Immobilization of Pectin Fragments on Solid Supports: Novel Coupling

Bioconjugate Chem. 2002, 13, 285−294

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Immobilization of Pectin Fragments on Solid Supports: Novel Coupling by Thiazolidine Formation Fanny Guillaumie,†,‡ Owen R. T. Thomas,‡ and Knud J. Jensen*,† Center for Process Biotechnology, Biocentrum-DTU, Building 223, Søltofts Plads, and Department of Chemistry, Building 201, Kemitorvet, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark. Received July 26, 2001; Revised Manuscript Received October 18, 2001

As a prerequisite to solid-phase and sequence analyses and for the study of the fine structure of pectin, we have developed oriented and chemoselective methodologies to couple model pectin fragments onto a solid support. Polyethylene glycol polyacrylamide (PEGA) resins were selected due to their excellent swelling properties in a wide range of solvents, including water, and their easy accessibility to enzymes. Following appropriate derivatization of amino-terminated PEGA resins, oligomers of R-D-galacturonic acid (GalA), up to the trimer, were anchored to the support through their reducing end. In addition to reductive amination, the strategies included the formation of an oxime bond, a glycosyl hydrazide, and a pyroglutamyl ring. Further, we developed a new immobilization approach based on the formation of a thiazolidine ring. All methods proved efficient and did not require modification of the GalA oligomers prior to coupling. In addition, very mild conditions and few steps for derivatization of the support were required. Immobilization by thiazolidine ring and oxime bond formation were the preferred methods, given the stability of the linkages formed, their compatibility with aqueous solvents, the few number of steps required, and their potential for application to larger pectin fragments. Thiazolidine and pyroglutamyl anchoring were developed further by the insertion of a disulfide bond which allowed release of the saccharides under mild, selective conditions.

INTRODUCTION

Pectin is a complex polysaccharide that occurs in the primary cell walls and the middle lamella of higher plants (1). The main feature of pectin is a linear chain of R-(1f4)-linked D-galacturonic acid units (the so-called ‘smooth region’) in which varying proportions of the carboxylic acid groups have been converted to methyl esters. The smooth region is interrupted by R-(1f2)linked L-rhamnose units (the so-called ‘hairy region’), carrying further neutral carbohydrate side chains, typically consisting of D-galactose and D-arabinose (1). The remarkable gelling properties of pectin have been widely exploited in the food industry for the production of jams, marmalades, and other fruit based products. Pectin has also been used as a stabilizer in dairy products (2) and for medical preparations as an antidiarrheal, detoxicant, or in the regulation and the protection of gastrointestinal tract (1). Pectin has been the subject of numerous studies, many of which focused on structure elucidation in pectin (39), binding of pectin with metal ions (10-13), synthesis of pectin fragments (14-17), and the development of new analytical methods for pectin structure elucidation (1820). However, despite all of these efforts, much still remains to be learned about structure-function relationships in pectin. Immobilization of pectin fragments on a solid support may prove to be an important technique in the study of * To whom correspondence should be addressed at the present address: Department of Chemistry, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C (Copenhagen), Denmark, Phone: + 45 3528 2430, Fax: + 45 3528 2398, E-mail: [email protected]. † Chemistry Department. ‡ Center for Process Biotechnology, Biocentrum-DTU.

fine structure-function relationships in pectin. For example, immobilization would allow solid-phase analysis of the pectin molecule to be carried out and would serve as a prerequisite to sequencing. Moreover, an immobilization strategy could be a useful tool for the determination of the factors influencing the formation of ‘egg-box’ cavities in pectin. The ‘egg-box’ model has been proposed for the gelation of low methyl esterified pectin (21). It involves the intermolecular binding of two non-esterified segments or two whole pectin chains with interstices in which calcium ions may pack and coordinate. A number of coupling methods have been developed for the synthesis of glycoconjugates and for the immobilization of oligosaccharides. Reductive amination was one of the first methods to be implemented in the preparation of glycoconjugates (22-24) and in the coupling of carbohydrates onto solid supports (25-27). The reactivity toward the reducing end of carbohydrates can be enhanced by substituting amino groups with hydrazide functions. This technique has recently proved to be efficient for the immobilization of simple oligosaccharides on an ELISA plate (28). The oxime linkage has also been used to prepare glycoconjugates (29) and to immobilize short oligosaccharides on ELISA plates (28). Furthermore, small oligosaccharides have been coupled to solid supports (30) and glycoconjugates have been prepared (31, 32) by formation of a glycosyl amide linkage. However, none of the above coupling chemistries have so far been applied to the immobilization of polyuronic acids such as pectin or heparin. The formation of a thiazolidine ring has found application in the synthesis of peptide dendrimers (33), but has not been applied to the preparation of glycoconjugates nor to the immobilization of oligosaccharides. All of the aforementioned strategies will introduce a stable linkage between the saccha-

10.1021/bc0155364 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/03/2002

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ride and the support. Accordingly, in this paper we report on the use of these methodologies for anchoring GalA1 oligomers onto solid supports. Further, to allow for release of immobilized structures, we have modified the thiazolidine and pyroglutamyl immobilization strategies by including a cleavable disulfide bond. Polyethylene glycol polyacrylamide (PEGA) resin was selected as the solid phase in these studies due to its compatibility with a wide range of solvents, including water, and its accessibility to enzymes with a molecular weight of up to 70 kDa. MATERIALS AND METHODS

The amino-functionalized PEGA resins (0.4 mmol/g, 150-300 µm, 11.19 wt % in MeOH or 0.2 mmol/g, 300-500 µm, 9.41 wt % in MeOH) used in this study were kindly supplied by Dr. Andrew Coffey of Polymer Laboratories, UK. The PEGA resins were weighed wet, unless stated otherwise. All chemical reagents and solvents were purchased from Sigma-Aldrich Denmark A/S (Vallensbaek Strand, Denmark). All amino acids were of the L-configuration. General. Melting points were uncorrected. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury 300 instrument equipped with a four-nuclei probe operating at 300.06 and 75.45 MHz, respectively, or on a Varian Unity Inova 500 equipped with a H-C-P probe operating at 499.87 and 125.69 MHz, respectively. FABMS analyses were performed on a VG TRIO-2 quadrupole mass spectrometer equipped with a combined electron impact/chemical ionization ion source. The samples were analyzed in the chemical ionization mode with ammonia as reagent gas. ES-MS analyses were conducted on a Micromass LCT mass spectrometer or on a ion trap Esquire-Bruker instrument at Danisco Cultor A/S (Copenhagen, Denmark). Elemental analyses were conducted at the Department of Chemistry, University of Copenhagen. Analytical HPLC was performed with a Waters chromatography system (600E pump, 996 photodiode array (PDA) detector, 717 Plus autosampler, Millenium32 control software) on a 3.0 × 50 mm Waters X-Terra RP18 column (3.5 µm particle size), using H2O (A) and CH3CN (B) as eluents. The following program was applied: after 0.1 min at 100% A, 0% B, a linear gradient (0-95% B) was applied over the next 5.9 min; the eluent composition was then maintained at 5% A, 95% B for a further 1 min before applying a step change to 100% A, 0% B over 0.1 min to regenerate the column; a flow rate of 1.20 mL/ min was employed at all stages. Individual peaks were analyzed by their UV-spectra. All solid-phase reactions were performed in plastic syringes equipped with a polypropylene filter. Before saccharide coupling, all the resins were washed with an increasing concentration of Et2O in CH2Cl2 (0%, 1 × 5 min; 25%, 1 × 5 min; 50%, 1 × 5 min; 75%, 1 × 5 min; 100%, 2 × 5 min) and dried in vacuo for 18 h to give dry PEGA resins suitable for weighing. 1 Abbrevations: H-Aoa-OH, aminooxy acetic acid; Boc, tertbutoxycarbonyl; Cys, cysteine; DIEA, diisopropylethylamine; DMF, dimethylformamide; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; GalA, R-D-galacturonic acid; (GalA)2, R-D-GalA-(1f4)-D-GalA; (GalA)3, R-D-GalA-(1f4)-R-D-GalA(1f4)-D-GalA; Glu, glutamic acid; HL, high loading; LL, low loading; PyBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide; HOBt, 1-hydroxybenzotriazole; PEGA, polyethylene glycol polyacrylamide; Pyr, pyridine; rt, room temperature; Trt, trityl.

Guillaumie et al.

Loading Determination by Trityl (Trt) Group Quantitation. The ‘indirect method’ was applied to the resins A, B, G, and H. Each coupling took place at room temperature and for 1 h. Derivatized resin was washed with CH2Cl2-DMF (9:1, 3 × 1 min, 1 × 5 min) and treated with TrtCl (5 equiv) with DIEA (10 equiv) in CH2Cl2-DMF (9:1) (34). Excess reagents and solvents were removed by filtration, the resin was washed with CH2Cl2 (5 × 1 min) and treated with TrtCl (5 equiv) and diisopropylethylamine (DIEA, 10 equiv) in CH2Cl2. This coupling step was repeated once. Solvents and unreacted materials were removed by filtration. The resin was then washed with CH2Cl2 (3 × 1 min) and treated with TFACH2Cl2 (1:1) in the presence of Et3SiH (6 equiv) (35). The filtrate was collected, and the resin was washed with two extra portions CH2Cl2. The filtrate was evaporated to dryness, redissolved into CH3CN (1 mL), analyzed by HPLC, and the absorbance of TrtH was read at 226 nm. The amount n (mmol) of Trt groups was calculated using a standard curve. The resin was washed with an increasing concentration of Et2O in CH2Cl2 (0%, 1 × 5 min; 25%, 1 × 5 min; 50%, 1 × 5 min; 75%, 1 × 5 min; 100%, 2 × 5 min), dried in vacuo for 18 h, and weighed (m). The ratio between n and m gave the loading of the resin in mmol/g. ‘Direct method’: Trt-protected resins E and F were washed with CH2Cl2 (3 × 1 min) and treated with TFA-CH2Cl2 (1:1) in the presence of Et3SiH (6 equiv) (35). In each case, the filtrate was collected, and the resin was washed with two extra portions CH2Cl2. The filtrate was evaporated to dryness, redissolved into CH3CN (1 mL), and analyzed by HPLC. The absorbance of TrtH was read at 226 nm and the amount n (mmol) of Trt groups was calculated using a standard curve. The resin was washed with an increasing concentration of Et2O in CH2Cl2 (0%, 1 × 5 min; 25%, 1 × 5 min; 50%, 1 × 5 min; 75%, 1 × 5 min; 100%, 2 × 5 min), dried in vacuo for 18 h, and weighed (m). The ratio between n and m gave the loading of the resin in mmol/g. Determination of Immobilization Efficiency by Anthrone Assay. Resins A, C, G, and H prior to immobilization and after immobilization were treated as follows. The resin was washed with an increasing concentration of Et2O in CH2Cl2 (0%, 1 × 5 min; 25%, 1 × 5 min; 50%, 1 × 5 min; 75%, 1 × 5 min; 100%, 2 × 5 min), and dried in vacuo for 18 h. A small portion of the resin (1-5 mg) was weighed in a 1.5 mL safelock plastic vial. Water (0.5 mL) was added, followed by anthrone solution (0.2% in concentrated H2SO4, 1 mL) (36). The samples were shaken vigorously for 3 min, incubated at 100 °C for 5 min, and cooled in an ice bath for 3 min. A 300 µL amount was transferred to an ELISA plate, and the optical density was read at 570 nm in a microtiter plate reader (Anthos reader 2001, Anthos Labtech Instruments, Salzburg, Austria). The difference in optical density between the pre- and the postimmobilization resins was used as a measure of the amount of GalA units immobilized. Determination of Immobilization Efficiency by the Ellman Test. Pre- and postimmobilization resins (resins E and F) were treated as follows. The resin was washed with an increasing concentration of Et2O in CH2Cl2 (0%, 1 × 5 min; 25%, 1 × 5 min; 50%, 1 × 5 min; 75%, 1 × 5 min; 100%, 2 × 5 min), dried in vacuo for 18 h. A small portion of resin (1-4 mg) was treated with a 0.01 M solution of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in aq Na2HPO4 buffer (0.1 M, pH 8.5, 400 µL) (37, 38). Water (600 µL) was added, and the sample was shaken for 20 min at room temperature and analyzed by reversed-phase HPLC in an isocratic mode using H2O

Immobilization of Pectin Fragments on Solid Supports

as the eluent. The absorbance of the released thiol anion was read at 412 nm, and the amount of resin-bound free thiol groups was determined using a calibration curve with H-Cys-OMe‚HCl as a standard. Preparation of Aminooxy-Terminated Resins (A). Reactions were carried out in a 20 mL syringe, and the washing volumes were 8 mL. Amino PEGA resin (3.8 g, 0.4 mmol/g) was washed with DMF (3 × 1 min), swollen in DMF (1 × 10 min), and washed with 10% DIEA in DMF (3 × 1 min) and DMF (3 × 1 min), respectively. Nβ-Boc-aminooxy acetic acid (Nβ-Boc-Aoa-OH, 100 mg, 0.52 mmol), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 271 mg, 0.52 mmol), and 1-hydroxybenzotriazole (HOBt, 70 mg, 0.52 mmol) were dissolved in DMF (7 mL). DIEA (157 µL, 0.92 mmol) was then added, and the solution was added to the resin. The syringe was shaken at room temperature for 1.2 h, after which a qualitative ninhydrin test was negative. Solvents and unreacted materials were filtered off, and the resin was washed with DMF (4 × 1 min) and CH2Cl2 (2 × 1 min, 1 × 5 min and 2 × 1 min). Removal of Boc groups was accomplished by treatment with TFA-CH2Cl2 (1:1, 8 mL) for 1 h at room temperature. Preparation of Carboxylic Acid-Terminated Resins (B). Reactions were performed in a 50 mL syringe, and the washing volumes were 15 mL. Amino PEGA resin (8.94 g, 0.4 mmol/g) was washed and swollen with the same solvents as for A. Succinic anhydride (160 mg, 1.6 mmol) and HOBt (162 mg, 1.2 mmol) were dissolved in DMF (13 mL), DIEA (308 µL, 1.8 mmol) was added, and the solution was added to the resin. The syringe was shaken at room temperature for 1.5 h. Excess reagents and solvents were filtered off, and the resin was washed with DMF (4 × 1 min) and MeOH (4 × 1 min). At this point, a ninhydrin test was negative. The resin was stored wet in MeOH at 5 °C until further use. Preparation of Hydrazide-Terminated Resins (C). A portion of resin B (1.98 g, 0.11 mmol) was transferred to a 10 mL syringe. The washing volumes were 4 mL. The resin was washed with DMF (1 × 1 min, 1 × 10 min, and 1 × 1 min). N-[(1H-Benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU, 173 mg, 0.46 mmol) and HOBt (62 mg, 0.46 mmol) were dissolved in DMF (3 mL). DIEA (88 µL, 0.51 mmol) was then added, followed by hydrazine monohydrate (28 µL, 0.57 mmol), and the mixture was added to the resin. After 2 h coupling at room temperature, solvents and unreacted compounds were removed by filtration, and the resin was washed with DMF (4 × 1 min) and MeOH (4 × 1 min). Preparation of Thiol-Terminated Resins (D). Resin B (3.30 g, 0.19 mmol) was transferred into a 20 mL syringe. Washing volumes were 6 mL. The resin was washed with DMF (1 × 1 min, 1 × 10 min, and 1 × 1 min). S-(2-Pyridylthio)cysteamine hydrochloride (39) (212 mg, 0.95 mmol), HBTU (288 mg, 0.76 mmol), and HOBt (103 mg, 0.76 mmol) were dissolved in DMF (6 mL). After addition of DIEA (146 µL, 0.86 mmol), the solution was added to the resin. After shaking for 2 h at room temperature, the resin was washed with DMF (4 × 1 min), H2O (4 × 1 min), and aq NaOAc buffer (0.1 M, pH 4.8, 2 × 1 min). Pyridine-2-thione was removed from the support by treatment with a solution of dithiothreitol (DTT) (147 mg, 0.95 mmol) in aq NaOAc buffer (7 mL). After 2 h at room temperature, solvents and excess reagents were filtered off, and the resin was washed with H2O (4 × 1 min) and then with MeOH (3 × 1 min).

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Preparation of Cysteine (Cys)-Terminated Resins (E). Synthesis of Boc-Cys(Trt)-NH-(CH2)2-S-S-Pyr. BocCys(Trt)-OH (566 mg, 1.22 mmol) was dissolved in CH3CN (10 mL). HBTU (512 mg, 1.35 mmol) and NEt3 (340 µL, 2.44 mmol) were then added, followed by S-(2pyridylthio)cysteamine hydrochloride (39) (771 mg, 1.22 mmol). Complete dissolution was accomplished by addition of water (1 mL). After overnight stirring at room temperature, a white precipitate formed. It was isolated by filtration and dried under reduced pressure (1st crop). The filtrate was diluted with EtOAc (40 mL), washed with 1 M aq HCl (3 × 15 mL), saturated aqueous NaHCO3 (3 × 15 mL), and brine (3 × 15 mL), dried over MgSO4, and concentrated to afford white crystals (2nd crop). Fractions 1 and 2 were identified as the title compound (753 mg, 98%), mp 131-134 °C. 1H NMR (CDCl3) δ 1.41 (s, 9 H, CH3), 2.56 (dd, 1 H, H-βa (Cys), J 5.2 Hz, J 12.5 Hz), 2.71 (dd, 1 H, H-βb (Cys), J 7 Hz, J 12.5 Hz), 2.86 (m, 2 H, CH2-S, J 1.8 Hz, J 5.8 Hz), 3.50 (m, 1 H, CH2-NH), 4.01 (dd, 1 H, H-R (Cys), J 0.8 Hz, J 5.4 Hz), 7.06-7.61 (m, 18 H, H arom.), 8.47 (d, 1 H, H-3′ (Pyr), J 4.9 Hz). 13C NMR (CDCl3) δ 28.58 (CH3, tBu), 34.59 (C-β, Cys), 37.62 (CH2-S), 38.87 (CH2-N), 52.17 (SCPh3), 67.34 (C-R, Cys), 121.19 (C-6′, Pyr), 121.38 (C-4′, Pyr), 127.07 (Trt), 128.45 (Trt), 129.79 (Trt), 137.15 (C3′, Pyr), 144.66 (Trt), 150.24 (C-3′, Pyr), 159.22 (CO, Boc), 170.64 (CO, Cys). ES-MS, calcd. for C34H37N3O3S3: 631.20. Found: m/z 670.11 [M + K]+, 654.13 [M + Na]+, 632.18 [M + H]+, 243.42 [Trt]+. Anal. Calcd for C34H37N3O3S3: C, 64.63; H, 5.90; N, 6.65; S,15.22. Found: C, 64.29; H, 5.82; N, 6.65; S, 14.91. Thiol-terminated resin D (376 mg, 29 µmol) was weighed in a 10 mL syringe. Washing volumes were 4 mL. Boc-Cys(Trt)-NH-(CH2)2-S-S-Pyr (72 mg, 0.114 mmol) was dissolved in MeOH (4 mL) and added to the resin. The resin was shaken at room temperature for 2 h and washed with MeOH (4 × 1 min) and CH2Cl2 (2 × 1 min, 1 × 5 min, 2 × 1 min). The resin was stored wet at 5 °C and Boc and Trt groups were cleaved with TFA-CH2Cl2 (1:1) in the presence of Et3SiH (15 equiv) prior to coupling. Preparation of Cys-Terminated Resins (F). ‘F high loading’ (F-HL): the reactions were performed in a 10 mL syringe, and the washing volumes were 4 mL. Aminoterminated PEGA resin (1.79 g, 0.4 mmol/g) was washed with DMF (3 × 1 min), swollen in DMF (1 × 10 min), and washed with DIEA-DMF (1:9, 3 × 1 min) and DMF (3 × 1 min), respectively. Boc-Cys(Trt)-OH (148 mg, 0.32 mmol) was coupled in the presence of PyBOP (125 mg, 0.24 mmol), HOBt (32 mg, 0.24 mmol), and DIEA (62 µL, 0.36 mmol) in DMF (4 mL) at room temperature. After 1 h, a qualitative ninhydrin test showed complete incorporation of the Cys derivative. The resin was then washed with DMF (3 × 1 min) and CH2Cl2 (2 × 1 min, 1 × 5 min, 2 × 1 min) and stored wet at 5 °C. Deprotection of Boc and Trt groups was accomplished with TFACH2Cl2 (1:1) in the presence of Et3SiH (15 equiv) immediately prior to sugar immobilization. ‘F low loading’ (F-LL): a parallel experiment was conducted using a low loading amino PEGA resin (2.13 g, 0.2 mmol/g) in a 10 mL syringe. Boc-Cys(Trt)-OH (74 mg, 0.16 mmol) was reacted with PyBOP (63 mg, 0.12 mmol), HOBt (16 mg, 0.12 mmol) and DIEA (31 µL, 0.18 mmol) in DMF (4 mL). The coupling was performed at room temperature and was complete within 1 h, according to a qualitative ninhydrin test. Washing conditions were similar to the ones described above. Boc and Trt groups were removed by TFA-CH2Cl2 (1:1) in the presence of Et3SiH (15 equiv) prior to immobilization (see below).


Preparation of Glutamic Acid (Glu)-Terminated Resins (G). Synthesis of Boc-Glu(OtBu)-NH-(CH2)2-S-SPyr. A solution of Boc-Glu(OtBu)-OH (249 mg, 0.82 mmol), HBTU (341 mg, 0.9 mmol), NEt3 (286 µL, 2.05 mmol), and S-(2-pyridylthio)cysteamine hydrochloride (39) (200 mg, 0.90 mmol) in CH3CN (8 mL) was stirred at room temperature. After 15 h, the mixture was diluted with EtOAc (35 mL), washed sequentially with 1 M aq HCl (3 × 12 mL), saturated aqueous NaHCO3 (3 × 12 mL), and brine (3 × 12 mL), dried over MgSO4, and concentrated to a foam identified as the title compound (352 mg, 91%). 1H NMR (CDCl3) δ 1.35 (s, 9 H, CH3), 1.37 (s, 9 H, CH3), 1.80 (m, 1 H, H-βa), 1.97 (m, 1 H, H-βb), 2.26 (m, 2 H, H-γ), 2.84 (t, 2 H, CH2-S, J 6.1 Hz), 3.46 (m, 2 H, CH2-NH), 4.03 (dd, 1 H, H-R, J 7.1 Hz), 7.08 (m, 1 H, H-4′ Pyr), 7.57 (m, 2 H, H-5′ Pyr, H-6′ Pyr), 7.82 (s, 1 H, NH), 8.44 (m, 1H, H-3′ Pyr). 13C NMR (CD3OD) δ 27.23 (CH3, Boc), 27.40 (C-β), 27.57 (CH3, tBu), 27.69 (Cγ), 31.57 (CH2-S), 37.91 (CH2-NH), 54.42 (C-R), 79.62 ((CH3)3-C, Boc), 80.63 ((CH3)3-C, tBu), 120.39 (C-6′, Pyr), 121.30 (C-4′, Pyr), 137.85 (C-5′, Pyr), 149.47 (C-3′, Pyr), 159.74 (CO, Boc), 172.74 (CO, Glu, side chain), 173.55 (CO, Glu). ES-MS, calcd. for C21H33N3O5S2: 471.19. Found: m/z 494.29 [M + Na]+, 472.31 [M + H]+, 416.33 [M - tBu + 2H]+, 372.37 [M - Boc + 2H]+, 316.35 [M - Boc - tBu + 3H]+. Resin D (275 mg, 29 µmol) was weighed in a 10 mL syringe. Washing volumes were 4 mL. Boc-Glu(OtBu)-NH-(CH2)2-S-S-Pyr (54 mg, 0.114 mmol) was dissolved in MeOH (0.7 mL), the volume was adjusted to 4 mL with MeOH, and the solution was added to the resin. During coupling for 2 h at room temperature, a yellow coloration was observed, indicative of the release of pyridine-2-thione. The resin was then washed with MeOH (4 × 1 min) and stored wet at 4 °C until further use. Preparation of Glu-Terminated Resins (H). Reactions were carried out in a 10 mL syringe. The washing volumes were 4 mL. The initial washing and coupling procedures were identical to the ones described above for the preparation of Cys terminated resins using Boc-Cys(Trt)-OH. In this case, Boc-Glu(OtBu)-OH (97 mg, 0.32 mmol) was anchored to amino PEGA resin (1.79 g, 0.4 mmol/g) in the presence of PyBOP (125 mg, 0.24 mmol), HOBt (32 mg, 0.24 mmol), and DIEA (62 µL, 0.36 mmol). According to a qualitative ninhydrin test, coupling was completed within 1 h at room temperature. The resin was then washed with DMF (3 × 1 min) and CH2Cl2 (2 × 1 min, 1 × 5 min, 2 × 1 min) and stored wet at 5 °C until deprotection and further derivatization. Model Reactions in Solution. Preparation of a Model Glycosyl Hydrazide. GalA (1.3 g, 6.14 mmol) was dissolved in MeOH (dried over 3 Å molecular sieves, 12 mL), and acetic acid hydrazide (500 mg, 6.75 mmol) (40) was added. The mixture was stirred at 65 °C for 7 h. Formation of a precipitate was observed. It was collected by centrifugation and dried under reduced pressure (1st crop). The filtrate was concentrated to a pale yellow solid in vacuo (2nd crop). Fractions 1 and 2 gave 1.53 g (99%) of the acyclic glycosyl hydrazide (E/Z ) 2:1, based on the assumption that δZ(H-1) > δE(H-1)). 1H NMR (DMSOd6) δ 1.85 (s, 1 H, CH3, Z), 2.05 (s, 2 H, CH3, E), 3.45 (d, 1 H, H-3, J 9.4 Hz), 3.80 (d, 1 H, H-4, J 9.4 Hz), 4.24 (m, 2 H, H-2, H-5), 7.35 (d, 0.66 H, H-1, E, J 5.7 Hz), 7.46 (d, 0.33 H, H-1, Z, J 5.9 Hz), 10.84 (s, 0.66 H, NH, E), 10.99 (s, 0.33 H, NH, Z). 13C NMR (DMSO-d6) δ 20.96 (CH3, E), 21.74 (CH3, Z), 70.33 (C-2), 70.62 (C-4), 71.98 and 72.06 (C-3), 72.66 and 72.77 (C-5), 149.35 (C-1, E), 152.03 (C-1, Z), 166.0 (C(O), Ac, Z), 172.27 (C(O), Ac, E), 176.04

Guillaumie et al.

(C-6). FAB-MS, calcd for C8H14N2O7: 250.08. Found: m/z 170 [M + 3H2O + 2NH4]2+, 153 [M + 3H2O + 2H]2+. Preparation of a Model Thiazolidine. H-Cys-OMe.HCl (1 g, 5.8 mmol) (41) was dissolved in H2O (10 mL). K2CO3 was added until neutral pH. The solution was extracted with Et2O (5 × 5 mL), and the combined extracts were dried over MgSO4 and concentrated in vacuo. Residual H-Cys-OMe (100 mg, 0.74 mmol) was dissolved in MeOH (5 mL), and a solution of GalA (157 mg, 0.74 mmol) in MeOH (5 mL) was added. The clear mixture (pH 4) was stirred at room temperature. After 24 h, TLC (eluent CH2Cl2-MeOH, 1:1) showed complete disappearance of the starting material. The solvent was then evaporated in vacuo. Purification on a short silica column using MeOH as eluent gave the thiazolidine derivative in 50% yield and as two diastereomers (R and S configurations at C-1, GalA). 13C NMR (D2O) δ 35.87 and 36.47 (C-β, Cys), 52.23 and 52.36 (CH3, Cys), 64.58 and 65.00 (C-R, Cys), 68.76 and 69.98 (C-1, GalA), 70.97-72.30 (C-2, C-3, C-4, GalA), 76.07 and 76.23 (C-5, GalA), 169.93 (C(O), Cys). FAB-MS, calcd for C10H17NO8S: 311.07. Found: m/z 294 [M + H - H2O]+, 276 [M + H - 2H2O]+. Immobilization Techniques. Immobilization via Reductive Amination. The reaction was performed in a 5 mL syringe, and the washing and reaction volumes were 2 mL. Hydrazide-terminated resin C (100 mg dry, 38 µmol) was washed with DMF (2 × 1 min, 1 × 5 min, 2 × 1 min). GalA (40 mg, 0.19 mmol) was dissolved in DMF-AcOH (99:1). The solution was then added to the resin, and following addition of NaBH3CN (24 mg, 0.38 mmol), the syringe was shaken at room temperature. After 24 h, the solvent and excess reagents were filtered off, and the resin was washed with DMF (4 × 1 min) and stored at 5 °C until quantitation. A similar procedure was applied to immobilize R-D-GalA-(1f4)-D-GalA ((GalA)2, 10 mg, 27 µmol) on resin C (14 mg dry, 5 µmol) in the presence of NaBH3CN (4 mg, 54 µmol). R-D-GalA-(1f4)R-D-GalA-(1f4)-D-GalA ((GalA)3, 5.9 mg, 11 µmol) was also coupled to resin C (6 mg dry, 2 µmol) using NaBH3CN (7 mg, 11 µmol). In these two cases, 2 mL syringes were used, and the washing and reaction volumes were 1 mL. The anthrone assay was used to determine the coupling efficiencies. Immobilization via Oxime Formation. The immobilization reaction was performed in a 5 mL syringe, and the washing and reaction volumes were 2 mL. Aminooxyterminated resin (128 mg dry, 44 µmol) was first washed with DMF-H2O (1:1, 1 × 1 min, 1 × 5 min, 2 × 1 min) and DMF-0.1 M aq NaOAc, pH 4.76 (1:1, 3 × 1 min). GalA (42 mg, 0.2 mmol) was dissolved in DMF-0.1 M aq NaOAc, pH 4.8 (1:1), and the solution was added to the resin. The syringe was placed in a sand bath at 40 °C for 24 h. The solvents and unreacted compounds were removed by filtration, and the resin was washed with H2O (4 × 1 min) and stored at 5 °C until quantitation. The same procedure was applied to immobilize (GalA)2 (9.5 mg, 26 µmol) on aminooxy-terminated resins A (14 mg dry, 5 µmol) and (GalA)3 (4.4 mg, 8 µmol) on resin A (5.6 mg dry, 2 µmol). In these latter cases, 2 mL syringes were used, and the washing and reaction volumes were reduced to 1 mL. Coupling efficiencies were determined by anthrone assay. Immobilization via Glycosyl Hydrazide Formation. Resin C (100 mg dry, 38 µmol) was weighed in a 5 mL syringe. Washing volumes were 2 mL. The resin was washed with MeOH (2 × 1 min, 1 × 5 min, 2 × 1 min). GalA (40 mg, 0.19 mmol) was dissolved in MeOH (2 mL), and the solution was added to the resin. The syringe was placed in a sand bath at 65 °C. After 20 h, the solvents


and excess reagents were removed by filtration. The resin was washed with MeOH (4 × 1 min), and stored at 5 °C until quantitation. Similar experiments were conducted in 2 mL syringes (the washing and reaction volumes were 1 mL) to immobilize (GalA)2 (10 mg, 27 µmol) and (GalA)3 (6.2 mg, 11 µmol) on resin C (14 mg dry, 5 µmol or 6 mg dry, 2 µmol, respectively). In the latter case, the reaction time was extended to 46 h. The reactions were quantified by the anthrone assay. Immobilization via Thiazolidine Formation. The coupling reactions were performed in 2 mL syringes, and the washing volumes were 1.5 mL. Resins E (20 mg dry, 0.6 µmol) was treated with TFA-CH2Cl2 (1:1, 1.5 mL) in the presence of Et3SiH (2 µL, 12 µmol) for 1 h at room temperature. It was then washed with CH2Cl2 (4 × 1 min) and with CH3CN-H2O-0.1 M aq NaH2PO4 buffer, pH 8.5 (1:1:1, 2 × 1 min). GalA (0.2 mg, 1 µmol) was dissolved into CH3CN-H2O-0.1 M aq NaH2PO4 buffer, pH 8.5 (1:1:1, 1.5 mL), and the solution (pH 4) was added to the resin. After 48 h at room temperature, the resins were washed with CH3CN (4 × 1 min) and stored at 5 °C until quantitation. The same procedure was repeated with resin F-HL (20 mg dry, 2 µmol) and resin F-LL (20 mg dry, 0.8 µmol) with Et3SiH (5 µL, 31 µmol and 2 µL, 12 µmol, respectively) and GalA (0.6 mg, 3 µmol and 0.2 mg, 1 µmol, respectively). A similar procedure was applied using the same amount of resins E, F-HL, and F-LL and (GalA)2 (0.4 mg, 1 µmol; 1 g, 3 µmol and 0.4 mg, 1 µmol, respectively) as a substrate. Similarly, (GalA)3 (1.4 mg, 3 µmol and 0.5 mg, 1 µmol, respectively) was coupled to Cys-terminated resins F-HL (20 mg dry, 2 µmol) and F-LL (20 mg dry, 0.8 µmol), respectively. The Ellman test was used to determine the coupling efficiencies. Immobilization via Pyroglutamyl Formation. Reactions were carried out in 2 mL syringes, and the washing volumes were 1.5 mL. Resin G (20 mg dry, 2 µmol) and resin H (20 mg dry, 6 µmol) were washed with CH2Cl2 (3 × 1 min, 1 × 3 min) and deprotected with TFA-CH2Cl2 (1:1, 1.5 mL) and Et3SiH (5 µL, 30 µmol; 11 µL, 70 µmol, respectively) at room temperature. After 1 h, the solvents and excess reagents were filtered off, and the resin was washed with CH2Cl2 (3 × 1 min) and DMF (3 × 1 min, 1 × 3 min). GalA (2 mg, 96 µmol and 5 mg, 23 µmol, respectively) and imidazole (1 mg, 14 µmol and 2.4 mg, 35 µmol, respectively) were dissolved in DMF (1 mL), and the solutions were added to the resins. The syringes were placed in a sand bath at 60 °C for 24 h. After filtration, the resins were washed with DMF (2 × 1 min). Solutions of PyBOP (7 mg, 14 µmol and 18 mg, 35 µmol, respectively) and imidazole (1 mg, 14 µmol and 2.4 mg, 35 µmol, respectively) in DMF (1 mL) were added to the resins. Coupling took place at room temperature for 4 h. Solvents and excess reagents were removed by filtration, and the resins were finally washed with DMF (3 × 1 min), H2O (2 × 5 min), and DMF (2 × 1 min) and stored at 5 °C until quantitation. In a parallel fashion, (GalA)2 (2 mg, 5 µmol and 4.4 mg, 12 µmol) was coupled to resin G (10 mg dry, 1 µmol) and resin H (10 mg dry, 3 µmol), respectively, using 2 mL syringes and washing with 1 mL solvent. Quantitation by anthrone assay provided data on the coupling efficiencies. Cleavage of Immobilized Pectin Fragment from the Support. The cleavage reactions were performed in 2 mL syringes, and the washing volumes were 1 mL. Resins E (4.3 mg) and G (7 mg), both derivatized with GalA, were washed with H2O (3 × 1 min) and 50 mM tris(hydroxymethyl)aminomethane buffer (pH 8, 3 × 1 min) and then treated with DTT (1 mg, 6 µmol) in the same buffer (pH 8, 0.8 mL) at room temperature. After


22 h, the filtrates from the cleavage reactions were collected and combined with H2O washes (2 × 1 min). H2O (5 mL) was then added to each filtrate, and the samples were freeze-dried. For resin E, ES-MS, calcd for C11H20N2O7S2: 356.07. Found: m/z 395.07 [M + K]+, 379.09 [M + Na]+, 281.20 [M - C2H6NS + H]+. For resin G, ES-MS, calcd for C13H20N2O8S: 364.09. Found: m/z 363.06 [M - H]-. RESULTS AND DISCUSSION

In this comprehensive study, we present the development of efficient methodologies for the stable, covalent, and chemoselective anchoring of oligomers of GalA onto a solid support. To ensure that the methods developed could also be used for longer pectin fragments, the oligogalacturonides were not derivatized and were immobilized through their reducing end, which provides a unique functionality in the molecule. Several functional groups were installed on amino-terminated PEGA resins by classical solid-phase manipulations prior to immobilization of small GalA oligomers (see Scheme 1). These functionalities included amino, hydrazide, Cys, and Glu moieties. For two of the supports, we also synthesized variants containing a disulfide bridge which, under mild reducing conditions, allows cleavage and release of the immobilized material into solution. Preparation of Activated Solid Supports. Resin A was prepared by coupling Nβ-Boc-Aoa-OH to amino PEGA resin (0.4 mmol/g). Free aminooxy groups were obtained upon treatment with TFA in CH2Cl2. The corresponding loading was determined via an ‘indirect method’, in which trityl groups were coupled to the aminooxy-terminated groups on the support and subsequently released into solution for quantitation (34). Cleavage of the Trt groups with TFA-CH2Cl2 (1:1) was optimized by trapping of the released trityl cation with Et3SiH to form TrtH (35). For the loading determination, it was essential to efficiently dry and accurately weigh functionalized PEGA resins. For this purpose, we developed a procedure in which the resin was allowed to collapse gradually using an increasing concentration of Et2O in CH2Cl2 followed by drying in vacuo (42). This ‘indirect method’ was applied to resin A and gave a loading of 0.34 mmol/g (Table 1). The first step in the preparation of a hydrazide support was coupling of succinic anhydride to amino-terminated PEGA resins (0.4 mmol/g, HL and 0.2 mmol/g, LL) using HOBt and DIEA in DMF. The obtained resin B (HL) was further derivatized to resin C by treatment with hydrazine monohydrate in the presence of standard coupling reagents. The loading of hydrazide resin C was 0.38 mmol/g, according to an ‘indirect’ trityl quantitation as described above (see Table 1). For the preparation of a thiol-functionalized support, S-(2-pyridylthio)cysteamine hydrochloride was synthesized following a literature procedure (39) and coupled to B (LL) under standard coupling conditions. Subsequent treatment with DTT at pH 4.8 cleaved the disulfide bridge to give free thiol groups of resin D (see Scheme 1). A yellow coloration was observed, indicating release of pyridine-2-thione into solution. Resin D was then reacted with synthesized Boc-Cys(Trt)-NH-(CH2)2-S-SPyr via a disulfide exchange reaction in MeOH. Final deprotection of the Boc and Trt groups in the presence of Et3SiH (35) afforded Cys-functionalized resin E. The release of Trt groups in the last deprotection step was also used to determine the loading of resin E (‘direct method’). It was found to be 0.03 mmol/g (Table 1). The low value of the loading may be attributed to the large


Guillaumie et al.

Scheme 1. Preparation of Various Activated Solid Supports from Amino-Terminated PEGA Resins (0.2 mmol/g or 0.4 mmol/g)a

a (a) (i) Nβ-Boc-Aoa-OH, PyBOP, HOBt, DIEA, DMF, rt, 1.2 h, (ii) TFA-CH Cl (1:1), rt, 1 h; (b) succinic anhydride, HOBt, DIEA, 2 2 DMF, rt, 1.5 h; (c) hydrazine monohydrate, HBTU, HOBt, DIEA, DMF, rt, 2 h; (d) S-(2-Pyridylthio)cysteamine hydrochloride, HBTU, HOBt, DIEA, DMF, rt, 2 h; (e) DTT, H2O, pH 4.8, rt, 2 h; (f) Boc-Cys(Trt)-NH-(CH2)2-S-S-Pyr, MeOH, rt, 2 h; (g) TFA-CH2Cl2 (1:1), Et3SiH, rt, 1 h; (h) (i) Boc-Cys(Trt)-OH, PyBOP, HOBt, DIEA, DMF, rt, 1 h, (ii) TFA-CH2Cl2 (1:1), Et3SiH, rt, 1 h; (i) BocGlu(OtBu)-NH-(CH2)2-S-S-Pyr, MeOH, rt, 2 h; (j) TFA-CH2Cl2 (1:1), rt, 1 h; (k) (i) Boc-Glu(OtBu)-OH, PyBOP, HOBt, DIEA, DMF, rt, 1 h, (ii) TFA-CH2Cl2 (1:1), rt, 1 h.

Table 1. Efficiencies for the Immobilization of Oligomers of GalA % coupling efficiencyb (uncertainty) coupling technique reductive amination glycosyl hydrazide oxime thiazolidine 1 thiazolidine 2 glycosyl amide 1 glycosyl amide 2

initial resina

loading (mmol/g)

C C A F E H G

0.38c 0.38c 0.34c 0.10d 0.03d 0.29c 0.12c

GalA 13)e

93 (( 117 (( 21)e 117 (( 23)e quant.g quant.g 97 (( 18)e 90 (( 7)f

(GalA)2 15)e

81 (( 89 (( 21)e 120 (( 31)e quant.g quant.g 71 (( 14)e 75 (( 12)f

(GalA)3 n.d. 75 (( 23)e 62 (( 15)e quant.g n.d. n.d. n.d.

a See Scheme 1. b Based on the initial resin loading. c By ‘indirect’ trityl quantitation. d By ‘direct’ trityl quantitation. e Anthrone assay; assumptions: 0.05 mmol/g uncertainty on the loading, 0.2 mg uncertainty on the mass. f Anthrone assay; assumptions: 0.02 mmol/g uncertainty on the loading, 0.2 mg uncertainty on the mass. g Quantitative based on negative Ellman test. n.d. ) not determined.

number of steps from the starting material amino PEGA resins and/or to an incomplete coupling of Boc-Cys(Trt)NH-(CH2)2-S-S-Pyr on thiol-functionalized support. Cysterminated support F, with a permanent linkage, was directly obtained from amino PEGA resins (0.4 mmol/g) by standard coupling of Boc-Cys(Trt)-OH followed by acidic treatment under similar conditions as described for E. Direct quantitation of the amount of Trt groups released in the last step gave a loading of 0.10 mmol/g (Table 1). Access to resin F was faster and easier than to resin E, but in the latter case, a cleavable linker was installed through the disulfide bond. A similar strategy to that used for E and F was applied to install Glu moieties on solid support. Resin D was functionalized with synthesized Boc-Glu(OtBu)-NH-(CH2)2S-S-Pyr and the protecting groups were further removed under acidic conditions to give G. The loading was 0.12 mmol/g, according to an ‘indirect’ trityl quantitation (Table 1). As in E, the disulfide bridge introduced a potentially cleavable linkage. Alternatively, Boc-Glu(tBu)-OH was coupled to amino-terminated PEGA resin

(0.4 mmol/g). Subsequent deprotection allowed access to Glu-terminated resin, H (loading 0.29 mmol/g, after ‘indirect’ trityl quantitation; Table 1). Resins A, C, E, F, G, and H were then available for immobilization of pectin fragments. Methods for the Immobilization of GalA Oligomers. Seppa¨la¨ and Ma¨kela¨ (22) reported on the use of hydrazide groups as an alternative to amino groups to enhance, via the R-effect, the reactivity during coupling with various neutral oligosaccharides. In case of solidsupported reductive aminations, coupling conditions were also optimized by careful choice of the solvent and the reagents (43). These observations were combined and applied to immobilize model pectin fragments, GalA and (GalA)2, on a hydrazide-terminated resin (see Scheme 2). Typically, the solid-phase reactions were performed in DMF-AcOH (99:1) using 5 equiv of oligogalacturonide and 10 equiv of NaBH3CN at room temperature for 24 h. For larger pectin fragments, an excess of resin should be used. The immobilization efficiency was determined by the anthrone assay, based on the initial loading of



Scheme 2. Methods for the Solid-Phase Immobilization of Model Pectin Fragmentsa

a (a) NaBH CN, DMF-AcOH (99:1), rt, 24 h; (b) MeOH, 65 °C, 20 h; (c) DMF-H O (1:1), pH 4.8, 40 °C, 24 h; (d) H O-CH CN 3 2 2 3 (2:1), pH 4, rt, 24 h; (e) (i) imidazole, DMF, 60 °C, 24 h, (ii) PyBOP, imidazole, DMF, rt, 4 h.

resin C. This colorimetric method was chosen for several reasons. First, it is a general and well-established method for the quantitation of carbohydrates, including uronic acids (36). Second, it takes place at very low pH, using concentrated H2SO4, and high temperatures are required for the color development of the reagent. Such acidic conditions would cleave the glycosidic linkages along the oligogalacturonide chains, liberating single galacturonic acid units. These could then be quantified by comparison with standards. Furthermore, the harsh conditions were believed to dissolve the PEGA resin, allowing direct analysis of a solution containing oligogalacturonides. Contributions from the support were accounted for by comparing resin samples prior to and after immobilization. Analysis of GalA immobilized resin gave a 93 ((13)% coupling efficiency, which shows that one saccharide unit reacted with anthrone (Table 1). The uncertainty on the coupling efficiency, which takes into account intermediate uncertainties, was calculated using standard methods (Table 1). In case of (GalA)2, the coupling efficiency was 81 ((15)%, when two saccharide units were cleaved and reacted with anthrone. These results prove that reductive amination with hydrazide is an effective method for the immobilization of unprotected GalA oligomers to solid supports. And, whereas conventional methods rely on affinity studies to estimate the degree of coupling (28, 30), the quantitation method developed hereby directly estimates the amount of pectin immobilized on the support. However, the data obtained are in contradiction with the literature (36, 44, 45). The mechanism for the reaction of anthrone with carbohydrates is complex and not fully elucidated: it involves the formation of strongly colored furfural derivatives when anthrone reacts with the aldehyde group at the reducing end of the carbohydrate. Since solid-phase anchoring of oligogalacturonides onto hydrazide-terminated resin under reducing conditions gives a modified saccharide, the reducing sugar is transformed into an open ring hydrazide. Thus, the anthrone assay was not

expected to give a positive response when analyzing GalA-immobilized resin and to show two saccharide units when analyzing (GalA)2 immobilized support. A second approach which we have implemented for the immobilization of underivatized GalA oligomers was based on the formation of an oxime bond. This type of linkage has recently become a popular method for preparing bioconjugates (46, 47), peptide dendrimers (48), glycopeptides (29), carbopeptides (49), and carboproteins (50). The efficiency of the method relies on the ease of formation and on the good stability of the oxime linkage up to pH 9. This well-established methodology appears not to have been used for the immobilization of polyuronic acids. Oligogalacturonides, ranging from the monomer to the trimer, were coupled to resin A through the aldehyde group at their reducing end. Oxime formation took place under mild conditions, in DMF-H2O (1:1), at 40 °C and pH 4.8 for 24 h (33). Incorporation of GalA oligomers was quantified by the anthrone assay. The coupling efficiencies, based on the initial loading of resin A, were 117 ((23)%, 120 ((31)% and 62 ((15)% for immobilized GalA, (GalA)2, and (GalA)3, respectively (Table 1). In each case, the contribution of the preimmobilization resin was subtracted. In contrast to reductive amination, the sugar structure was preserved after coupling; an equilibrium between the closed ring and open ring forms might occur at the surface of the support. This efficient technique requires few derivatization steps on solid-phase and its compatibility with aqueous solvents makes it very attractive when using larger pectin fragments with limited solubilities. The direct coupling between the reducing end of various neutral oligosaccharides with hydrazide groups has recently been investigated in carbohydrate recognition studies (51) and in the synthesis of surfactants (52), demonstrating the stability of the glycosyl hydrazide linkage under neutral to basic conditions. We decided to include this technique in our study, as an alternative to other linkage types. Prior to solid-phase immobilization,


Guillaumie et al.

Scheme 3. Solution Synthesis of Glycosylhydrazide and Thiazolidine Derivatives of GalAa

a

(a) MeOH, 65 °C, 7 h; (b) MeOH, rt, 24 h.

a model study was conducted in solution (see Scheme 3). Following the conditions described by Auge´ and LubinGermain (52), GalA was coupled to acetic acid hydrazide (40) in MeOH at 65 °C for 7 h to give the corresponding glycosyl hydrazide in 99% yield. NMR analysis revealed the presence of two acyclic glycosyl hydrazides, most likely the E and Z isomers (2:1). These optimized conditions were then applied to the coupling of GalA, as well as its dimer and trimer to hydrazide functionalized resin C (Scheme 2). The reaction time was extended to 20 h to compensate for the slower reaction rates generally encountered with solid supports. Quantitation by the anthrone assay showed 117 ((21)%, 89 ((21)% and 75 ((23)% immobilization efficiencies for GalA, (GalA)2, and (GalA)3, respectively (Table 1). The coupling yield decreased with increasing size of the oligogalacturonides. Although an unsaturated hydrazone compound was isolated after solution synthesis, an equilibrium between this form and the closed ring glycosyl hydrazide might occur at the surface of the support. Despite its high coupling efficiency and its ease of preparation, this third approach suffers from poor stability of the linkage at low pH. Acid present in the media could indeed protonate the glycosidic nitrogen at the reducing end and cleave the glycosidic bond. A well-documented methodology for the covalent linkage of oligosaccharides to other molecules or matrixes is based on the formation of a glycosylamine derivative. The reaction is reversible, and a further acylation step is required to stabilize the glycosylamine (53). This latter step can proceed by treatment with acetic anhydride (30). Alternatively, a method for the preparation of glycoconjugates has recently been developed by Que´tard et al. (31, 32) in which various neutral oligosaccharides were reacted with glutamic acid derivatives to form the corresponding glycosylamines. In each case, stabilization of the linkage proceeded via an intramolecular acylation by the activated carboxyl group on the side chain of the glutamyl moiety. The potential of this elegant technique has not been exploited to a large extent, and we wish to report here its application to solid-phase immobilization of model pectin fragments. The designed solid-phase strategy included the introduction of two different spacers with a terminal Glu residue on the resin. Glu-functionalized resin H would allow for permanent immobilization, whereas resin G contained a linker cleavable in the form of a disulfide bridge. The attachment of GalA and (GalA)2 proceeded according to the conditions described by Que´tard et al. (31, 32). Typically, the reducing end of the oligomer of GalA was reacted with the amino group of the glutamyl acid in DMF in the presence of imidazole for 24 h to form a glycosylamine (Scheme 2). The reactivity was improved by increasing the temperature to 60 °C. After removal of the excess reagents, the coupling agent PyBOP was added, and the intramolecular acylation of the secondary amine and concomitant

pyroglutamyl ring formation was allowed to proceed at room temperature for 4 h. Quantitation of the reaction was performed using the anthrone assay. Immobilization of GalA and (GalA)2 on a short spacer resin (H-LL) proceeded with an efficiency of 97 ((18)% and 71 ((14)%, respectively (Table 1). Moreover, GalA and (GalA)2 were immobilized on long spacer resin (G-HL) with equally high efficiency: 90 ((7)% and 75 ((12)% respectively (Table 1). In this latter case, an uncertainty of 0.02 mmol/g on the resin loading was assumed. Resin H-LL functionalized with GalA was treated with DTT. Subsequent cleavage of the disulfide bond released a pyroglutamyl derivative of GalA as the major compound upon MS analysis. Finally, both early (54) and recent (55) studies reported on the formation of a thiazolidine ring when reacting a Cys derivative with an aldehyde. Various thiazolidine compounds have so far been prepared from carbohydrates (56-60), and the stability of the linkage over a large range of pH (3-9) has recently been illustrated in the synthesis of peptide dendrimers (33). However, this very efficient methodology has apparently not been extended to the preparation of larger glycoconjugates nor to immobilization on solid supports. We therefore decided to include the formation of a thiazolidine ring in our general strategy for the solid-phase immobilization of pectin fragments. First, optimization of the available conditions was required, and model reactions were conducted using GalA and Cys methyl ester (H-Cys-OMe, Scheme 3). According to earlier studies, the reaction between an 1,2-aminothiol and an aldehyde proceeds very rapidly at pH 4-5 (54, 55). Such acidic conditions were also preferred in order to avoid disulfide formation of the Cys residue. Reaction of GalA with H-Cys-OMe in MeOH gave the expected thiazolidine in 50% yield. These conditions were then modified for immobilization on a solid support. GalA, (GalA)2, or (GalA)3 dissolved in H2OCH3CN (2:1) at pH 4 and were coupled to resins E or F at room temperature for 24 h (Scheme 2). The anthrone assay was not suitable for on-resin quantitation due to a very high absorbance of the preimmobilization resin. Support bound thiol nucleophiles could theoretically react with the carbonyl group of anthrone. The Ellman test is, however, a well-established assay for the quantitation of free thiol groups (37, 38, 61). It was used in our study as an alternative to the anthrone assay for this particular reaction. Conditions for the application of the Ellman test to our system were optimized in terms of sample preparation and analytical technique. Postimmobilization resins were analyzed for their content of free thiol groups, by treatment with the Ellman reagent, DTNB. In all cases, a negative test was obtained, corresponding to a quantitative coupling of the GalA oligomers (see Table 1). Disulfide formation between Cys moieties could in principle also account for a negative test, but this was ruled out by a control experiment on the preimmobiliza-


tion resins, which showed excellent response to the Ellman test. This last strategy therefore proved very efficient for the anchoring of pectin fragments on solid support. In addition, a very stable linkage was installed in a few steps. Finally, this coupling method was validated by conducting a cleavage test on resin E functionalized with GalA; reduction of the disulfide linkage with DTT released the corresponding thiazolidine derivative. Extension of this new coupling chemistry to immobilization of other oligosaccharides and the synthesis of glycoconjugates can easily be envisaged. CONCLUSION

We have prepared new hydrazide, aminooxy, and glutamyl-functionalized PEGA supports designed for chemoselective immobilization of oligomers of GalA by hydrazide, oxime, and pyroglutamyl formation, respectively. As an alternative to these well-established immobilization chemistries, we also prepared Cys-functionalized PEGA resins to immobilize GalA oligomers by novel thiazolidine formation. In addition to supports which allow permanent and covalent anchoring of saccharides, we also developed Glu- and Cys-functionalized supports in which a cleavable disulfide linkage was inserted. This strategy allows release of the saccharide derivatives under mild selective conditions. The loading of each functionalized support was determined either by an ‘indirect’ trityl quantitation, based on a couplingcleavage strategy, or by a ‘direct’ trityl quantitation, when trityl groups were part of the synthetic pathway. All supports allowed for efficient immobilization of oligomers of GalA, i.e., unprotected uronic acids, under mild conditions and without modification of the oligosaccharide prior to immobilization. Most of the coupling chemistries were first validated in solution. The two preferred immobilization methods in this study were oxime formation and formation of a thiazolidine. We anticipate that the novel thiazolidine coupling chemistry may also be applied to the immobilization of other saccharides. These methods are currently being used in our laboratories in the solid-phase analysis and the sequencing of pectin fragments. ACKNOWLEDGMENT

We wish to thank the following: Dr. Andrew Coffey (Polymer Laboratories, UK) for the gift of PEGA resins; Dr. Tove Christensen (Danisco Cultor, Denmark) for supplying GalA oligomers; Mr. Clive Phipps Walter of Danisco Cultor, Denmark, and Associate Professor Jørgen Øgaard Madsen and Mrs. Anne Hector from the Department of Chemistry, Technical University of Denmark, for performing various MS analyses. Finally, we are grateful to: Dr. Gerrit Limberg for initiating this project, the Danish Technical Science Research Council for financial support through a ‘Centerkontrakt’ grant, and the Alfred Benzon Foundation for a Bøje Benzon fellowship to K.J.J. LITERATURE CITED (1) Voragen, A. G. J., Pilnik, W., Thibault, J.-F., Axelos, M. A. V., and Renard, C. M. G. C. (1995) Pectins. Food polysaccharides and their applications (A. M. Stephen, Ed.) pp 287339, Marcel Dekker Inc., New York. (2) May, C. D. (1990) Industrial pectins: sources, production and applications. Carbohydr. Polym. 12, 79-99. (3) Rombouts, F. M., and Thibault, J.-F. (1986) Enzymic and chemical degradation and the fine structure of pectins from sugar-beet pulp. Carbohydr. Res. 154, 189-203.

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BC0155364

Immobilization of Pectin Fragments on Solid Supports: Novel Coupling

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