Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy
Received on October 29, 2001; revised on January 22, 2002; accepted on January 22, 2002.
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Abstract |
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Key words: glycopolymer/Linear B disaccharide/transglycosylation/xenotransplantation
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Introduction |
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The prerequisite for a successful xenotransplantation is the prevention of the anti-Gal Ab/-Gal interaction or, even better, the removal from the human plasma of the antibodies responsible of this interaction. The ideal goal could be to develop immunoaffinity columns of an
-Gal oligosaccharide capable of being used in an extracorporeal immunosorbent system to deplete anti-
-Gal Abs from the host. The
-Gal antigen should be covalently linked through a linking arm to a biocompatible solid support. The latter could be either in the form of a continuous large surface or as spherical particles. The main problem to solve is the availability of significant amounts of the
-Gal antigen. Chemical approaches for the synthesis of oligosaccharides have been developed extensively, but the available chemical methods are not completely stereospecific and need multireaction steps, which complicates the scaling up (Boons, 1996
). Transglycosylation synthesis are more promising and interesting because their activities can be modulated and they use noncomplicated sugars as glycosyl donors (Nilsson, 1988a
).
Modification of biologically active compounds with polymers is one of the methods for altering and controlling their biodistribution and, very often, toxicity (Monfardini and Veronese, 1998). One peculiarity of carbohydrateprotein and carbohydratecarbohydrate recognition is that the high affinity and specificity of these interactions are obtained by polyvalency. At the level of isolated molecules (monovalent interaction) the affinity (if any) is very low; it can be dramatically increased on linking several units to a single macromolecular carrier. The importance of the sugar density within the glycoconjugate for the specific interactions between the ligand (the oligosaccharide) and the receptor (the protein) is known as cluster effect. It was proposed for the first time by Lee (1992)
. The multivalent nature of cell surface carbohydrate may act cooperatively to increase the overall binding avidity of these interactions. Molecular design of water-soluble polymers containing biologically active oligosaccharides as side chains is gaining interest as one of the most effective methods to increase the density of that molecule. Glycopolymers (Sigal et al., 1996
) and glycodendrimers (Roy et al., 1993
) have been used to show that inhibitory potencies of glycosides are increased when carbohydrates are properly presented in a multivalent form. Conjugation of
-Gal epitopes onto a polyacrylamide backbone has been reported (Wang et al., 1999).
Poly(styrene co-maleic anhydride) (SMA) is a synthetic copolymer with interesting chemical and biological properties (Klumperman, 1994). The anhydride groups can be hydrolyzed to give poly(styrene co-maleic acid) (HSMA); they can also easily react with low molecular compounds to give modified SMA polymers. As a drug carrier, SMA has been used to deliver neocarzinostatin, a potent but very toxic antitumor protein having a short plasma half-life. Conjugation with SMA increases its plasma half-life and reduces protein toxicity (Maeda et al., 1985
). Recently, SMA has been used for immobilization of Laminin peptide YIGSR, resulting in an increase of the antimetastatic effect of the latter (Mu et al., 1999
).
In this study, we report that a significant improvement of the transglycolytic synthesis of the Linear B disaccharide as well as a speed-up of the purification procedures can be achieved. Moreover, anchoring the p-aminophenyl derivative of the synthesized disaccharide to HMSA chains affords the synthesis of the Linear B amide branched polymer (Figure 1), the interesting biological activity of which is also reported and discussed.
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Results |
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The characterization of Gal1-3Gal
-pNP was performed by ion spray mass spectrometry and by 13C- and 1H-nuclear magnetic resonance (NMR) spectroscopy. The positive mass spectrum showed peaks at 486.1 and 502.0 amu corresponding to [M+Na]+ and [M+K]+ adducts, respectively.
The assignments of 13C- and 1H-NMR are reported in Table I. All the structural data confirm the identity of the obtained molecule with Gal1-3Gal
-pNP. In fact, taking into account the NMR chemical shift values reported for monomeric
-galactose (Morris and Hall, 1981
), a considerable downfield shift is observed for the sugar carbon at position 3 (i.e., from 70 ppm to the present 75 ppm) whereas carbon at position 4 remains practically unchanged (i.e., 6970 ppm). Likewise, carbon 6 remains equally unchanged. Furthermore, an expected, albeit small upfield shift is observed for the resonance of carbon 2 (from 69 ppm to the present 67 ppm), carbon 2 being more strongly influenced than carbon 4 by the hindering 13 and anomeric linkages.
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Preparation, purification, and characterization of SMALinear B disaccharide
To obtain a reactive polymer to be used for glycosylation, the anhydride group of SMA was hydrolyzed in carbonate buffer to give the corresponding HSMA, the carboxylic groups of which can be activated by water-soluble N'-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC). Once activated, carboxylic groups easily react in mild conditions with the amine of Gal1-3Gal
-pNH2P to afford SMALinear B glycopolymer in which the oligosaccharide portion is linked to the polymers through an amide bond. The degree of substitution was evaluated by elemental analysis. The composition was C = 56.0%, H = 5.41%, N = 2.2%, corresponding to a value of the degree of substitution (with respect to the anhydride monomer) equal to 0.99 (i.e., about one disaccharide unit every SMA repeating unit). Infrared spectroscopy confirmed the presence of the amide bond in the glycopolymer derivative. In the spectrum of SMALinear B derivative (not reported) a new band corresponding to the C = O stretching of the amide linkage develops at 1637 cm1, which is absent in the spectrum of underivatized HSMA.
Binding of human serum and human IgG and IgM to SMALinear B polymer
The evaluation of the binding of the SMALinear B polymer to anti-Gal antibodies was accomplished by enzyme-linked immunosorbent assay (ELISA) with whole AB serum or with affinity-purified IgG and IgM. Taking into account the value of the degree of substitution, a concentration of SMALinear B of 1 mg/ml corresponded to an effective Linear B disaccharide concentration of 1.5 mM. The data summarized in Figure 3 show that for both the whole serum and the isolated IgG and IgM there is a significant and specific interaction with the glycopolymer, whereas no signal has been obtained with solution of underivatized HSMA at the same molar concentration of free carboxylic groups (data not shown).
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Discussion |
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Chemical synthesis of -epitope usually requires lengthy protection and deprotection reaction sequences (Jacquinet et al., 1981
; Koike et al., 1987
; Matsuzaki et al., 1993
; Reddy et al., 1994
; Gege et al., 2000
).
The present results show that a relatively high yield in the Gal1-3Gal transglycolytic synthesis can be achieved in a comparatively short reaction time scale. The high regioselectivity of the transglycolytic synthesis already described in literature (70100%: Nilsson, 1987
; Nilsson and Fernández-Mayoralas, 1991
; Matsuo et al., 1997
) had its negative counterpart in the very low values of yield (i.e., 314%) as well as in the high incubation time required (2452 h). Moreover, the use of organic cosolvent such as N,N-dimethylformamide was required, which might complicate the reaction and purification scale-up and produce undesirable levels of contamination.
Recently a transglycolytic synthesis of Linear B disaccharide and of its PEG derivatives has been described (Matsuo et al., 1997). The yield reported was 11% with a regioselectivity of 85.5%. Again the experimental procedure required the use of N,N-dimethylformamide as an organic cosolvent. A comparative survey of the previous and of the present methods is given in Table II. The significant improvement of the present procedure (as indicated by the values of the last column of Table II) was achieved by studying the effect of pH and temperature on both yield and regioselectivity. pH and temperature can be very important for the modulation of the yield and the regioselectivity (Vetere and Paoletti, 1996
; Vetere et al., 1997
). In none of the previous works was their effect analyzed in detail.
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The conversion of the free Linear B disaccharide into its p-nitrophenylamine derivative was obtained with a very simple way. The amine derivative of the Linear B disaccharide so obtained is a good reactive intermediate for the synthesis of (neo)glycoconjugates and/or biocompatible polymers like SMALinear B.
Synthesis of the SMALinear B disaccharide conjugate
Polymers with a high density of sugar ligands seem to be effective not only as biomimetic models of glycoconjugates but also for therapeutic and/or diagnostic purposes in the biomedical field. Biocompatibility of the polymer backbone is then an essential requirement if therapeutic use of glycopolymers is to be achieved. The glycopolymer SMALinear B, the synthesis of which is reported herein, can be considered as an example of this kind of tool. The unreacted carboxylic groups provide to SMALinear B the anionic character that produces optimal interactions with cells. The latter ones are enhanced also by the hydrophobic nature of the phenyl moiety of the styrene comonomer. On practical grounds, the maleic anhydride unit guarantees for a strictly alternating structure also in a polymerization reaction mixture with, say, divinylbenzene, to get crosslinked material for the preparation of solid supports.
Biological activity of the SMALinear B disaccharide conjugate
The interaction of the SMALinear B with both whole human serum and partially purified anti-Gal Ab has been well demonstrated by ELISA experiments. The specificity of interaction is high enough to expect a practical use of this glycopolymer. This conclusion is confirmed the results of Figure 4, which show that this glycopolymer is able to efficiently inhibit the cytotoxic effect of human serum on pig kidney cell line PK15. The high protective effect (> 90%) of SMALinear B allows us to envisage the potential use of this glycopolymer as a therapeutical tool as an injectable anti--Gal antibodies plasma remover. Interestingly enough and in agreement with previous reports (Parker et al., 1996
; Nagasaka et al., 1997
), the synthesized glycoconjugate has shown a high antibody inhibition effect despite its Gal
1-3Gal
disaccharide sequence, different from the natural one (Gal
1-3Galß). This points to a predominant antigenic role of the Gal
1-3Gal moiety at the nonreducing terminal, diminishing the importance of the anomeric configuration at the reducing end.
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Materials and methods |
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Kinetic analysis of the synthesis of Gal1-3Gal
-pNP
Four hundred fifty milligrams of Gal-pNP were dissolved in 4.5 ml water. From this solution three groups of four aliquots each were formed. Buffer solutions (500 µl) were added to each aliquot (500 µl) of each group. The buffer composition was 100 mM sodium acetate for experiments at pH 5.0 (2x), 100 mM potassium phosphate for experiments at pH 6.5 (2x) and 100 mM TrisHCl for experiments at pH 8.0 (2x). One unit (20 µl) of coffee bean
-D-galactosidase was added to each aliquot. The aliquots of each pH were incubated for 8 h at 25°C, 37°C, and 55°C, respectively. During incubation, every 60 min 100 µl of each sample were collected, added to 900 µl distilled water, and heated in a boiling water bath for 10 min and then immediately cooled in ice. After centrifugation at 11,000 rpm for 5 min the clear supernatants were analyzed by a Jasco HPLC system consisting of a BIP-I pump equipped with a UVIDEC-100-V UV-visible detector (
= 280 nm) and an Spherisorb-ODS column (5 µm, 250 x 4.6 mm ID). The column was eluted isocratically, at a flow rate of 1 ml/min, using a solution of acetonitrile:water (10:90).
Preparation and purification of Gal1-3Gal
-pNP
Five hundred milligrams of Gal-pNP were dissolved in 10 ml 50 mM potassium phosphate pH 6.5; 10 U (200 µl) of coffee bean
-D-galactosidase were added. After incubation at 37°C for 8 h, the mixture was heated in a boiling water bath for 10 min to inactivate the enzyme and then immediately cooled in ice. After centrifugation at 11,000 rpm for 5 min the supernatant was initially purified by solid phase extraction on a Sep-Pak C18 (Vac 35 cc) cartridge. Free galactose was eluted by washing with water. Less polar compounds like Gal
-pNP and transgalactosylation products were eluted with methanol. The methanol-eluted fraction was dried under reduced pressure, redissolved in 5 ml of water and then purified by gel permeation chromatography on two serial columns (2.0 x 100 cm) of Bio-Gel P2 equilibrated in water. The elution was followed by UV, reading the absorbance at 280 nm, to detect the presence of Gal
-pNP and the transgalactosylation products. The analysis of the latter ones was made by use of the same HPLC system as described. The separation of the regioisomers of the transgalactosylation reaction was obtained by use of the same HPLC system as before but equipped with a semi-preparative column Econosil C18 10U eluted under isocratic conditions using acetonitrile:water (5:95) at a flow rate of 3 ml/min.
Preparation and purification of Gal1-3Gal
-pNH2P
One hundred milligrams of Gal1-3Gal
-pNP were dissolved in 10 ml of a 2% CuSO4 solution and stirred with zinc dust (Fukase et al., 1996
; Matsuo et al., 1997
). The course of the reaction was followed by thin-layer chromatography developed with 6:2:1 n-propanol:1 M ammonia:water and detected with 0.2% orcinol in 2 M H2SO4. After 2 h the reduction completed, and the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was dried under reduced pressure, redissolved in 5 ml of water, and then desalted by gel permeation chromatography on a column (2.0 x 100 cm) of Bio-Gel P2 equilibrated in water. The elution was followed by UV, reading the absorbance at 280 nm, to detect the presence of Gal
1-3Gal
-pNH2P. The fractions containing the Gal
1-3Gal
-pNH2P were pooled and freeze-dried to give 93 mg of an amorphous solid corresponding to 99% of recovery of the reduced product.
Preparation and purification of SMALinear B disaccharide
SMA 1000P was first hydrolyzed to give the HSMA form. One hundred milligrams of SMA were dissolved in 0.8 ml of dimethyl sulfoxide and added drop-wise to 48 ml of a 0.5 M NaHCO3/Na2CO3 buffer solution (pH 9.0), and the solution was stirred for 3 h. The solution was dialyzed exhaustively against MilliQ water and lyophilized yielding the hydrolyzed polymer (HSMA). HSMA (43.8 mg) were dissolved in 6 ml of 0.3 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 4.5 and EDC and NHS were added in a molar ratio of EDC/COOH and of NHS/EDC of 2.5 and 0.2, respectively. The mixture was stirred for 10 min, and then 1 ml of a 72 mg/ml solution in 0.3 M MES buffer (pH 4.5) of Gal1-3Gal
-pNH2P (for a final molar ratio of COOH/Gal
1-3Gal
-pNH2P = 2) was added. The solution was stirred at room temperature overnight, dialyzed against a 0.1 M bicarbonate solution for 1 night, and then dialyzed against MilliQ water. The solution was lyophilized to obtain 103 mg conjugated polymer.
Structural identification methods
1H- and 13C-NMR experiments were performed on a Bruker AC 200 spectrometer equipped with a 5 mm multinuclear probe.
1H measurements were performed at 300°K, and the signal chemical shifts were referred indirectly to acetone (2.225 ppm). All samples were dissolved in D2O with a typical concentration of 15 mg/ml. The spectral width was 3500 Hz, and the digital resolution was 0.25 Hz/pt; the acquisition time was 2.3 s.
The assignment of 1H signals was made by means of a 2D correlated spectroscopy experiment with the presaturation of residual DHO signal. 2D spectrum was acquired using 192 scans per series with 1 and 0.5 K data points in F2 and F1, respectively, with zero-filling in F1. Centered sine-bell multiplication functions were applied prior to Fourier transformation.
Decoupled 13C spectra were obtained at 313°K, and the signal chemical shifts were referred indirectly to tetramethylsilane using 1,4 dioxane as internal standard. 13C spectra width was 16.000 Hz with a digital resolution of 1 Hz/pt. The full assignment of 13C signals was made by means of a heteronuclear (1H13C) 2D chemical shift correlation experiment. The applied pulse sequence gave proton decoupling in each dimension. Spectral widths of 2080 and 280 Hz, 2048 and 256 data points were used in F2 and F1, respectively.
The mass spectra were recorded on an API-I PE SCIEX quadrupole mass spectrometer equipped with an articulated ion spray and connected to a syringe pump for the injection of the samples. The instrument was calibrated using a polypropylene glycol (PPG) mixture (3.3 x 105 M PPG 425, 1 x 104 M PPG 1000, and 2 x 104 M PPG 2000), 0.1% acetonitrile, and 2 mM ammonium formate in 50% aqueous methanol. The samples were dissolved in 50% aqueous acetonitrile at a final concentration of 0.2 x 104 M. Ammonium acetate 0.6 x 104 M and 0.25 x 103 M was used as ionizing agent in the positive and negative ion modes, respectively. The injection flow rate was 0.3 µl/min. The analyses were conducted in the positive mode with an ion spray voltage of 5000 V and an OR of 90 V. The spectra were recorded using a step size of 0.1 amu.
Determination of C, H, and N content was performed on SMALinear B derivative. Infrared spectra of purified HSMA and SMALinear B samples in KBr were recorded with a Jasco FT/IR 200 spectrometer. Both polymers were reprecipitated with isopropanol from an aqueous solution.
Purification of antiLinear B human IgG and IgM
Fifty milliliters of AB human plasma were dialyzed against 1 L of phosphate buffered saline (PBS) and loaded on a affinity column of Bdi-PAA (10 x 2.5 cm) equilibrated in PBS. The column was washed with PBS until the optical density at 280 nm of the eluate was less than 0.005. Then the adsorbed antibodies were eluted with 1% NH4OH. The fractions positive for reading at 280 nm were pooled, neutralized dialyzed against 1% PBS, and freeze-dried. Lyophilized antibodies were resuspended in distilled water.
Interaction of human serum and human IgG and IgM to SMALinear B polymer
The binding of human antibodies to SMALinear B polymer was evaluated by ELISA. One hundred microliters of 1 mg/ml solution of SMALinear B in 0.1 M Na2CO3/NaHCO3 buffer (pH 9.6) were pipetted in a 96-well plate and incubated overnight at 4°C. The plate was washed twice with PBS, and wells were blocked for 1 h with a blocking solution consisting of PBS containing 4% defatted milk, 0.2% bovine serum albumin, 0.4 M NaCl, and 0.2% Tween 20. The plate was then washed three times with PBS, and 100 µl of whole human serum or mixture of partially purified human IgG and IgM antiLinear B were added to the wells and incubated for 1 h at room temperature. The wells were washed twice with PBS, and 100 µl of 1:1000 diluted alkaline phosphataseconjugated monoclonal goat Abs anti-human IgG and IgM were added. Following incubation for 1 h, the wells were washed four times with PBS and twice with a solution of 0.1 M diethanolamine (pH 9.6). Finally 100 µl of a 1 mg/ml solution of pNPP in 0.1 M diethanolamine buffer (pH 9.6) were added and plate incubated 1 h at 37°C in the dark. The reaction was quenched adding 25 µl 3 M NaOH, and absorbance was determined at 410 nm.
Testing for inhibition of cytotoxicity by SMALinear B
PK15 cell were seeded on microscope slides in a 24-well plates and cultured until confluence in Dulbeccos modified Eagle medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml of penicillin, and 100 µg/ml of streptomycin at 37°C in an atmosphere of 5% CO2. For testing the inhibitory effects of SMALinear B, 1 mg glycopolymer was dissolved in 1 ml human serum and incubated at room temperature for 1 h under gentle agitation. HSMA (0.5 mg) was dissolved in 1 ml serum as a control to attain the same molar concentration of free carboxylic groups, and then treated as described.
Human sera, untreated and preincubated with SMALinear B and with HSMA. were added to PK15 cells and incubated for 1 h at 37°C. Incubation was followed by washing of cells with 2 x 1 ml PBS and staining with a mixture of two fluorescent probes according to manufacturers protocol. This staining procedure allowed for a clear distinction between live cells (in green) and dead cells (in red).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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