Heavy Metal Binding to Heparin Disaccharides. I. Iduronic Acid is the Main Binding Site DENNIS M. WHITFIELD,',* JEANCHOAY,' and BIBUDHENDRA SARKAR't

'Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1x8, and Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1 A8, and 'Sanofi Recherche, 9, Rue du President Salvador Allende, 94256 Gentilly-Cedex, France

SYNOPSIS

As model compounds for Ni (I1) -binding heparin-like compounds isolated from human kidneys (Templeton, D. M. & Sarkar, B. (1985) Biochem. J. 230 35-42.), we investigated two &saccharides-4-0- ( 2-O-sulfo-a-~-idopyranosyluronic acid) -2,5-anhydro-D-mannitol, disodium salt (la), and 4-0- ( 2-O-sulfo-a-~-idopyranosyluronic acid) -6-O-sulfo-2,5-anhydro-D-mannitol, trisodium salt & ()I -that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (la) was active whereas the disulfate (&) was inactive in a high-performance liquid chromatography ( HPLC ) binding assay with the tracer ions 63Ni(11) 54Mn( I1 ) , 65Zn(11), and "Cd( 11).This result is in accord with the isolation of two 67Cu(11) and 63Ni(11) binding fractions from a complete pool of nitrousacid-derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (la)and the other as a tetrasulfated trisaccharide by high resolution FAB-MS, NMR and HPLC-PAD. Similarly, two synthetic disaccharides-methyl, 2-0-sulfo-4-0-( a-L-idopyranosyluronic acid) -2deoxy-2-sulfamido-a-D-glucosamine, trisodium salt [ IdopA2S (a1,4) GlcNSaMe, @ I , and 2-0-sulfo-4-0-( a-L-idopyranosyluronic acid) -2-deoxy-2-sulfamide-6-O-sulfo-a-D-glucosamine, tetrasodium salt [ IdopA2S (a1,4)GlcNSGSaMe, Z b ] -were shown to bind tracer amounts of 63Niand 6"Cu using chromatographic assays. Subsequently, 'H NMR titrations of la,lb,2a, and & with Zn ( O A C )were ~ analyzed to yield 1:l Zn( 11)-binding constants of 472 k 59, 698 +- 120, 8,758 ? 2,237 and 20,100 k 5,598M-', respectively. The values for 2a and & suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 'H and 13Cchemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn( 11) ions.

INTRODUCTION Several workers have investigated the possibilities of metal complexation by carbohydrates, but the structural factors necessary for strong complexation remain unclear. It is our premise that oligosaccharides should form the strongest complexes due to their flexibility and number of binding Biopolymers. Vol. 32, 585-596 (1992) 0 1992 John Wiley & Sons, Inc.

CCC 0006-3525/92/0605S5-12$04.00

* Present address: Carbohydrate Research Centre, Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8. t To whom correspondence should be addressed.

sites. Most monosaccharide^^-^ do not have sufficient binding sites to fill a metal coordination sphere, whereas polysa~charides~~~ are often too rigid to form the required coordination geometries. The study of metal complexation to oligosaccharides was given further impetus by the observation that a low-molecular-weight fraction from human kidney was capable of binding radioactive 63Ni( 11)and 67Cu( 11).7 Subsequent studies have shown this fraction to contain sulfate, iduronic acid, glucuronic acid, and glucosamine, strongly suggesting the presence of heparin-like fragments: To investigate this novel finding, we studied the structural requirements for metal ion binding to model compounds containing iduronic acid. Our initial studies with monosaccharides re585

586

WHITFIELD, CHOAY, A N D SARKAR

lated to heparin strongly implicated the importance of the iduronic acid residues and not the glucuronic acid or the sulfated 2-acetamido-2-deoxy-glucosamine residue^.^ For example, we have shown that the monosaccharide sodium, methyl-a-l-idopyranosiduronate ( 5 ),binds Zn (11) > Pb (11) > Cd (11) > Ca( 11) % K ( I ) by observation of 13C and 'H nuclear magnetic resonance (NMR) chemical shifts (6) and 'H NMR coupling constants ( J )and nuclear Overhauser enhancement (NOE) changes. This NMR data was interpreted to indicate that the 'C4 chair conformation of the IdopA ring was stabilized by cations and that Zn ( 11)bound to the carboxylate and the ring oxygen. In this report, we will confirm the importance of idopyranosiduronic residues for binding to heavy metals like Ni (II), Zn (11), Cu (11), Mn(II), and Cd(I1) in disaccharides related to heparin.

MATERIALS AND METHODS Materials

Radioactive 63NiC12and aqueous counting scintillant were purchased from Amersham Corporation (Oakville, Ontario). 54MnC12,109CdIz,and 65ZnC12were obtained from New England Nuclear Corporation (Lachine, PQ). These last two isotopes were converted to acetates before use by dissolving the stock solution in acetate buffers. 67CuC12was supplied by Los Alamos National Laboratory (Los Alamos, NM) .Buffers were of analytical grade and the water was low conductivity from an ion-exchange system. N-Ethylmorpholine ( Aldrich-Terochem, Toronto, Ontario) was redistilled before use. Ultrapure HCl (J. T. Baker, Toronto, Ontario) was used for adjusting the pH of buffers. Heparin-derived disaccharides were obtained from Sanofi RechercheCentre Choay, France: disaccharide pool (IC 85 1670, batch P 58 E X H ) , 4-0- (2-O-sulfo-a-~-idopyranosyluronic acid-2,5-anhydro-D-mannitol, disodium salt (la) (IC 85 1670 C, batch BC 10-190 acid) -6C ) ,4-0- (2-O-sulfo-a-~-idopyranosyluronic O-sulfo-2,5-anhydro-D-mannitol, trisodium salt (IC 85 1670 A, Batch BC 10-190 F ) . Disaccharides 2-0-sulfo-4-0-( a-L-idopyranosyluronic acid) -2deoxy-2-sulfamido-a-~-glucosamine, trisodium salt LIdopA2S (a1,4)GlcNSaME, 2 a ] and 2-0-sulfo4 - 0 - (a-L-idopyranosyluronic acid) -2 -deoxy-2sulfamido-6-O-a-D-glucosamine, tetrasodium salt [ IdopA2S ( 4 4 ) GlcNSGSaMe, 2 b ] were synthesized at Sanofi Recherche-Centre Choay, France [ Jaurand, G., Tabeur, C. & Petitou, M. (to appear) 1.

(a)

Metal Binding Assays

To a plastic centrifuge tube (1.5 mL) was added sequentially aqueous aliquots of the radioactive tracer, the oligosaccharide, and the buffer, N-ethylmorpholine-HC1, pH 7.0-7.5, or ammonium bicarbonate, pH 7.6. This solution was equilibrated overnight in the cold room, centrifuged in a desk top centrifuge, and aliquots taken either for high-performance liquid chromatography (HPLC ) or gel permeation chromatography (GPC 1. HPLC was performed on a Waters 510 HPLC equipped with an automated gradient controller and 481 spectrometric detector using a Waters 1-60 gel permeation column (4.5 X 30 cm) in either 20 mmol ammonium bicarbonate, pH 7.6, or 10 mmol Nethylmorpholine HC1+ 50 mmol NaC1, pH 7.4 buffers. A flow rate of 1 mL min-l and UV spectral monitoring at 210 nm was used. One-minute fractions were collected and counted for radioactivity using a liquid scintillation @-counter(Beckman LS 8100) for 63Ni or a y-counter (Beckman Gamma 8000) for 67Cu,65Zn,54Mn,and lo9Cd. Gel permeation was performed on a Biogel P-2 (Bio-Rad, Mississauga, Ontario) column (4.5 X 70 cm) or a G-10 superfine (Pharmacia) column (4.5 X 70 cm) using water as eluant. The void volume was determined using blue dextran and the column volume with NaCl ( AgN03 test) fractions containing 50 drops were collected and analyzed for radioactivity. Carbohydrate was measured either using the phenol/ sulfuric acid test or the dimethylmethylene blue ( Serva-Terochem, Toronto, Ontario) test."

FPLC A Pharmacia (Etobicoke, Ontario) FPLC system using a fixed 214-nm wavelength detector and a Pharmacia MonoQ HR 5 / 5 strong anion exchange column was used. The flow rate was 1.0 ml min-'. Initial separations used 20 mmol Tris-HC1 (Fisher, Don Mills, Ontario), pH 7.4, and linear gradients from 0-1M NaC1. Subsequently, the starting buffer A was 5 mmol KH2P04,pH 4.8, and the final buffer B was 5 mmol KHzP04,pH 4.8, plus 133.3 mmol NazS04.A typical gradient was A for 0-8 min, to 85 15% B over 4 min, hold at 85% A 15% B for 10 min, increase to 100% B over 20 min, and then hold a t 100% B for 20 min. As much as 10 mg of oligosaccharides can be separated in one run under these conditions. However, it should be noted that extra salt in the injection sample seriously deteriorates the separation. As well, the length of equili-

+

+

HEAVY METAL BINDING TO DISACCHARIDES. I

bration in the starting buffer and the amount of sample injected alter the retention times. Thus, it is necessary to optimize the gradient for every sample. NMR and Mass Spectroscopy

'H NMR (300.133 MHz or 500.136 MHz) and 13C NMR (75.469 MHz) spectra were recorded with a Bruker AM 300- or 500-MHz Spectrometer at the Carbohydrate Research Centre, University of Toronto. They were obtained a t 300 K in D2O (99.98, Merck, Sharpe and Dohme, Montreal, Quebec) with a trace of acetone ('H 2.225 ppm relative to internal DSS or 13C30.5 ppm relative to dioxane at 66.8 ppm) as internal standard. Samples for NMR analyses in DzO were passed through a Chelite N (Bio-Rad, Mississauga, Ontario) ion exchange resin in H 2 0to remove paramagnetic impurities, lyophilized, and then lyophilized three times with DzO. NaP04 buffer, pH 7.5, and Zn ( OAc)z. ( HzO)zwere lyophilized from D20 before use. Zinc acetate was added as small aliquots (5-20 pL) of aqueous solutions (300-500 mM) directly to the NMR tube ( 5 mm) using a micropipette or in some cases directly as a solid. The stability constant determination was made using the sodium salts of l a (7.2 mg), & (6.4 mg), (1.6 mg), & (3.0 mg), and Zn ( O A C ) ~This . salt did not promote precipitation and allowed for direct determination of the Zn( II)/sugar mol ratio by integration of the acetate signal and the sugar resonances with interpulse delays > 10 s to allow for complete relaxation. The initial sugar concentration was calculated from its analytical weight and the solution volumes. Its value was checked by comparing it to the value determined from the Zn ( O A C )concentration ~ and the mol ratio determined by NMR; discrepancies were less than 10%. Errors in measured chemical shifts are estimated to be k 3 Hz and errors in integration are estimated to lead to errors in mol ratios of +-5%. Equilibrium constants were determined by curve fitting the NMR data using a self-written basic program NMRTITM that iterates for the equilibrium constant and the chemical shift of the 1:l complex. Corrections for Zn (11)binding to the acetate counter ion were made using the basic program ALPHA.lZ Both programs were run on a 386 20-MHz PC. Overhauser enhancements were measured in the lDdifference mode- The partially relaxed spectrum were run using the Bruker inverSion-recoverY Program with variable delays. Twodimensional 'H--'H COSY experiments were run using standard Bruker software and the 13C-lH 2D

587

COSY spectra were obtained in the inverse mode.13 TOCSY and ROESY 2D experiments used the decoupler for spin locking. Typical conditions were 32 scans per 512 experiments, 1.5- to 2.0-s delays with 90' pulses of about 90 ps for ROESY and 50 ps for TOCSY, and mixing times of 600 ms for the ROESY. 2D spectra were processed using the HARE software package ( Hare Research, Woodinvale, WA ) . Spectral simulations used the program LACOON. Fast atom bombardment mass spectra (FAB-MS) were recorded in the negative ion mode with a VG Analytical ZAB-SE spectrometer a t the Carbohydrate Research Centre, University of Toronto. The samples, which were dissolved in water and loaded onto to the target containing triethanolamine matrix, were bombarded by Xe atoms generated using an Ion-Tech Gun (8 keV, 1.2 mA anode current). In low-resolution mode, mass spectra were acquired using a VG11/250 data system under multichannel analyzing mode (MCA) at unit resolution and exponentially (magnetic) scan between m/z 1500 to 100. The exact mass values were determined either using accelerating voltage scan or peak matching at high resolution (6-8000, 10%valley definition). In the accelerating voltage scan, the matrix containing the sample was also mixed with polyethylene glycol (PEG, 2% concentration). Mass spectra of a narrow range (100 amu) with three inclusive PEG internal reference ions were acquired to determine the exact mass values of the sample ions. In the peak matching

T i . min

Figure 1. SAX-FPLC (Tris-HC1 and C1- gradient) 0.D. (214 nm) trace of a 63Ni (11) -binding heparin &saccharide fraction previously purified by recycling twice through HPLC (1-60 gel permeation) followed by P-2 gel permeation, keeping the radioactive fractions. The peak with the radioactivity corresponds to la;note the approximately 30-s delays between the detector and the collector.

588

WHITFIELD, CHOAY, AND SARKAR

Table I 500-MHz 'H NMR Data for Oligosaccharides Za, Zb, 2a,2b, 3, and 4

Residue

H1

H1'

H2

H3

H4

H5

H6

H6'

JlZ

J11'

Jlz.

J23

J34

J45

J56

J56'

CH3 J66

la IdopA2S AnManOH

-

5.114 (2.8) 3.760 (3.4)

(0.8)" 3.692 (-12.4)

3.939 (5.8)

4.019 (3.9) 4.175 (6.7)

56.5 (2.0) 0.0 (3.3)

(1.1)a 0.3 (-12.4)

18.5 -5.2 (5.7)

27.4 (3.6) 13.8 (7.1)

5.144 (2.4)

(0.8)'

(3.2)

3.765 (-12.5)

4.211 -

4.011 (3.8) 4.128 (5.3)

4.581 (2.2) 4.082 (6.3)

-

(1.3)b 3.792 (3.6)

3.738 (5.6)

-

(-12.4)

+

la

Zn(OAc)z shifts in Hz IdopA2S

AnManOH lb IdopA2S AnManOH6S

lb

4.233 -

3.692 (5.6)

4.041 (4.2) 3.986 (6.6)

15.5 (3.7) 26.4 (5.6) 4.015 (4.1) 4.178 (5.1)

38.3 (2.3) 4.8 (6.2)

(0.8)' -5.2 (3.6)

4.593 (2.4) 4.194 (6.0)

(0.8)' 4.283 (2.9)

50.7 (2.2) -0.5 (5.8)

(o.9)b -0.2 (1.9)

-

-

-

4.254 (5.8)

4.205 (-11.0)

3.9 (5.6)

(-11.4)

+

Z ~ ( O A Cshifts ) ~ in Hz IdopA2S

AnManOH6S

70.4

(1.7) 3.7 (3.2)

-

(0.9)' -1.7 (-12.5)

-

-6.5 (5.5)

19.9 (3.4) 10.9 (7.0)

36.1 (3.5) 23.0 (4.6)

-

2a IdopA2S GlcNS 2a

+

5.119 (2.3) 5.024 (3.6)

-

4.226 3.255

-

-

(0.7)"

Zn(OAc)z shifts in Hz IdopA2S

4.023 (3.8) 3.660 (10.1)

3.972 (3.9) 3.710 (8.6)

41.0 (2.8) 38.8 (10.1)

48.0 (3.0) 22.8 (8.8)

4.030 (3.6) 3.736 (10.1)

3.975 (3.7) 3.675 (8.8)

-

67.4 (2.8) 15.7 (10.1)

40.2 (3.1) 38.9 (8.8)

3.549 4.420 -

3.696 (7.8) 4,263 (3.0)

3.875 (6.6) 3.928 (6.3)

48.0 -21.1

GlcNS

-

4.746 (2.5) 3.761 (9.6)

(0.7)' 3.841 (2.2)

76.8 (1.8) 15.0 (9.6)

5.9 (2.2)

4.721 (2.2) 4.004 (9.7)

(0.6)' 4.340 (2.3)

-

3.891 (4.3) -8.6 (4.0)

3.405 (-12.3) 2.7 -

(-12.1)

2b IdopA2S GlcNS6S 2b

5.142 (2.6) 5.020 (3.6)

-

4.248 3.285

-

-

(0.8)" -

45.7 -18.0

-

(0.5)"

-

-

4.301 (4.9)

3.418 -

(-11.3)

+

Zn(OAc)z shifts in Hz IdopA2S GlcNS6S

115.0 (1.7) -11.7 (3.5)

94.3 (2.0) 6.5 (9.6)

-

-

13.8 (2.1)

-

-25.2 (4.9)

2.9 (-11.5)

3 IdopA GlcNAcCOOH

-

-

4.523 (4.5) 4.111 (4.6)

-

-

-

-

4.282 (3.0)

4.170 (7.7)

-

2.086 (-10.6)

HEAVY METAL BINDING T O DISACCHARIDES. I

Table I

589

(Continued)

Residue

H1

H 1'

H2

H3

H4

H5

H6

H6'

Jl2

J11'

Jiz*

523

J34

54s

Js6

JS6'

CH3 JW

4

IdopA2S

GIcNSGS IdoA5SOH

5.191 (2.6) 5.420 (3.7)

-

(0.9)" -

4.31' 3.257 -

-

-

4.32'

-

-

-

4.101 (3.5) 3.738 (10.1) 4.32" -

3.985 (3.4) 3.786 (9.1) 4.239 (2.7)

4.818 (2.6) 4.025 (9.3) 4.565 (7.8)

(0.9)b 4.27' -

4.007 (4.7)

-

4.34" 3.936 (6.7)

-

(-11.9)

Chemical shifts in ppm and coupling constants in Hz. a 513. 524.

' Overlapped resonances marked with the same footnote letter.

mode, samples were introduced into the mass spectrometer via a wobble probe that had a dual target containing CsI as reference and the other containing the sample and triethanolamine.14 HPLC-PAD High-performance liquid chromatography with a Dionex Bio LC system with pulsed amperometric detection (HPLC-PAD) of the hydrolyzed monosaccharides was performed using a Dionex HPLCAS6 separating column (250 X 4 mm i.d.) with a guard column, Dionex HPLC-AG6 ( 5 0 X 4 mm i.d.) equipped with a 25-mL sample loop system, and a flow rate of 1.0 mL/min. Conditions were: gradient elution, eluant A (50 mM NaOH) and eluant B (50 mM NaOH/150 mM NaOAc mixture); detector settings, E l = 0.05 V, E2 = 0.60 V, E3 = -0.80 V, t l = 120 ms, t2 = 120 ms, t3 = 300 ms; working electrode, gold; reference electrode, silver-silver chloride; output range, 3K n Amp fill scale; chart speed, 0.5 cm/min. The eluants were protected from the atmosphere with an He module degasser. A Spectra-Physics ( SP4270) integrator was used to analyze the output. Samples with mannitol as internal standard were prepared by hydrolysis ( 1 M HCI 4 h at 100°C) followed by lyophilization. The samples were redissolved in deionized water and directly injected in the HPLC."

RESULTS AND DISCUSSION Purification and Metal-Binding Analysis of Heparin Fragments

Our first experiments used a complete pool of disaccharides derived from heparin by nitrous acid using the HPLC assay for metal binding; two were

cleavage followed by reduction and p ~ r i f i c a t i o n . ~ ~ ~ ' ~ This pool contains mostly 1,4-linked disaccharides with the 2,5-anhydro-~-mannitol residue at the reducing termini and either p-D-glucopyranosiduronic acid or a-L-idopyranosiduronic acid at the nonreducing termini. The heterogeneity is greatly increased by varying degrees of 0-sulfation. Initial HPLC and gel permeation binding assays (see experimental ) demonstrated specific metal binding with "Ni (II) and 67Cu(11), i.e., radioactivity was recovered in tubes corresponding to O.D. associated with some but not all carbohydrates. However, these chromatographic techniques were unable to completely separate the binding components from the nonbinding ones. Therefore, an ion-exchange procedure similar to some published procedures was used." Figure 1 shows an FPLC trace of the radioactive fractions, previously isolated by HPLC followed by gel permeation. In this case, chloride gradients in a TrisHC1 buffer on a MonoQ column were used. As shown in Figure 1, one of these peaks also retained some radioactivity, demonstrating that it is the metal binding component.8 This binding on the anion exchange column indicates the strength of the interaction since the quaternary ammonium-charged sites of the resin were not able to displace the 63Ni(11)ions. The NMR spectrum of a fraction corresponding to this peak demonstrated that the major species is la. Subsequently, a system using sulfate gradients in dilute KHzPOl buffer on a MonoQ column was developed? Aliquots of the complete disaccharide pool were applied. All fractions were desalted using a Sephadex G-10 column in water, keeping sugar-positive fractions. Subsequently, fractions were tested

690

WHITFIELD, CHOAY, AND SARKAR

OR

?

AH

bH Figure 2. lb, R = H -

bS0;

bso; Chemical structures of R = H la,R =

a,and R = -SO3 3.

-so3

active. One was the fraction corresponding to compound and the second was the most charged compound, 4. By high-resolution FAB-MS, an exact mass

(961.8898) was obtained for 4,which corresponds to the formula, C18H25N029S4Na5. This corresponds to a tetrasulfated trisaccharide with two more protons than expected for two iduronic acid and one glucosamine residues. Furthermore, the fragment ions at m/z 662, 660, and 319 are consistent with one sulfate on each of the two terminal residues and two sulfates on an internal hexosamine. Thus, the following structure for the trisaccharide was proposed, 4 - 0 - ( 2-O-sulfo-a-~-idopyranosyluronic acid) -3-0-( 2-deoxy-2-sulfamido-6-0-sulfo-a-Dglucopyranosyl ) -5-0-sulfo-D-idonic acid, hexasodium salt, 4.The presence of glucosamine and iduronic acid were confirmed by HPLC-PAD analysis of the acid hydrolysates of 4.15It should be noted that NaBH4 reduction of 2-0-sulfo-~-iduronic acid gives 5-0-sulfo-D-idonic acid. This structure was substantiated by NMR analysis (see Table I ) . The three spin systems of the monosaccharides were established by 'H COSY and TOCSY 2D spectra. The connectivitives between the monosaccharides were determined by 1D NOE difference spectra. Other workers have reported side reactions in the nitrous acid reduction of 2-amino-2-deoxy-pyranosides but these involve competing rearrangement~.l~-~l Compound 4 is proposed to form by acid hydrolysis of the IdopA2S-a-1,4-bond in heparin and then subsequent borohydride reduction.

limo, min

Figure 3. SAX-FPLC ( KH2P0, and SO4gradient) O.D. (214 nm) trace of impure fraction of la.The major O.D. peak is 3 while the other large peak is la.Approximately 10 mg of sample was injected.

HEAVY METAL BINDING TO DISACCHARIDES. I

With la,the apparent selectivity as based on the percent recoveries of added radioactivity is: Ni(100%)> Zn(58%)> Mn(37%)> Cd(5%).The disulfated compound, &, which corresponds to the major disaccharide found in heparin, was tested and was inactive ( < 1 % ) with all metals tested. The structures of & and & are shown in Figure 2. But the active fraction containing lawas impure. Further purification by the sulfate gradient method (MonoQ) produced the separation shown in Figure 3. This result demonstrates the efficiency of the sulfate gradient method as la had already been purified by FPLC using chloride gradients on a MonoQ column. The major O.D. peak was desalted and subjected to FAB-MS, NMR, and the HPLC metal binding assay. To our surprise, this compound was not the expected disaccharide. Its structure is still uncertain, but examination of its 'H NMR spectrum reveals an acetate resonance and two monosaccharide units; one is unsulfated iduronic acid and the other has a six-proton spin system C2HC3HC4HC5HC6Hz. The large coupling constants for the IdopA residue (see Table I) suggest population of either the 4C1or 'So conformation.'' An 'H ROESY 2D spectrum demonstrated an IdopA-a-1,4-linkage but no crosspeaks to the acetate methyl were observed. High-resolution FAB-MS gave an exact mass of 492.0666 that corresponds to a formula of C14H22N016S'Na3. This data is consistent with the structure 4-0- (a-L-idopyranosyluronic acid) -6-Osulfo-2-deoxy-2-acetamido-D-gluconic acid, trisodium salt, 3. This structure requires that the aldehyde group of the ring-opened form has been oxidized to a carboxylic acid. This compound is likely an artifact of the nitrous acid reaction and is probably not found in native heparin. The metal binding assay with 3 and 63Ni(11)was very weakly positive. Close examination of the spectra of the mixture (see X s in Figure 4a) shows the presence of this compound 3 in the mixture and so it is not an artifact of the purification process. These results show the danger of relying on O.D. for the purification of acidic sugars like & and &I since these compounds have negligible O.D. at convenient wavelengths. It is the acetamide carbonyl of 3 that contributes most of the absorption observed at 214 nm in Figure 3. The other major O.D. peak showed metal binding activity. Spectral analysis revealed this to be compound la. A tentative conclusion that can be drawn from these studies is that an IdopA-a-group is essential for binding. These results prompted a more detailed NMR study of these two disaccharides, & and &I.

591

M6M6

12

I1

M4 I314 M2

M3

M1'

M1

I

MS

.

54

52

50

48

46

44

42

40

38

7

36

34

3.8

3.6

PPM

5.4

5.2

5.0

4.8

4.6

4.4

4.2

4.0

PPM

Figure 4. ( a ) 'H NMR spectra (500.137 MHz) of & with (top) and without (bottom) the addition of 3.1 equivalents of Zn ( OAc ) z . The lines between the spectra indicate the chemical shift changes. The resonances marked with X s correspond to 3. ( b ) 'H NMR spectra (500.137 MHz) of with (top) and without (bottom) the addition of 4.2 equivalents of Z ~ ( O A C )The ~ . lines between the spectra indicate the chemical shift changes.

592

WHITFIELD, CHOAY, AND SARKAR

These results also prompted us to study binding to the synthetic disaccharides from which they are derived. Metal Binding to Synthetic Heparin Disaccharides

Heavy metal binding to two synthetic disaccharides, namely, that have D-glucosamine residues at the reducing termini was investigated for structures, see Figure 2. Chromatographic assays using an HPLC assay (Waters 1-60 gel permeation column) and 63Niwas positive for both and &. That is radioactivity was recovered in fractions that also contained sugar. Similarly, 67Cuwas shown to bind to both and by finding comigration of radioactivity and sugar on a Sephadex G-10 gel permeation column (see Material and Methods for details). These findings are similar to those found with the kidney-derived oligosaccharides and disaccharide la.'

m,

a

Analysis by NMR

These positive metal binding results prompted us to study diamagnetic Zn (11) binding by titrating the oligosaccharides with Zn ( O A C )in ~ an NMR tube and monitoring the 'H and 13CNMR spectra. Zn (11) was chosen as probe ion because it is diamagnetic and was shown to produce the largest shifts with the monosaccharide 6, A preliminary NMR experiment involved adding excess Zn ( O A C ) ~as, a solid, directly to a phosphate-buffered solution of in D,O. This led to marked spectral changes very similar to those discussed below, but some unidentified precipitate also formed and therefore all subsequent spectra used unbuffered solutions. The apparent pH of all disaccharide solutions was >5.5 before or after addition of the Z ~ ( O A C )well ~ , above the pKA of 3.19 f 0.1 of methyl a-L-idopyranosiduronic acid, 5.' Thus, all binding is to completely ionized carboxylates or sulfates.

Table I1 75.5-MHz 13C NMR Data for Disaccharides la, lb, 2a,and 2b ~

Residue

la IdopA2S AnManOH la

c1

c2

c3

c4

c5

99.54 61.65

75.65 81.80

70.04 75.72

69.64 85.18

69.42 82.97

C6

175.35 61.31

+

Zn(OAc)zshifts in Hz IdopA2S AnManOH

1b IdopA2S AnManOH6S

-3.9 -9.3 99.82 61.05

-66.3 12.5 75.23 83.33

-52.7 -26.3 69.53 75.38

-32.8 -48.0 69.72 85.36

-47.0 -22.8 69.25 79.73

-73.2 5.6 176.05 68.32

+

1b Zn(OAc)2shifts in Hz IdopA2S AnManOH6S

-17.5 -5.0

-25.4 -9.4

-54.5 -15.9

-60.9 -76.1

-52.5 -25.5

-11.4 -12.7

2a IdopA2S GlcNS

2a

75.08 58.20

69.67 70.02

69.48 78.14

69.26 70.83

176.45 60.34

+

Zn(OAc)zshifts in Hz IdopA2S GlcNS

2b IdopA2S GlcNS6S 2b

99.43 98.51 58.8 -7.3 99.37 98.43

-108.8 23.9 75.30 57.97

-73.7 1.4 69.75 70.15

-77.6 29.8 69.75 78.04

-82.3 -6.5 69.86 68.69

-27.8 -9.5 176.61 67.20

+

Zn(OAc)2shifts in Hz IdopA2S GlcNS6S Chemical shifts in ppm.

61.5 5.6

-119.1 29.6

-108.0 2.2

-69.5 22.8

-104.6 5.3

-43.0 -18.2

CHB

HEAVY METAL BINDING T O DISACCHARIDES. I

The initial and final spectra from NMR titrations of la and & with aliquots of the diamagnetic metal ~ shown in Figures 4a and 4b. The salt Zn ( O A C )are corresponding chemical shifts and coupling constants are shown in Table I. As is readily apparent, marked spectral changes for the IdopA2S resonances are observed for both disaccharides. The AnManOH resonances for H3 and H4 also show shifts, but only small shifts for the other AnManOH resonances are observed. The addition of Zn ( I1) also produced I3C NMR shifts to the IdopA2S resonances and to C3 and C4 of the AnManOH residue, as shown in Table 11. The 'H spectra were assigned by conventional homonuclear decoupling at 500 MHz. For la,all signals were sufficiently well dispersed that assignments were readily made, except that IdopA2S H3 and H4 overlapped in the metal free spectra. This overlap causes virtual coupling that necessitated the use of spin simulation using the program LACOON to determine the coupling constants for this resid ~ e . Zn 2 ~( 11)binding lifted this degeneracy, abwing for first-order assignments. For lb,several peaks overlap in the region of 4.3-4.15 ppm. Fortunately, specific partially relaxed spectrum allowed for the assignment of these resonances. This technique takes advantage of the differential relaxation times of the different protons. For example, the methylene protons AnManOH H6 and H6' and the aglyconic proton AnManOH H4 all have shorter relaxation times than the other protons. Once the 'H spectra were assigned, the 13C spectra were assigned using 'H-detected 13C-'H COSY 2D spectra. For both disaccharides, a shift toward the 'C4 conformation is suggested by the smaller 'H-'H coupling constants.' However, even the metal free spectra are consistent with a predominant 'C4 conformation with some 2Soor 4C1conformers present (see Table I). A previous analysis of the coupling constants of & was interpreted as a 78 : 22 mixture of the 'C4 and 4C1 conformation^.^^ This interpretation is different from the single conformation proposed by Huckerby et al.,25who first reported the NMR data for &, although the same NMR data are entirely consistent with those of ours. Since the major effects are to the IdopA2S resonances, it is tempting to suggest that this is the main binding site with metal coordination at the carboxylate. Metal binding to sugar carboxylates has been reported p r e ~ i o u s l y . ' Also, ~ ~ ~ the H6 and C6 resonances of the AnManOH are scarcely affected and therefore it seems unlikely that these groups-6OH or 6-0-sulfate-are directly involved in metal binding.

593

Figure 5 shows the results of the same NMR experiment with disaccharide & and the chemical shifts in the Zn(I1) free spectra all set to 0 Hz. Marked chemical shifts for all IdopA2S resonances were observed, with only small shifts for most GlcNS6S resonances. The maximum chemical shifts along with the starting chemical shifts and the coupling constants are shown in Table I. Since the 'H spectra of both @ and & are essentially first order, the assignments were readily made by conventional decoupling techniques. Comparing & to &J shows that the IdopA2S shifts are comparable but there are significant differences in the GlcNS ( 6 s ) shifts. For &,the GlcNS6S H4 and H6 resonances exhibit the largest shifts, whereas for H3 and H4 show the largest shifts. The initial and final 'H NMR spectra for & and & are shown in Figures 6a and 6b, respectively. The Zn ( 11)-induced shifts are marked by the lines joining the resonances in Figure 6ab. 13CNMR spectra of the initial and final samples for the titration were also measured. Again, the carbon resonances of the IdopA2S carbons show marked shifts and the C4 and C2 resonances of GlcNS ( 6 s ) for both @ and & show shifts as does the C6 carbon of &. All Zn (11)-induced 13C shifts are reported in Table I1 with the chemical shifts of the Zn( 11)free compounds. The 13Cresonances were assigned using 'H-detected 13C-lH COSY spectra. 140

120

s

100

20

1-

__

0

. -2 . .

05

I

I

I

I

I

I

15

2

25

3

Zinc Acetate I Sugar Figure 5. Chemical shifts in Hz during a 'H NMR titration of & by Zn( O A C ) ~The . Zn(I1) free spectra has its chemical shifts defined as 0 Hz. (m), IdopA2S H1; (+) IdopA2S H2; ( * ) , IdopABS H5; ( 0j , GlcNSGS H1; ( x), GlcNS6S H3; ( 0) ,GlcNS6S H4; (A),GlcNSGS H6; (0) , GlcNSGS H6.

594

WHITFIELD, CHOAY, AND SARKAR

Gi

I1

HDO

I5

12

GS'GSG.5

13 14

ion binds weakly to Zn (11) and this was corrected for by using the known binding constants?' The corrected, including dilution effects, [ Zn (11)] was then used to fit the titration data by iterating for the chemical shift of the complex dcomp and the equilibrium constant, assuming 1 : 1stoichiometry. The data was assumed to fit the equation dabs = ( [ZnSug]/[Sugli) X dcomp, where dabs is the observed chemical shift in Hz, [ ZnSug] is the concentration of the complex, and [ Sugli is the initial sugar concentration. The results of such a fit are shown in Figure 7 for selected resonances of compound In Figure 7, the curves are calculated and the points are the experimental ones. The equilibrium constants are reported in Table I11 as averages of all the resonances for which fits were determined +SD. Also included in Table I11 is the previously determined constant for monosaccharide 6. Since the complexation constants for and & are at least one order of magnitude larger than for 5, la,and l b , then and zf2 must be chelating the Zn (11). Metal binding to the monosaccharide 5 stabilized the 'C4 conformation. Since the IdopA2S residues in la and & are already mostly in the 'C4 conformation, this factor is likely the cause of the higher binding to la and & as compared to 5, rather than chelation. Since 5 is not sulfated on the 2-OH as in

a.

! ! 54

52

50

48

46

44

42

40

38

36

34

32

34

32

PPM

GI

I1

15

10

G6'12 G613

14G5

CH

G4 G3

I

1! 54

52

50

48

46

44

42

40

38

36

PPM

Figure 6. ( a ) 'H NMR spectra (500.137 MHz) of with (top) and without (bottom) the addition of 2.85 equivalents Zn(OAc)*. ( b ) 'H NMR spectra (500.137 MHz) of with (top) and without (bottom)the addition of 2.93 equivalents of Z ~ ( O A C ) ~ .

To quantitate the titration data, the amount of added Zn (11)was determined by integrating the acetate methyl resonance against several sugar resonances whose concentration is known. The acetate

0

0.5

1

1.5

2

2.5

Zn(I1) / sugar Figure 7. Calculated chemical shifts in H z (solid curves) for the 'H NMR titration of by Zn (11) compared to experimental points. The estimated error in chemical shifts is k 3 H z and in the mol ratios of +5%. The Zn (11)free spectra has its chemical shifts defined as 0 Hz. The [ Zn (11) ] have been corrected for binding to acetate. Points are shown for: (m), IdopA2S H1; ( A ) , IdopAZS H3; ( *) , IdopAZS H5; and ( X ) , GlcNSGS H4.

HEAVY METAL BINDING TO DISACCHARIDES. I

Table I11 Zinc Binding Constants for L-Iduronic-Acid-Containing Oligosaccharides

K M-' k SD Compound

(# of resonances)

c~MeIdopA(5) IdopA2S(c~1,4)AnManOH( la) IdopA2S(a1,4)AnManOH6S(l b )

177k 15 (5) 472+ 59 (7) 698+ 120 (5) IdopA2S(crl,4)GlcNSaOMe(2u) 8,758 ? 2,237 (6) IdopA2S(al,4)GlcNS6SaOMe(2b) 20,1005 k 5,598 (7)

595

charide heparin pools and found specific metal binding activity (unpublished observations). These findings are consistent with the reports of a highaffinity Zn (11) binding site on porcine intestinal heparin34and a high-affinity Ni (11)binding site on purified bovine glomerular basement membrane 35 by equilibrium dialysis techniques. Our results strongly implicate L-iduronic acid residues as part of any such heavy metal binding sites. and esThe combined data for disaccharides pecially strongly support the hypothesis that these molecules can chelate metal ions. Thus, disaccharides and & are plausible models for the iduronic acid and glucosamine containing heavy metal binding oligosaccharides isolated from kidneys. Furthermore, these groupings are good candidates for the Zn ( 11),34,36,37 C U ( I I ) , ~ and ~,~~ Fe( 111)33 binding sites of heparin. For example, it has been shown that for oxygen-based ligands a linear relationship exists between the logarithms of the ligand-metal ion binding constants and the hydroxide-metal ion binding constant^.^' If this relation holds for and then these sugars should bind Cu (11) and Fe( 111) better than they bind Zn ( 11).

a

a

l a and lb,this suggests that this 2-0-sulfate group is not important for binding. A satisfactory treatment of the NMR data requires molecular modeling of the IdopA ring inversion^^^-^^ and the interglycosidic conformations. Ragazzi et aL31 showed that at least the lC4, 4C1,and 'So conformations must be considered. Very little has been reported about the conformations of the IdopA2s-a-1,4-AnManOH glycosidic linkage or the AnManOH conformation. The results of such calculations for la, lb, 2a, and are presented in the following paper. Our analysis of Zn ( I1) binding to 5 led us to propose a model with binding to the carboxylate oxygen and the ring oxygen. If this model is correct and if the Zn (11) coordination sphere is tetrahedral, then the two other sites must be occupied by water or acetate ligands. The discrepancy between almost equal 1: 1binding between la or j& and Zn (11) and the marked preference for binding to & in the radioactive tracer studies (metal : sugar < 1 : 100) can be explained by assuming that the radioactive tracer studies involve 1 : 2 or higher stochiometries for binding. Thus, and not should be able to form stable multivalent complexes with heavy metal ions. In support of this interpretation is the recent observation that Fe( 111)32 and Cu (11)33 bind to intact heparin a t low metal to polymer mol ratios but at the same mole ratios lb does not bind Cu (11).At higher concentrations of metal, did show Cu (11) binding.

CONCLUSION The disaccharide la has been shown to bind some heavy metals in radioactive tracer assays, with a preference for the harder metals Ni ( 11) % Cd (11). One-to-one binding to Zn(I1) leads to marked NMR changes of the IdopABS ring for la,lb, 2a, and We have also tested tetrasaccharide and hexasac-

a.

a

a,

The authors thank Drs. A. Grey and H. Pang for their assistance in NMR and MS analyses, respectively. This work was supported by the Medical Research Council of Canada, B. Sarkar M T 1800, and Sanofi Inc., France. Funding for the Carbohydrate Research Centre, University of Toronto, is from the Medical Research Council of Canada MT 6499.

REFERENCES 1. Kohn, R. (1987) Carbohydr. Res. 160,343-353. 2. Riahi, H. T. (1986) J. Inorg. Biochem. 26, 23-33. 3. Aruga, R. (1981) Bull. Chem. SOC.Jpn. 54, 12331235. 4. Cook, I. B., Magee, R. J., Payne, R. & Ternai, B. (1986) Aust. J . Chem. 39,1307-1314. 5. Lages, B. & Stivala, S. S. (1973) Biopolymers 12,127143. 6. Mukherjee, D. C., Park, J. W. & Chakrabarti, B. (1978) Arch. Biochem. Biophys. 191,393-399. 7. Templeton, D. M. & Sarkar, B. (1985) Biochem. J. 230, 35-42. 8. Predki, P. F., Whitfield, D. M. & Sarkar, B. (1992) Biochem. J. 281,835-841. 9. Whitfield, D. M. & Sarkar, B. (1991) J . Inorg. Biochem. 41,157-170. 10. Kobata, A. (1971) Adu. Enzymol. 28,262-271. 11. Farndale, R. W., Buttle, D. J. & Barrett, A. J. (1986) Biochim. Biophys. Acta 883, 173-177. 12. Ginzburg, B. (1976) Talantu 23, 149-153.

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WHITFIELD, CHOAY, PND SARKAR

13. Bax, A., Griffey, R. H. & Hawkins, B. L. (1983) J. Magn. Reson. 55,301-315. 14. Reinhold, V. N., Carr, S. A., Green, B. N., Petitou, M., Choay, J. & Sinay, P. (1987) Carbohydr. Res. 161, 305-313. 15. Whitfield, D. M., Stojkovski, S., Pang, H., Baptista, J. & Sarkar, B. (1991) Anal. Biochem. 194,259-267. 16. Bienkowski, M. J. & Conrad, H. E. (1985) J. Biol. Chem. 260, 356-365. 17. Huckerby, T. N., Sanderson, P. N. & Nieduszynski, I. A. (1986) Carbohydr. Res. 154, 15-27. 18. Green, E. D. & Baenziger, J. U. (1986) Anal. Biochem. 158,42-49. 19. Shively, J. E. & Conrad, H. E. (1976) Biochemistry 15, 3932-3950. 20. Erbing, C., Lindberg, B. & Svensson, S. (1973) Acta Chem. Scand. 27,3699-3704. 21. Horton, D. & Philips, K. D. (1973) Carbohydr. Res. 30,367-374. 22. Ferro, D. R., Provasoli, A., Ragazzi, M., Casu, B., Torri, G., Bossennec, V., Perly, B., Sinay, P., Petitou, M. & Choay, J. (1990) Carbohydr. Res. 195,157-167. 23. Brisson, J. R. & Carver, J. P. (1982) J. Biol. Chem. 257,11207-11209. 24. Casu, B., Petitou, M., Provasoli, M. & Sinay, P. (1988) Trends Biochem. Sci. 13, 221-225. 25. Huckerby, T. N., Sanderson, P. N. & Nieduszynski, 1. A. (1985) Carbohydr. Res. 138,199-206. 26. Cook, W. J. & Bugg, C. E. (1977) in Metal-Ligand Interactions in Organic Chemistry and Biochemistry, part 2, (Pullman, B. & Goldblum, N., Eds., D. Reidel Publishing Company, Dordrecht-Holland, pp. 231256.

27. Martell, A. E. & Smith, R. M. (1974) in Critical Stability Constants, Plenum Press, New York. 28. van Boeckel, C. A. A., van Aest, S. F., Wagenaars, G. N., Mellema, J. R., Paulsen, H., Peters, T., Pollex, A. & Sinnwell, V. (1987) Recl. Trav. Chim. Pays-Bas, 1 0 6 , 19-29. 29. Sanderson, P. N., Huckerby, T. N. & Nieduszynski, A. (1987) Biochem. J. 243,175-181. 30. Ferro, D. R., Provasoli, A., Ragazzi, M., Torri, G., Casu, B., Gatti, G., Lormeau, J. C., Sinay, P., Petitou, M. & Choay, J. (1986) J.Am. Chem. SOC.108,67736778. 31. Ragazzi, M., Ferro, D. R. & Provasoli, A. (1986) J. Comp. Chem. 7,105-112. 32. Rej, R. N., Holme, K. R. & Perlin, A. S. (1990) Can. J. Chem. 88,1740-1745. 33. Rej, R. N., Holme, K. R. & Perlin, A. S. (1990) Carbohydr. Res. 207,143-152. 34. Woodhead, N. A., Long, W. F. & Williamson, F. B. (1986) Biochem. J. 237,281-284. 35. Templeton, D. M. (1987) Toxicology 43, 1-15. 36. Sato, C. S. & Gyorkey, F. (1976) J. Biochem. 80, 883-886. 37. Parrish, R. F. & Fair, W. R. (1981) Biochem. J. 193, 407-410. 38. Grushka, E. & Cohen, A. S. (1982) Anal. Lett. 15, 1277-1288. 39. Evers, A., Hancock, R. D., Martell, A. E. & Motekaitis, R. J. (1989) Inorg. Chem. 28,2189-2195.

Received June 4, 1991 Accepted October 3, 1991