7] which demonstrate that Pb and Cd bind with D-galacturonic acid but Zn only binds weakly. It was therefore of some interest to see whether or not the LSD-methyl glycoside (2) bound these metals. A similar set of experiments was performed for (2) as for (I). In this case only very small shifts were observed after addition of Zn. The largest shifts for (2) were found for H5 and C5 suggestive of coordination to the carboxylate (see Tables 2 and 3). Our data are consistent with previous NMR studies of the sodium salt of D-glucuronate [30]. There is no evidence for any perturbation of the normal 4C, ring conformation of (2) in the presence or absence of added metal ions (compare Table 2 and Fig. 8).
162
D. M. Whitfield and B. Sarkar
1.1 -
Hl
-Ii5
-I+6
1.0
-AY
0.0
5
0.0
c 0.7
0.06
Mole
FIGURE 3. ‘H NMR T, relaxation of increasing Ni.
%
4
3
2
1
0
NI
times (normalized
to the metal free T,) in the presence
TABLE 2. ‘H NMR Spectral Data for (I), (2), and (3), 6 ppm (Hz) Sampie
(0 (2)Na (2)Zn’ (3)H (3)Na (3)Zn
Hl(J,,)
WJ,,)
HXJ,)
5.13q8.4) 4.382(8.0) 0.08(8.0) 4.821(3.4) 4.681(4.9) 4.727(4.6)
4.013(10.3) 3.297(101) O.M(lOl) 3.598(5.6) 3.466(6.8) 3.492(6.5)
3.737(8.9) 3X5(61) 0.08(61) 3.854(X0) 3.71q6.4) 3.737(6.1)
WJ,,)
HXJ,,)
3.619(9.8) 3.887(2. I) 3.505(61) 3.726(61)’ 0.08(61): 0.08(61) 4.705 4.003(3.2) 4.414 3.853(4. I) 4.423 3.874(3.8) ________-____l_l_l_
WJ66.) 4.399( - 11.4) --
WJ,,) 4.2610 --
._ -_
-
‘Chemical shifts relative to Na(2) are given in ppm. *The spectrum is not in first order The notation 1 refers to the number of lines in the multiplet.
TABLE 3. 13C NMR Spectral Data for (2) and (3), 6 ppm Sample
Cl
c2
c3
c4
C5
C6
(.Wa
103.62 0.0 102.14 101.77 101.91
73.30 --0.01 69.61 70.95 70.64
76.00 -0.01 70.72 12.25 71.85
72.18 -0.01 70.08 71.04 70.77
76.19 -0.02 69.31 70.77 70.b4
176.26 0.0 173.62 176.37 176.42
(2)Zn’ (3)H (3)Na (3)Zn ‘Chemical
shifts relative to Na( 2) are given in ppm
2)
METAL BINDING
TO HEPARIN
MONOSACCHARIDES
163
,
FIGURE 4.
‘H NMR inversion recovery experiment of Na (2) in the presence of (top) and absence (bottom) of 0.5% Ni.
That the carboxylate is indeed the principal binding site has been substantiated by differential line broadening of H5 as compared to the other ring protons in the presence of small amounts of Ni for (2). Figure 4 demonstrates that Ni reduces the T, of H5 more than any other proton as would be anticipated for binding to the carboxylate. Figure 4 (no Ni) shows a trace for a particular T value in an inversion-recovery experiment with all the resonances inverted whereas in Figure 4 ( + Ni) the H5 resonance has reverted while the remaining resonances are inverted. However, in accord with previous reports any binding must be weak.
Methyl-a-L-idopyranosiduronic
Acid (3)
There are no reports of metal binding studies with this monosaccharide. Huckerby et al. [4] have reported that added monovalent ions had little effect on the NMR spectra of some heparin derived oligosaccharides and Van Boeckel et al. [14] have reported that 3 M NaCl stabilized the ‘C, ring conformation of the 2-0-sulpho-L-iduronate residues in different heparin derived oligosaccharides. This last result is in accord with our results presented below. Figure 5 shows the ‘H NMR spectra of the sodium salt of methyl-cY-l-idopyranosiduronic acid (I), in the presence and absence of added Zn ions. All five resonances are shifted downfield and more importantly, all the vicinal coupling constants change. Figure 6 shows the C2, C3, C4, and C5 resonances of the corresponding
164
D. M. Whitfield and B. Sarkar
FIGURE 5. ‘H NMR Spectrum of Na (3) in the presence of (top) and abSCnCC (bottom) of 1.3 equivalent Zn (OAc), in I&O. 13C NMR spectra where the assignments are based on 13C-‘H COSY spectra. Metal induced chemical shifts of l-3 ppm are observed whereas the Cl, CH,, and C6 (COO) resonances shift less than 0.5 ppm (1 .O ppm = 75 Hz). These results are clearly consistent with a metal induced conformational change of the pyran ring. Following after Sinay et al., [31] the large coupling constants for (3) in the absence of added metal are consistent with a mixture of the ‘So and ‘C, ring conformations with perhaps some 4C *. Zinc apparently shifts this equilibrium toward the lC, conformation as all the coupling constants are much smaller. Ball and stick representations of these conformers are shown in Figure 7 and calculated coupling constants are shown in Figure 8. Further confirmation of this hypothesis comes from Nuclear Overhauser Enhancements (NOE) experiments, in which the NOE between H5 and H2 (irradiate H5) is monitored [32]. A large value for this NOE is expected if the *S, conformation is highly populated. For (3) this NOE decreased from 0.32 rt 0.6 to 0.18 +- .04 (relative to H4, average of 4 determinations), along with other small WOE changes in the presence of Zn. Calculated values are 4C L (-0.167), ‘SC, (0.66). and ‘C, (0.06). Similarly, the NOE between Hl and H3 (irradiate Hl) is sensitive to the ring conformation. For (3) this NOE changed from 1.28 f .26 to 0.62 I . I2 (relative to H2, average of 3 determinations) after addition of Zn. Calculated values are ‘C , (4.20), *So (2.90), and ‘C, ( - 0.14). Both of these results are consistent with a change from about 50:5O ?S,:‘C,) for the sodium salt to about 25:75 for the
METAL BINDING TO HEPARIN MONOSACCHARIDES
R
n
70
165
00
FIGURE 6. ‘k NMR spectrum of Na (3) with none (bottom), 0.5 equivalent (middle) and 1.8 equivalent (top) ZnCl, in D,O. Our results can be explained without invoking any population of the 4C, conformation. It should be noted that a full search of the potential energy surface including counter ions and solvent is necessary in order to accurately calculate populations. Therefore we do not attempt to estimate populations quantitatively. Metal binding to ligands can induce 13C chemical shifts by at least two mechanisms, namely direct metal-ligand electronic effects or by induced ligand conformational changes. It is usually difficult to separate these two mechanisms. In this case, the pronounced chemical shifts are for carbons not likely to be directly involved in metal coordination and thus can be attributed to induced conformational changes (see complex.
FIGURE
7. Ball and stick representation of three conformers of Na (3).
W4Cl
166
D. M. Whitfield and B. Sarkar ---
12 Ns
Na
Na
Na
Na
Na
‘Cd +
+
+
+
+
+
H
Zn
Pb
Cd
Ca
K
4
5
1
7
3
6
7
t
zn t
zn +
Na
Zn
Ns
15
1’
Na
Na
8
4
zn ‘so
17
!.’
-‘Cl
?4
FIGURE
8. ‘H NMR coupling constants of (3). Entries 1. 1.3.and 1-I are from MM2 for ‘C,, ‘S,, and 4C,. Entries 2-12 are (31, Na(3) -t 1.8 equivalent ZnCl,, Na (3) -t 1.3 equivalent Pb (OAc), , Na iSj + 1.3 equivalent CdCi2, Na (3) + E.9 equivalent CaCIZ, Na (3) + 1.8 equivalent KCl, Na (3), Na(3) ,t 36 equivalent NaOAc. Zn !3) + 8 6 equivalent Zn(OAc),, Zn(3), Zn(3) + 5 4 equivalent NaOAc. Figs. 5 and 6). This hypothesis is substantiated by comparison to the small metal induced shifts of the C5 epimer (2). Also the ‘3C resonance of the carboxylate of (3) was not observable in the presence of 0.5 mol% Ni sulphate, strongly suggesting coordination to the carboxylate [33]. This compares with the addition of > 3 molR Ni to (I) to produce substantial line broadening. An apparent 1:l binding constant for Zn for (3) was found to be 177 i- 30 M ’ by curve fitting. The Zn binding to the acetate counter ions wab corrected for by using the known binding constants (see Experimental) 1341. This value is slightly higher than those reported for uranic acids above, suggesting chelation The shifts for (2) were too small to make such a calculation reliable. Figures 8- 10 show a comparison between the addition of various metal ions to rhe sodium salt of (3). At the same approximate mole ratios, the magnitude of the induced shifts is Zn > Pb > Cd > Ca & K (entries 3.-9 in Fig. 8, 2-X in Figs. 9 and 10). The shifts induced by Zn. Ca, Cd, and excess Na were ail in the same directions and therefore it is tempting to assume that the binding to all these Ions is very similar. The coupling constants and shifts, especially the “C (see Fig. 10, entry 3) changes for Pb are substantially different and suggest a different mode of Interaction. Previous workers [7, 281 have interpreted their data for Pb binding in terms of covalent bond formation and this may be the case here as well j 151, ‘indeed. Pb
METAL BINDING
TO HEPARIN
MONOSACCHARIDES
167
100
i! r
5 a _o
80
E a, 6 f
60
I
2
3
4
5
6
7
8
9
10
11
FIGURE 9. ‘H NMR chemical shifts of (3). Entries 1- 11 are the same as 2- 12 in Figure 7.
binding to the carboxylate of D-glucuronic acid has been demonstrated by IR spectroscopy by Tajmir Riahi [36]. Also shown in Figures 8-10 (entries lo-12 in Fig. 8, 11-13 in Figs. 9 and 10) are NMR data for the Zn salt of (3) and this salt in the presence of added Na-acetate or Zn-acetate. The Zn salt itself has NMR spectra comparable to the addition of 0.5 equivalent of Zn to the sodium salt. This is not unexpected as this must be a 1:2 complex and would be very different from 1: 1 complexes. Indeed, addition of more Zn produces shifts like the additions to the Na-salt and addition of Na requires a large excess to produce an effect. This experiment was a control to rule out any complications that the presence of Na may have had on the binding by Zn. Zinc binding is suggested to be principally electrostatic, resulting in a reduction of electron density on the carbohydrate oxygens. Such a diminution in electron density would reduce the repulsive interactions in the ‘C, conformation, notably the 1,3-diaxial interactions between 01 and 03, and between 02 and 04. This result, although speculative, leads us to propose a model where the metal ions are bound to carboxylate and to 05. Molecular modeling experiments with Zn and (3) suggest that this arrangement can be accommodated while maintaining 200 +- 10 pm bond length to both oxygens [37]. Such an arrangement requires that the O-C6-C505 dihedral angle is near 0”. This value is indeed found in several x-ray crystal structures of uranic acid salts [38-421. All of those compounds exhibit chelation to their counter-cations via the carboxylate oxygen and 05. Alternate chelation to 04 is
Na
$40
Na
+..--~.---
Na
--._ :’
FIGURE
Na
Na
Na
5
f:
Na
*__.~ ‘I
Zn
Zn
__“__~_..-___ll__-.~_.
4
1
:I,
-_-..
:1
10. liC ~MRche~~~~ shiftsof (3). Entries I - 11 are the same as 2-. I in Figure 7.
less likely due to these obse~ations. These s~~nlations are in accord with the observations of Angyal et ai., concerning metal binding to D-galacturonate 1431. Also shown in Figures 8-10 (entry 2 in Fig. 8, 1 in Figs. 9 and 10) are the NMR data for the neutral free acid (3). These are very similar to the values for the Zn complex of the sodium salt and support the electrostatic model proposed above except for the marked shift for C6 upon protonation, not observed in the Zn complex. In order to be certain that in all experiments the carboxylic acid was completely ionized, we determined the apparent pK, to be 3.19 4 0. ! on the pH* scale. All spectra of salts were indeed recorded 2+ pli* units above this pK, (see Table 1). This pK, is markedly different from the value of 5, t reported for hduronic acid in heparin [#I. Our value is in accord with the literature values of 3.28 and 3.23 for D-glucuronic acid and 3.5 1 and 3.47 for D-galacturonic acid (45, 461. The discrepancy between the values for the polymer and the monosaccharides is probably due to the ~lyelectrolyt~ nature of heparin as the reported valnc is an ~~~pare~t pK, 1471. CONCLUSION We were not able to demonstrate metal binding to the O-sulphate group of (1) under conditions in which we did observe binding to (2) and (3). We could not quantitate the difference in binding strengths between izj and (3) but it is suggested that because (3) is more flexible it is able to adopt a geometry which creates a metal
METAL, BINDING TO HEPARIN MONOSACCHARIDES
169
binding site and hence has higher affinity for metals. This site is suggested to be the carboxylate oxygen and the ring oxygen 05 with the remaining sites occupied by anions or water. The conformational flexibility of L-iduronic acid has recently been proposed to be important for the biological activity of heparins [31]. The metal induced conformational shifts changes reported in this work could then represent a control mechanism for these biological functions. The authors would like to thank Dr. A. Grey of the NMR laboratory of the Carbohydrate Research Centre for assistance with the NMR spectra. The work was supported by the Medical Research Council of Canada, B. Sarkar, MT 1800, and to Carbohydrate Research Centre, MT 6499 and MA 9646.
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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
(1982). M. L. Dheu-Andries and S. Perez, Carbohydr. Res. 124, 324 (1983). R. Kohn, Carbohydr. Res. 160, 343 (1987). I. B. Cook, R. J. Magee, R. Payne, and B. Temai, Aust. J. Chem. 39, 1307 (1986). H. A. T. Riahi, J. Znorg. Biochem. 26, 23 (1986). R. Aruga, Bull. Chem. Sot. Jpn. 54, 1233 (1981). D. M. Templeton, Biochim. Biophys. Acta 926, 94 (1987). D. R. Ferro, A. Provasoli, M. Ragazzi, G. Torri, B. Casu, G. Gatti, J. C. Jacquinet, P. Sinay, M. Petitou, and J. Choay, J. Am. Chem. Sot. 108, 6773 (1986). M. Ragazzi, D. R. Ferro, and A. Provasoli, J. Comput. Chem. 7, 105 (1986). C. A. A. van Boeckel, S. F. van Aelst, G. N. Wagenaars, J. R. Mellema, H. Paulsen, T. Peters, A. Pollex, and V. Sinnwell, Reel. Trav. Chim. Pays-Bus 106, 19 (1987). D. M. Whitfield, G. Bimbaum, H. Pang, J. Baptista, and B. Sarkar, submitted for publication. T. Chiba and P. Sinay, Carbohydr. Res. 151, 379 (1986). A. Bax, R. H. Griffey, and B. L. Hawkins, J. Magn. Reson. 55, 301 (1983). C. A. G. Haasnoot, F. A. A. M. De Leeuw, and C. Altona, Tetrahedron 36, 2783
(1980). 19. J.-R. Brisson and J. P. Carver, Biochemistry 22, 3671 (1983). 20. S. Stojkovski, D. M. Whitlield, R. J. Magee, B. D. James, and B. Sarkar,
J. Znorg.
Biochem. 39, 125 (1990). R. P. Millane, A. K. Mitra, and S. Amott, J. Mol. Biol. 169, 903 (1983). J. Mattai and J. C. T. Kwak, Biochim. Biophys. Acta 677, 303 (1981). P. Dais, Q-J. Peng, and A. S. Perlin, Carbohydr. Res. 168, 163 (1987). D. Lamba, W. Mackie, B. Sheldrick, P. Belton, and S. Tanner, Carbohydr. Res. 180, 183 (1988). 25. A. Pusino, D. Droma, P. Decock, P. Dubois, and H. Kozlowski, Znorg. Chim. Acta
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138, 5 (1987). M. W. G. De Bolster, 26. E. B. V. Appelman-Lippens, Visserluirink, Znorg. Chim. Acta 108, 209 (1985).
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L. Erre, G. Micera, P. Piu, and C. Gessa, Inorg. Chim. .4cta 46. 249 (1980). C. Makridou, M. Cromer-Morin, and 3. P. Scharff, Bull. Sot. Chim. France 5. 59 (1977). R. 0. Gould and A. F. Rankin, J. Chem. Sot. Chem. Comm., 489 (1970). L. W. Jaques, J. B. Macaskill, and W. Weltner Jr., .I. Phys. Chem. 83, 1412 (1979). B, Casu, M. Petitou, M. Provasoli, and P. Sinay. TrendsBiochem. Sci. 13. 221 (1988).
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29. 30. 31. 32. M. Ragazzi, D. R. Ferro, B. Perly, G. Torri. B. Casu, P, Sinny. M. Petitou. and J. Choay, Curbohydr. Res. 165, Cl (1987). 33. J. P. Laussac and B. Sarkar. Can. /. Chem. 58, 2055 (1980). 34. R. M. Smith and A. E. Martell, Critical Stability Constants. Plenum Press. New York, 1975, Vol. 3 35. A. Cesaro. F. Delben, A. Flaibani, and G. Paoietti, Carbohydr. Re.s. 181. 13 (19881. 36. H. A. T. Riahi, Bull. Chem. Sac. Jpn. 52, 1281 (1989). 37. L. Lebioda and B. Stec, J. Am. Chem. Sot. 111, 8511 (1989~. 38. S. E. B. Gould, R. 0. Gould. D. A. Rees. and W E. Scott, 3. Chenz. Tot. Perkin
Trans 2, 237 (1975). 39. T. Taga, T. Kaji, and K. Osaka, BUN. Chem. Sot. Jpn. 58, 30 (1985). 40. S. Thanomkul, J. A. Hjortas, and H. Sorum, Acta Crysr. B32. 920 (1976). 41. F. MO, T. J. Brobak, and I. R. Siddiqui, Carbohydr. Res. 145, 13 (1985). 42. L. Delucas, C. E. Bugg. A. Terzis, and R. Rivest, Carbohydr. Res. 41. 19 (1975). 43. S. J. Angyal, D. Greeves, and L. Littlemore, Curbohydr. Res. 174. 121 (1988). 44. J. W. Park and B. Chakrabarti, Biochem. Biophys. Res. Commun. 78. 604 (1977). 45. R. Kohn and P. Kovac. Chem. Zvesti 32, 478 11978). 46. H. Holvik and H. Hoiland, J. Chem. Thermodyn. 9, 345 (1977). 47. T. M. Fyles. J. Chem. Sot. Faraday Tram I82, 617 ~1986,. Received April 4, 1990; accepted June 26, 1990
ABSTRACT In order to ascertain which residues in heparin may be responsible for its metal bindin capacities we have investigated metal binding to some of its component monosaccharides by ‘H and $3C NMR. The diamagnetic Zn ion and the paramagnetic Ni ion were used as probes. 4-Methylumbelliferyl-2deoxy-2acetamido-6-O-sulpho-D-glucosamine was used as a model for 0-sulphates. Only weak interactions with the sulphate group were found. The 4C, ring conformation of sodium methyl-fl-D-glucopyranosiduronate was not perturbed by binding to its carboxylate and little evidence exists for chelation. By contrast, the ring conformation of the sodium methyl-ar-l-idopyranosiduronate is affected by the addition of Zn > Pb > Cd > Ca * K ions. The sodium salt is suggested to be an equilibrium mixture of the ‘So and ‘C, ring conformations. Cation binding to the carboxylate group shifts this equilibrium towards the ‘C, conformation and suggests additional binding to 05 or, less likely, 04. This effect appears to be electrostatic in nature, as excess Na and protonation produce similar shifts. Lead complexation is different from the other ions and suggests some covalent character. The control of the ring conformation of iduronic acid by metal ions may have biological implications for the action of heparin and heparin-like compounds.
INTRODUCTION D-glucosamine, D-glucuronic acid, and L-iduronic acid, the C-5 epimer of Dsuch glucuronic acid, are common constituents of mammalian mucopolysaccharides as heparin [l]. These monosaccharides are frequently sulphated. These polymers have been implicated as potential metal binding sites for both toxic and essential metals [2, 31. In spite of this, little work has been published on metal binding to small oligomers including the monosaccharides [4, 51. The anionic sulphate and carboxylate groups are the most probable binding sites but suitably oriented sugar oxygens can also coordinate [6]. Indeed, the carboxylate Address reprint requests to: Dr. Bibudhendra Sarkar, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada. Journal of Inorganic Biochemistry, 41, 157-170 (1991) 0 1991 Elsevier Science Publishing Co,, Inc., 655 Avenue of the Americas, NY, NY 10010
157 0162-0134/91/$3.50
158
D. M, Whitfeld and B. Sarkar
(2-l FIGURE
1.
Structures of il). (2), and (3)
group of uranic acids has been shown to be a binding site for metals by Kohn [7] and others [8-lo]. Thus it seemed plausible that the uranic acids in heparin, including L-iduronic acid, could bind metals. The binding of metals to O-sulphate groups of carbohydrates has been scarcely studied [ 1 I]. NMR experiments [ 121 and semi-empirical calculations [ 13] by a number of roups [ 141 have clearly demonstrated that L-iduronic acid can adopt at least the and ‘C, ring conformations depending on ita substituents. We have synthesized the a,@-methyl glycosides of L-iduronic ~c:iJ and shown that the ring conformation of the a-anOmcr ( 1) is sensitive to pH, whereas the $-anomer is essentially in the ‘C, ring conformation [ 15. 161. In this c0mmanication WC rcpoi-t the investigation on the conformation of the pyran ring oi ~-1 -idurmx acrd in the presence of added metal ions like Tn. ‘ui. Cd. Ph, anti C;: EXPERIMENTAL Chemicals 4-Methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulpho-D-glucosamine (I) was from Toronto Research Chemicals (Toronto, Ontario). Methyl-P-D-glucopyranosiduronic acid (2) and methyl-cr-L-idopyranosiduronic acid (3) were synthesized as described previously 1151. For structures and numbering see Figure i The 1 and at this point no further additions were made. The stability constant determination was made using the sodium salt of (3) and Zn acetate. This salt did not promote precipitation and
160
D. M. Whitfield and B. Sarkar
allowed for direct determination of the Zn/sugar mole ratio by integration of the acetate signal and the sugar resonances, with interpulse delays > 10s to allow for complete relaxation. The complete list of experiments is tabulated in Table 1. Equilibrium constants were determined by curve fitting the NMR data using a basic program NMRTITM which iterates for the equilibrium constant and the chemical shift of the 1: 1 complex. Corrections for Zn binding to the acetate counter ion were made using the basic program ALPHA. Both programs were run on a 386-20 MHz PC. Molecular
Modeling MM2 calculations were performed on a 386-20 MHz PC using the software package CHEMCAD (Austin, Texas). Coupling constants were calculated from the MM2 H-H torsion angles using Equation 8E of Haasnoot et al. [ 181. The coordinates for these structures were then entered into the program CCM using the program KC both of which were run on a MicroVAXII computer at the Carbohydrate Research Centre (University of Toronto) ]19]. This program uses a full distance matrix approach to calculate NOE. Only NOE ratios are presented since the requisite correlation times for absolute NOE are unknown. The structures were printed using the program PLUTO.
RESULTS
AND DISCUSSION
4-Methylumbelliferyl-2-deoxy-2-acetamid~6-O-sulpho-D-~uco~mine
(I)
Since the 0-sulphate group of carbohydrates is completely ionized above pH 2 it could be anticipated that electrostatic binding to metal cations would take place. Thus we chose to study binding to the readily available monosaccharide 4-methylumbelliferyl-2-deoxy-2-acetamido-6-0-sulpho-D-glucosamine ( I). As in previous studies [20], we have chosen to use glycosides in order to avoid complications arising from mutarotation of reducing sugars. For structures and numbering see Figure 1. A number of groups have reported metal binding to polymeric sulphated carbohydrates (211 including heparin [22]. Since the extent of binding is proportional to the charge density in most cases, this binding has been ascribed to polyelectrolyte effects [23]. There is little evidence for site binding to sulphate in solution although some crystal structures like potassium 6-0-sulpho-B-D-glucopyranose do indeed show coordination 1241. The free amino D-glucosamine has been shown to bind Cu. Ni. and Co [25] but the 2-acetamido-2-deoxy-D-glucosamine derivative did not 1261. Thus the 0-sulphate group in (1) is a potential binding site. Figure 2 shows the ‘H chemical shifts produced by the addition of Zn acetate to (I). It is readily apparent that only small shifts are observed. The largest shifts are observed for the umbelliferyl aglycone which suggests that the lactone group successfully competes with the sulphate group as a Zn binding site. Figure 3 shows the results of T, measurements of (I) with increasing Ni sulphate concentrations. Because the H6 and HS resonances are slightly more efficiently relaxed than the HI resonance by the addition of Ni, it is tempting to suggest an interaction with the sulphate. However, as w-ith the Zn induced shifts. the lactonr also appears to be a binding site Thus, at least for {I), there appears to be only weak or nonexistent interactions with the sulphate group.
METAL BINDING TO HEPARIN MONOSACCHARIDES
-20
!
161
1 tit
~(2
~3
~4
HS
148
H8’
A0
A6
AS
A3
Resonances FIGURE 2. ‘H NMR chemical shifts for K (I) plus 1.3 equivalent for numbering).
Methyl-/3-D-Glucopyranosiduronic
Zn (OAc),
(see Fig. 1
Acid
Nickel has been shown by ESR to bind to the carboxylates of polygalacturonic acids [27] but no complex was found by potentiometry [28] with the reducing Dglucuronate. This same group reports values for 1: 1 complexes of 42 M- ’ for Pb and 13 M-’ for Cd with D-glucuronate and values of 100 M-’ for Pb and 14 M-’ for Cd with D-galacturonate. The corresponding values for Ca binding to (1: 1) D-glucuronate are 32 and for D-galacturonate 64 M- ’ [29]. These results concur with those of Kohn [7] which demonstrate that Pb and Cd bind with D-galacturonic acid but Zn only binds weakly. It was therefore of some interest to see whether or not the LSD-methyl glycoside (2) bound these metals. A similar set of experiments was performed for (2) as for (I). In this case only very small shifts were observed after addition of Zn. The largest shifts for (2) were found for H5 and C5 suggestive of coordination to the carboxylate (see Tables 2 and 3). Our data are consistent with previous NMR studies of the sodium salt of D-glucuronate [30]. There is no evidence for any perturbation of the normal 4C, ring conformation of (2) in the presence or absence of added metal ions (compare Table 2 and Fig. 8).
162
D. M. Whitfield and B. Sarkar
1.1 -
Hl
-Ii5
-I+6
1.0
-AY
0.0
5
0.0
c 0.7
0.06
Mole
FIGURE 3. ‘H NMR T, relaxation of increasing Ni.
%
4
3
2
1
0
NI
times (normalized
to the metal free T,) in the presence
TABLE 2. ‘H NMR Spectral Data for (I), (2), and (3), 6 ppm (Hz) Sampie
(0 (2)Na (2)Zn’ (3)H (3)Na (3)Zn
Hl(J,,)
WJ,,)
HXJ,)
5.13q8.4) 4.382(8.0) 0.08(8.0) 4.821(3.4) 4.681(4.9) 4.727(4.6)
4.013(10.3) 3.297(101) O.M(lOl) 3.598(5.6) 3.466(6.8) 3.492(6.5)
3.737(8.9) 3X5(61) 0.08(61) 3.854(X0) 3.71q6.4) 3.737(6.1)
WJ,,)
HXJ,,)
3.619(9.8) 3.887(2. I) 3.505(61) 3.726(61)’ 0.08(61): 0.08(61) 4.705 4.003(3.2) 4.414 3.853(4. I) 4.423 3.874(3.8) ________-____l_l_l_
WJ66.) 4.399( - 11.4) --
WJ,,) 4.2610 --
._ -_
-
‘Chemical shifts relative to Na(2) are given in ppm. *The spectrum is not in first order The notation 1 refers to the number of lines in the multiplet.
TABLE 3. 13C NMR Spectral Data for (2) and (3), 6 ppm Sample
Cl
c2
c3
c4
C5
C6
(.Wa
103.62 0.0 102.14 101.77 101.91
73.30 --0.01 69.61 70.95 70.64
76.00 -0.01 70.72 12.25 71.85
72.18 -0.01 70.08 71.04 70.77
76.19 -0.02 69.31 70.77 70.b4
176.26 0.0 173.62 176.37 176.42
(2)Zn’ (3)H (3)Na (3)Zn ‘Chemical
shifts relative to Na( 2) are given in ppm
2)
METAL BINDING
TO HEPARIN
MONOSACCHARIDES
163
,
FIGURE 4.
‘H NMR inversion recovery experiment of Na (2) in the presence of (top) and absence (bottom) of 0.5% Ni.
That the carboxylate is indeed the principal binding site has been substantiated by differential line broadening of H5 as compared to the other ring protons in the presence of small amounts of Ni for (2). Figure 4 demonstrates that Ni reduces the T, of H5 more than any other proton as would be anticipated for binding to the carboxylate. Figure 4 (no Ni) shows a trace for a particular T value in an inversion-recovery experiment with all the resonances inverted whereas in Figure 4 ( + Ni) the H5 resonance has reverted while the remaining resonances are inverted. However, in accord with previous reports any binding must be weak.
Methyl-a-L-idopyranosiduronic
Acid (3)
There are no reports of metal binding studies with this monosaccharide. Huckerby et al. [4] have reported that added monovalent ions had little effect on the NMR spectra of some heparin derived oligosaccharides and Van Boeckel et al. [14] have reported that 3 M NaCl stabilized the ‘C, ring conformation of the 2-0-sulpho-L-iduronate residues in different heparin derived oligosaccharides. This last result is in accord with our results presented below. Figure 5 shows the ‘H NMR spectra of the sodium salt of methyl-cY-l-idopyranosiduronic acid (I), in the presence and absence of added Zn ions. All five resonances are shifted downfield and more importantly, all the vicinal coupling constants change. Figure 6 shows the C2, C3, C4, and C5 resonances of the corresponding
164
D. M. Whitfield and B. Sarkar
FIGURE 5. ‘H NMR Spectrum of Na (3) in the presence of (top) and abSCnCC (bottom) of 1.3 equivalent Zn (OAc), in I&O. 13C NMR spectra where the assignments are based on 13C-‘H COSY spectra. Metal induced chemical shifts of l-3 ppm are observed whereas the Cl, CH,, and C6 (COO) resonances shift less than 0.5 ppm (1 .O ppm = 75 Hz). These results are clearly consistent with a metal induced conformational change of the pyran ring. Following after Sinay et al., [31] the large coupling constants for (3) in the absence of added metal are consistent with a mixture of the ‘So and ‘C, ring conformations with perhaps some 4C *. Zinc apparently shifts this equilibrium toward the lC, conformation as all the coupling constants are much smaller. Ball and stick representations of these conformers are shown in Figure 7 and calculated coupling constants are shown in Figure 8. Further confirmation of this hypothesis comes from Nuclear Overhauser Enhancements (NOE) experiments, in which the NOE between H5 and H2 (irradiate H5) is monitored [32]. A large value for this NOE is expected if the *S, conformation is highly populated. For (3) this NOE decreased from 0.32 rt 0.6 to 0.18 +- .04 (relative to H4, average of 4 determinations), along with other small WOE changes in the presence of Zn. Calculated values are 4C L (-0.167), ‘SC, (0.66). and ‘C, (0.06). Similarly, the NOE between Hl and H3 (irradiate Hl) is sensitive to the ring conformation. For (3) this NOE changed from 1.28 f .26 to 0.62 I . I2 (relative to H2, average of 3 determinations) after addition of Zn. Calculated values are ‘C , (4.20), *So (2.90), and ‘C, ( - 0.14). Both of these results are consistent with a change from about 50:5O ?S,:‘C,) for the sodium salt to about 25:75 for the
METAL BINDING TO HEPARIN MONOSACCHARIDES
R
n
70
165
00
FIGURE 6. ‘k NMR spectrum of Na (3) with none (bottom), 0.5 equivalent (middle) and 1.8 equivalent (top) ZnCl, in D,O. Our results can be explained without invoking any population of the 4C, conformation. It should be noted that a full search of the potential energy surface including counter ions and solvent is necessary in order to accurately calculate populations. Therefore we do not attempt to estimate populations quantitatively. Metal binding to ligands can induce 13C chemical shifts by at least two mechanisms, namely direct metal-ligand electronic effects or by induced ligand conformational changes. It is usually difficult to separate these two mechanisms. In this case, the pronounced chemical shifts are for carbons not likely to be directly involved in metal coordination and thus can be attributed to induced conformational changes (see complex.
FIGURE
7. Ball and stick representation of three conformers of Na (3).
W4Cl
166
D. M. Whitfield and B. Sarkar ---
12 Ns
Na
Na
Na
Na
Na
‘Cd +
+
+
+
+
+
H
Zn
Pb
Cd
Ca
K
4
5
1
7
3
6
7
t
zn t
zn +
Na
Zn
Ns
15
1’
Na
Na
8
4
zn ‘so
17
!.’
-‘Cl
?4
FIGURE
8. ‘H NMR coupling constants of (3). Entries 1. 1.3.and 1-I are from MM2 for ‘C,, ‘S,, and 4C,. Entries 2-12 are (31, Na(3) -t 1.8 equivalent ZnCl,, Na (3) -t 1.3 equivalent Pb (OAc), , Na iSj + 1.3 equivalent CdCi2, Na (3) + E.9 equivalent CaCIZ, Na (3) + 1.8 equivalent KCl, Na (3), Na(3) ,t 36 equivalent NaOAc. Zn !3) + 8 6 equivalent Zn(OAc),, Zn(3), Zn(3) + 5 4 equivalent NaOAc. Figs. 5 and 6). This hypothesis is substantiated by comparison to the small metal induced shifts of the C5 epimer (2). Also the ‘3C resonance of the carboxylate of (3) was not observable in the presence of 0.5 mol% Ni sulphate, strongly suggesting coordination to the carboxylate [33]. This compares with the addition of > 3 molR Ni to (I) to produce substantial line broadening. An apparent 1:l binding constant for Zn for (3) was found to be 177 i- 30 M ’ by curve fitting. The Zn binding to the acetate counter ions wab corrected for by using the known binding constants (see Experimental) 1341. This value is slightly higher than those reported for uranic acids above, suggesting chelation The shifts for (2) were too small to make such a calculation reliable. Figures 8- 10 show a comparison between the addition of various metal ions to rhe sodium salt of (3). At the same approximate mole ratios, the magnitude of the induced shifts is Zn > Pb > Cd > Ca & K (entries 3.-9 in Fig. 8, 2-X in Figs. 9 and 10). The shifts induced by Zn. Ca, Cd, and excess Na were ail in the same directions and therefore it is tempting to assume that the binding to all these Ions is very similar. The coupling constants and shifts, especially the “C (see Fig. 10, entry 3) changes for Pb are substantially different and suggest a different mode of Interaction. Previous workers [7, 281 have interpreted their data for Pb binding in terms of covalent bond formation and this may be the case here as well j 151, ‘indeed. Pb
METAL BINDING
TO HEPARIN
MONOSACCHARIDES
167
100
i! r
5 a _o
80
E a, 6 f
60
I
2
3
4
5
6
7
8
9
10
11
FIGURE 9. ‘H NMR chemical shifts of (3). Entries 1- 11 are the same as 2- 12 in Figure 7.
binding to the carboxylate of D-glucuronic acid has been demonstrated by IR spectroscopy by Tajmir Riahi [36]. Also shown in Figures 8-10 (entries lo-12 in Fig. 8, 11-13 in Figs. 9 and 10) are NMR data for the Zn salt of (3) and this salt in the presence of added Na-acetate or Zn-acetate. The Zn salt itself has NMR spectra comparable to the addition of 0.5 equivalent of Zn to the sodium salt. This is not unexpected as this must be a 1:2 complex and would be very different from 1: 1 complexes. Indeed, addition of more Zn produces shifts like the additions to the Na-salt and addition of Na requires a large excess to produce an effect. This experiment was a control to rule out any complications that the presence of Na may have had on the binding by Zn. Zinc binding is suggested to be principally electrostatic, resulting in a reduction of electron density on the carbohydrate oxygens. Such a diminution in electron density would reduce the repulsive interactions in the ‘C, conformation, notably the 1,3-diaxial interactions between 01 and 03, and between 02 and 04. This result, although speculative, leads us to propose a model where the metal ions are bound to carboxylate and to 05. Molecular modeling experiments with Zn and (3) suggest that this arrangement can be accommodated while maintaining 200 +- 10 pm bond length to both oxygens [37]. Such an arrangement requires that the O-C6-C505 dihedral angle is near 0”. This value is indeed found in several x-ray crystal structures of uranic acid salts [38-421. All of those compounds exhibit chelation to their counter-cations via the carboxylate oxygen and 05. Alternate chelation to 04 is
Na
$40
Na
+..--~.---
Na
--._ :’
FIGURE
Na
Na
Na
5
f:
Na
*__.~ ‘I
Zn
Zn
__“__~_..-___ll__-.~_.
4
1
:I,
-_-..
:1
10. liC ~MRche~~~~ shiftsof (3). Entries I - 11 are the same as 2-. I in Figure 7.
less likely due to these obse~ations. These s~~nlations are in accord with the observations of Angyal et ai., concerning metal binding to D-galacturonate 1431. Also shown in Figures 8-10 (entry 2 in Fig. 8, 1 in Figs. 9 and 10) are the NMR data for the neutral free acid (3). These are very similar to the values for the Zn complex of the sodium salt and support the electrostatic model proposed above except for the marked shift for C6 upon protonation, not observed in the Zn complex. In order to be certain that in all experiments the carboxylic acid was completely ionized, we determined the apparent pK, to be 3.19 4 0. ! on the pH* scale. All spectra of salts were indeed recorded 2+ pli* units above this pK, (see Table 1). This pK, is markedly different from the value of 5, t reported for hduronic acid in heparin [#I. Our value is in accord with the literature values of 3.28 and 3.23 for D-glucuronic acid and 3.5 1 and 3.47 for D-galacturonic acid (45, 461. The discrepancy between the values for the polymer and the monosaccharides is probably due to the ~lyelectrolyt~ nature of heparin as the reported valnc is an ~~~pare~t pK, 1471. CONCLUSION We were not able to demonstrate metal binding to the O-sulphate group of (1) under conditions in which we did observe binding to (2) and (3). We could not quantitate the difference in binding strengths between izj and (3) but it is suggested that because (3) is more flexible it is able to adopt a geometry which creates a metal
METAL, BINDING TO HEPARIN MONOSACCHARIDES
169
binding site and hence has higher affinity for metals. This site is suggested to be the carboxylate oxygen and the ring oxygen 05 with the remaining sites occupied by anions or water. The conformational flexibility of L-iduronic acid has recently been proposed to be important for the biological activity of heparins [31]. The metal induced conformational shifts changes reported in this work could then represent a control mechanism for these biological functions. The authors would like to thank Dr. A. Grey of the NMR laboratory of the Carbohydrate Research Centre for assistance with the NMR spectra. The work was supported by the Medical Research Council of Canada, B. Sarkar, MT 1800, and to Carbohydrate Research Centre, MT 6499 and MA 9646.
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(1980). 19. J.-R. Brisson and J. P. Carver, Biochemistry 22, 3671 (1983). 20. S. Stojkovski, D. M. Whitlield, R. J. Magee, B. D. James, and B. Sarkar,
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Trans 2, 237 (1975). 39. T. Taga, T. Kaji, and K. Osaka, BUN. Chem. Sot. Jpn. 58, 30 (1985). 40. S. Thanomkul, J. A. Hjortas, and H. Sorum, Acta Crysr. B32. 920 (1976). 41. F. MO, T. J. Brobak, and I. R. Siddiqui, Carbohydr. Res. 145, 13 (1985). 42. L. Delucas, C. E. Bugg. A. Terzis, and R. Rivest, Carbohydr. Res. 41. 19 (1975). 43. S. J. Angyal, D. Greeves, and L. Littlemore, Curbohydr. Res. 174. 121 (1988). 44. J. W. Park and B. Chakrabarti, Biochem. Biophys. Res. Commun. 78. 604 (1977). 45. R. Kohn and P. Kovac. Chem. Zvesti 32, 478 11978). 46. H. Holvik and H. Hoiland, J. Chem. Thermodyn. 9, 345 (1977). 47. T. M. Fyles. J. Chem. Sot. Faraday Tram I82, 617 ~1986,. Received April 4, 1990; accepted June 26, 1990
