Synthesis and Macrophage Activation of Lentinan-Mimic Branched

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Synthesis and Macrophage Activation of Lentinan-Mimic Branched Amino Polysaccharides: Curdlans Having N-Acetyl-D-glucosamine Branches Keisuke Kurita,*,† Yuriko Matsumura,† Hiroki Takahara,† Kiyoshige Hatta,‡ and Manabu Shimojoh*,‡ † ‡

Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, Musashino-shi, Tokyo 180-8633, Japan Research and Development Center, Toyo Suisan Kaisha, Ltd., Shiohama, Koto-ku, Tokyo 135-0043, Japan

bS Supporting Information ABSTRACT: N-Acetyl-D-glucosamine branches were incorporated at the C-6 position of curdlan, a linear β-1,3-D-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a β-1,3-Dglucan having D-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection
deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a D-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.

’ INTRODUCTION Certain branched polysaccharides show important biological, physiological, and medicinal functions including antitumor and immunostimulating activities.1
6 For example, branched glucans such as lentinan7 and schizophyllan,8 extracted from mushrooms and having β-1,6-glucose branches on the β-1,3-D-glucan backbone, have been used clinically in cancer chemotherapy as immunoadjuvants. However, it is difficult to provide these branched β-glucans in quantity. Furthermore, their solubility is generally poor, and lentinan is almost insoluble in water. These factors have undoubtedly limited both the therapeutic applications and basic studies despite their potential. A clue to solving these problems may be found in establishing synthetic approaches to branched polysaccharides because the presence of proper sugar branches on polysaccharide backbones is critical for expressing distinctive biological activities.9 Introduction of branches into linear polysaccharides will therefore be a key to facilitate the studies on the development of polysaccharide drugs. Our interest has been focused on the regioselective and controlled chemical modifications to introduce sugar branches, such as D-galactose,10 D-mannose,11 D-maltose,12 Dglucosamine,13 and N-acetyl-D-glucosamine13 into linear polysaccharides chitin and chitosan with the view of synthesizing tailored branched polysaccharides. The resulting nonnatural branched polysaccharides exhibited various unique properties including solubility, moisture absorption, lysozyme susceptibility, and bactericidal activity. Recently, we reported the efficient r 2011 American Chemical Society

production of tumor necrosis factor (TNF-R) and nitric oxide (NO) by direct stimulation of macrophages with chitins having N-acetyl-D-glucosamine branches at the C-6 position,14 and such activity offers encouraging prospects of the nonnatural branched polysaccharides, especially those containing amino sugars, as immunomodulating agents. These results prompted us to study further the structure
activity relationship to develop better immunoadjuvants for chemotherapy than lentinan for actual clinical use. To elucidate the influence of amino sugar branches on the activity, it is necessary to establish a procedure for introducing N-acetyl-Dglucosamine units into curdlan, the main chain of lentinan, and a possibility of transformation reaction was shown briefly in a preliminary communication.15 Here we report the results of detailed studies on the synthetic manipulations to branched curdlans and on the macrophage activation by the products.

’ EXPERIMENTAL SECTION General. IR spectra were recorded on a Shimadzu FTIR-8900 instrument by the KBr method. 1H and 13C NMR spectra were obtained with a JEOL JNM-LA400D in deuterated dimethyl sulfoxide (DMSOd6) at 30 °C or in D2O at ambient temperature at 400 and 100 MHz. Received: March 15, 2011 Revised: April 27, 2011 Published: April 28, 2011 2267

dx.doi.org/10.1021/bm200353m | Biomacromolecules 2011, 12, 2267–2274

Biomacromolecules Elemental analysis was performed on a Perkin-Elmer 2400 II instrument. Solvents such as DMSO, N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF), and pyridine were purified in usual manners and stored over molecular sieves. All chemicals were of reagent grade and used without further purification. Molecular Weight Measurement. GPC for organosoluble substances was conducted with a Shimadzu LC-10AD (column, Shodex GPC KD-806M; solvent, DMAc containing 5% LiCl; flow rate, 0.5 mL/min) at 40 °C, pullulan standards being used for molecular weight calibration. Conditions for water-soluble substances: column, Shodex OHpak SB-G þ Shodex OHpak SB-804HQ; solvent, CH3CO2H (0.07 mol/L)/ CH3CO2Li (0.05 mol/L)/H2O; flow rate, 0.5 mL/min; temperature 40 °C; standards, pullulans.16 Viscometry was carried out with a rotational viscometer (Visconic ED, Tokyo Keiki) and an Ubbelohde viscometer (no. 2613-001, Shibata Scientific Technology) to determine the intrinsic viscosity and thereby molecular weight as described for chitin.17 Triphenylmethylation of Curdlan. To a solution of 1.00 g (6.17 mmol) of curdlan in a mixed solvent of 30 mL of pyridine and 40 mL of DMSO was added 17.19 g (0.0617 mol) of chlorotriphenylmethane (trityl chloride). After the solution was stirred at 80 °C for 24 h under a nitrogen atmosphere, the solution was poured in 200 mL of methanol to precipitate the product. It was collected by filtration, washed with 250 mL of methanol, and dried to give 2.23 g (89%) of 6-O-trityl-curdlan as a white powdery material. The degree of substitution (ds) was 1.00, as calculated from the C/N value of elemental analysis. IR (KBr): ν 3470 (O
H), 3057 (C
H), 1150
1000 (pyranose), and 764, 746, and 704 cm
1 (arom). 1H NMR (DMSOd6): δ 3.3
4.9 (pyranose) and 7.1
7.3 (phenyl). Anal. Calcd for C25H24O5: C, 74.24; H, 5.98. Found: C, 74.46; H, 5.92. Chemical Analysis of 6-O-Trityl-Curdlan. The ds for trityl was also confirmed from the weight of triphenylmethanol obtained by hydrolysis with sulfuric acid, as reported for tritylated cellulose.18 In brief, 0.5003 g of 6-O-trityl-curdlan was added to 5 mL of concentrated sulfuric acid portionwise, and after dissolution was complete in 30 min, 45 mL of deionized water was added dropwise. The white precipitate was collected by filtration on a sintered glass filter, washed with deionized water until neutral, and dried. The amount of triphenylmethanol was 0.3223 g, which corresponded to the ds of 1.00. Phenylcarbamoylation of 6-O-Trityl-Curdlan. 6-O-Tritylcurdlan (1.00 g, 2.47 mmol) obtained above was dissolved in 10 mL of pyridine by heating, and 1.45 g (12.4 mmol, 2.5 equiv) of phenyl isocyanate was added at room temperature. The solution was heated to 100 °C for 24 h in nitrogen with stirring. After cooling to room temperature, the same amount of phenyl isocyanate was added, and the solution was stirred for an additional 24 h at 100 °C. The product was precipitated in a mixture of 320 mL of methanol and 20 mL of saturated aqueous sodium chloride. It was filtered, washed with 300 mL of deionized water and with 150 mL of methanol, and dried to give 1.38 g (87%) of 2,4-di-O-phenylcarbamoyl-6-O-trityl-curdlan as a pale tan powder. The ds values for trityl and carbamoyl were 1.0 and 2.0 as calculated from the C/N value of the elemental analysis. IR (KBr): ν 3400 (N
H), 3060 (C
H), 1740 (CdO), 1205 (ester), 1150
1000 (pyranose), and 758 and 692 cm
1 (arom). 1H NMR (DMSO-d6): δ 3.6
4.7 (pyranose), 6.8
7.2 (phenyl), and 7.72 and 8.51 (NH). Anal. Calcd for C39H34N2O7 3 0.1H2O: C, 72.68; H, 5.35; N, 4.35. Found: C, 72.62; H, 5.21; N, 4.40.

Detritylation of 2,4-Di-O-phenylcarbamoyl-6-O-trityl-Curdlan. To 10 mL of dichloroacetic acid was added 1.00 g (1.56 mmol) of 2,4-di-O-phenylcarbamoyl-6-O-trityl-curdlan portionwise over a period of 10 min for dissolution. The solution was stirred at room temperature for 2 h and poured in 500 mL of ice water slowly. The precipitate was collected by filtration, washed with methanol overnight, and dried to give 0.502 g (86%) of 2,4-di-O-phenylcarbamoyl-curdlan as a pale tan

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powder. The ds for carbamoyl was 2.0, as calculated from the C/N value of the elemental analysis. IR (KBr): ν 3400 (O
H, N
H), 1724 (CdO), 1223 (ester), 1150
1000 (pyranose), and 758 and 692 cm
1 (arom). 1H NMR (DMSO-d6): δ 3.3
4.4 (pyranose), 7.0
7.4 (phenyl), and 8.40 and 8.88 (N
H). Anal. Calcd for C20H20N2O7 3 0.8 H2O: C, 57.91; H, 5.25; N, 6.75. Found: C, 57.89; H, 5.35; N, 6.71.

Preparation of the Oxazoline Donor from D-Glucosamine. The oxazoline derivative was prepared by peracetylation of D-glucosamine, followed by treatment with trimethylsilyl trifluoromethanesulfonate, as reported.19 It was purified by column chromatography on silica gel with toluene/ethyl acetate/triethylamine (100/200/1) to give a pale tan oil: yield, 83%. Glycosylation of 2,4-Di-O-phenylcarbamoyl-Curdlan. To a solution of 2.46 g (7.5 mmol) of the above-obtained oxazoline in 50 mL of 1,2-dichloroethane were added 1.00 g (2.5 mmol) of 2,4-di-Ophenylcarbamoyl-curdlan and 0.1 g (0.43 mmol) of (þ)-10-camphorsulfonic acid. The mixture was stirred at 80 °C for 24 h in nitrogen, and the resulting solution was poured in 300 mL of diethyl ether. The precipitate was collected by centrifugation and washed with 300 mL of a mixture of methanol and deionized water (1:1) overnight. After drying, 1.56 g of the glycosylated product was obtained. The ds for the protected N-acetyl-D-glucosamine branches was 0.70, as determined by the peak area ratio of acetyl/phenyl in the 1H NMR spectrum, and the yield was 98% based on the ds value. The ds was also calculated from the C/N value of the elemental analysis to be 0.71. IR (KBr): ν 3350 (O
H, N
H), 1747 (CdO), 1670 (amide I), 1225 (ester), 1150
1000 (pyranose), and 758 and 692 cm
1 (arom). 1H NMR (DMSO-d6): δ 1.63
2.00 (acetyl), 3.7
5.1 (pyranose), 6.97
7.39 (phenyl), and 7.90 and 8.70 (N
H). Anal. Calcd for (C34H39N3O15)0.71 (C20H20N2O7)0.29 3 0.5H2O: C, 55.91; H, 5.41; N, 5.90. Found: C, 55.89; H, 5.32; N, 5.90.

Synthesis of Curdlan Having N-Acetyl-D-glucosamine Branches. The glycosylated product obtained above, 0.60 g, was dissolved in 30 mL of 1,4-dioxane, and a solution of 0.60 g of sodium in 30 mL of methanol was added dropwise, resulting in a white precipitate. The mixture was heated to 60 °C for 2 h with stirring in nitrogen, dialyzed with deionized water for 2 days, and freeze-dried to give 0.156 g of the product, curdlan having N-acetyl-D-glucosamine branches. The ds determined by the C/N value of the elemental analysis was 0.74, and the yield was thus calculated to be 53%. IR (KBr): ν 3340 (O
H, N
H), 1651 (amide I), 1558 (amide II), and 1150
1000 cm
1 (pyranose). 1H NMR (D2O): δ 1.88 (acetyl) and 3.3
4.4 (pyranose). 13 C NMR (D2O): δ 23.0 (CH3), 61.3 (C-20 ), 68.1 and 68.7 (C-6,60 ), 70.4 (C-4), 73.8 (C-40 ), 74.4 (C-2,30 ), 76.4 (C-5, C-50 ), 84.7 (C-3), 102.6 and 103.2 (C-1,10 ), and 175.8 (CdO). Anal. Calcd for (C14H23NO10)0.74(C6H10O5)0.26 3 2H2O: C, 41.09; H, 6.83; N, 2.98. Found: C, 41.07; H, 6.64; N, 2.98. Cell Culture and Reagents. Mouse macrophagelike cell line RAW264.7 was obtained from American Type Culture Collection, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS). L929 cells were obtained from Riken Cell Bank and cultured in Eagle’s minimum essential medium (MEM) containing 2 mM Lglutamine and 10% heat-inactivated FBS. B16, Vero, Mv.1.Lu, and WI-38 cells were also obtained from Riken Cell Bank. B16, Vero, and WI-38 cells were cultured in MEM containing 2 mM L-glutamine and 10% heat-inactivated FBS. Mv.1.Lu cells were cultured in MEM containing 2 mM L-glutamine, 1% (v/v) nonessential amino acids, and 10% heat-inactivated FBS. Lentinan (ds 0.4) was purchased from Ajinomoto. The vial contained 1 mg of lentinan along with 100 mg of D-mannitol and 2 mg of dextran 40. Under aseptic conditions, 2 mL of ultrapure water was added to the vial, and the mixture was shaken vigorously prior to use. All reagents such as DMEM, MEM, L-glutamine, phosphatebuffered saline (
) (PBS(
)), FBS, recombinant murine TNF-R, 2268

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Biomacromolecules lipopolysaccharide (LPS, from Escherichia coli 0111:B4), and recombinant mouse interferon-γ (IFN-γ) were purchased from the same suppliers as mentioned in the previous paper.14 Limulus Amebocyte Lysate Assays. The endotoxic activity of branched curdlan solutions (1 mg/mL) was determined by quantitative kinetic assay based on the reactivity of Gram negative endotoxin with Limulus amebocyte lysate (LAL) at 37 °C, using test kits of Limulus ES-J Test Wako (Wako Pure Chemical Industries).14,20 Determination of TNF-r Secretion in RAW264.7 Cells. The assay of TNF-R secretion in RAW264.7 cells was carried out by the same method as that previously described.14 In brief, the cells were incubated with DMEM containing 10% heat-inactivated FBS for 24 h, and a branched curdlan solution (final concentration, 50 μg/mL) or lentinan solution (final concentration, 50 μg/mL) was added. After incubation for 8 h, TNF-R secretion was measured using the L929 cell bioassay procedure.21 Assay for NO Secretion in RAW264.7 Cells. NO secretion was determined, as described in the previous paper.14 RAW264.7 cells were incubated with DMEM containing 10% heat-inactivated FBS for 24 h, and a branched curdlan solution (final concentration, 50 μg/mL) or lentinan solution (final concentration, 50 μg/mL) was added. The amount of NO was measured with the Griess reagent system (2% sulfanilamide, 0.2% naphthylethylene diamine dihydrochloride, and 5% phosphoric acid).22 Effect of IFN-γ on NO Secretion. RAW264.7 cells (5  105) were treated with LPS (2 ng/mL), IFN-γ (20 units/mL), or both. The cells (5  105) were also treated with a branched curdlan (ds 0.32, 50 μg/mL), IFN-γ (20 units/mL), or both. Similarly, they were treated with lentinan (50 μg/mL), IFN-γ (20 units/mL), or both. After 24 h at 37 °C, NO in the supernatant was determined using the Griess reagent.14 MTT Assay for Cytotoxic Effect. Cell-mediated reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Dojindo Lab.) was assayed in a previously reported manner14 based on Mosmann’s method.23 In brief, a cell suspension (3.0  103) was cultured overnight at 37 °C in an atmosphere containing 5% CO2. An equal volume of the branched curdlan of ds 0.32 (final concentration, 500 μg/mL) or lentinan (final concentration, 500 μg/mL) was added to each well, and the cells were incubated for 24 h at 37 °C. A stock MTT solution (5 mg/mL in PBS(
), 10 μL) was added, and after incubation for an additional 4 h, the reduction of MTT was determined.

’ RESULTS AND DISCUSSION To examine the influence of the chemical structure of branches on the activity and thereby to develop more potent immunomodulating agents than lentinan (1), we replaced the side branch D-glucose of lentinan by N-acetyl-D-glucosamine in view of the various distinctive biological activities of the amino monosaccharide as well as its oligomers and polymers.24 Curdlan was thus converted to the target hybrid molecule, branched curdlan (2) (Scheme 1) by a series of controlled chemical modification reactions. Curdlan Acceptor. As suggested from our previous studies on the site-specific introduction of sugar branches into chitin and chitosan,10
13 curdlan should be protected appropriately to give branched derivatives with well-defined molecular structures. Although diphenylcarbamoylated curdlan was reported in the literature,25 the structure was not fully supported. To synthesize branched polysaccharides by glycosylation, it is crucial to use a structurally unambiguous acceptor having a free hydroxyl at C-6 and protected hydroxyls at C-2 and C-4. Curdlan was thus first treated with trityl chloride in pyridine/DMSO to give trityl-curdlan (3) (Scheme 2), and the ds was 1.0, as determined by elemental analysis and quantitative chemical analysis of triphenylmethanol

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Scheme 1. Lentinan (1) and Branched Curdlan (2)

Scheme 2. Synthesis of Acceptor 5a

(i) TrCl, pyridine, 90 °C; (ii) Ph-NdCdO, pyridine, 100 °C; (iii) dichloroacetic acid, room temperature.

a

obtained by acidic hydrolysis of 3. IR (Figure 1) and 1H NMR (Figure 2) spectroscopies also confirmed the structure. The remaining C-2 and C-4 hydroxyl groups were then protected by phenylcarbamoylation in pyridine, but the ds values after the reaction with excess phenyl isocyanate (5 equiv) at 100 °C for 24, 48, and 72 h were almost in a similar level, 1.81
1.85. The reaction was therefore repeated two times with 2.5 equiv of isocyanate for 24 h to attain the ds 2.0. The IR spectrum of the fully substituted derivative (4) in Figure 1 showed bands at 3400 and 1740 cm
1 characteristic of carbamoyl NH and CdO. In the NMR spectrum in Figure 2, two weak peaks due to NH were observed at 8.40 and 8.88 ppm besides strong aromatic peaks. Subsequent deprotection at C-6 was effected with dichloroacetic acid to give the acceptor (5), and the IR spectrum showed strong OH bands and weak aromatic CH bands (Figure 1). All of these transformation reactions proceeded smoothly in solution and quantitatively with regard to the ds, leading to the synthesis of structurally well-defined 5 for glycosylation. Glycosylation Reaction. Glycosylation of 5 with the oxazoline (6) derived from D-glucosamine was conducted in 1,2-dichloroethane with camphorsulfonic acid (CSA) as the catalyst, giving rise to the glycosylated product (7) (Scheme 3). The progress of 2269

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reaction could be followed by 1H NMR spectroscopy (acetyl (1.6
2.0 ppm)/phenyl (6.9
7.4 ppm) peak ratio) and elemental analysis (C/N ratio), and the values were close to each other. The glycosylation reaction of 5 proceeded quite facilely compared with that of phthaloylated chitosan,10
13 and substantial substitution was achieved within a few hours at 80 °C, as listed in Table S1 in the Supporting Information. For instance, the ds was ∼0.5 after 1 h with an equivalent amount of 6. Judging from the substitution degrees, however, 24 h of reaction was considered preferable. To examine the influence of the donor amount, glycosylations were carried out with various donor/acceptor ratios under

carefully controlled reaction conditions for 24 h, and the results are shown in Figure 3. The ds increased steadily with the amount of donor, and with 10 equiv of 6, the ds reached 1.0. The result is in sharp contrast with the maximum ds values of around 0.65 in the glycosylation of phthaloylated chitosan,10
13 implying that the β-1,3 backbone is much more accessible than β-1,4. The yields of 7 were satisfactory in the range of 80
95%. Figures 1 and 2 include typical IR and NMR spectra of 7. In the IR spectrum, the OH bands were weak, and a band ascribable to the amide of side branches appeared at 1670 cm
1, which became strong with an increase in the ds. Molecular weight characteristics of curdlan and the derivatives were examined by GPC and viscometry.26 As summarized in Table S2 (Supporting Information), the viscosity-average

Figure 1. IR spectra of curdlan and the derivatives.

Figure 2. 1H NMR spectra of the curdlan derivatives in DMSO-d6.

Scheme 3. Synthesis of Branched Products 7 and 2a

a

(i) CSA, ClCH2CH2Cl, 80 °C; (ii) Na, MeOH/dioxane, 60 °C. 2270

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Figure 5. Conversion of glycosylated derivatives 7 (dotted bars) having different ds values to the corresponding 2 (hatched bars). Figure 3. Influence of the amount of 6 on the degree of substitution of 7.

Figure 6. TNF-R secretion in RAW264.7 cells (5  105) incubated with branched curdlans 2 or lentinan (LNT) (50 μg/mL) at 37 °C for 8 h. (The data shown are the means ( SE of three individual experiments.)

Figure 4. 1H and 13C NMR spectra of 2 (ds 0.74) in D2O.

molecular weight Mv of curdlan by rotational and Ubbelohde viscometers was around 900 000, and the number-average molecular weight Mn by GPC was 356 000. Tritylation turned out to degrade the curdlan main chain because of the harsh reaction conditions, and the Mn of 3 was 19 000. After phenylcarbamoylation and detritylation, the Mn of 5 was 9600. Subsequent glycosylation did not reduce the molecular weight significantly. The polydispersities of 5 and 7 were narrow, probably owing to the reprecipitations during the multistep synthetic procedure. Curdlans Having N-Acetyl-D-glucosamine Branches. The O-phenylcarbamoyl and O-acetyl groups of 7 were selectively removed to afford the target product 2. Because 7 was soluble in 1,4-dioxane but only swelled in methanol, it was subjected to transesterification with sodium in methanol/1,4-dioxane. After dialysis and freeze-drying, 2 was obtained as a pale tan fibrous material. The structure of 2 was supported by elemental analysis and spectroscopies. The IR spectrum indicated the absence of Oprotective groups (Figure 1). In the 1H NMR spectrum, a sharp singlet N-acetyl peak was observed besides the pyranose peaks, and the peaks due to phenyl groups disappeared (Figure 4). The 13 C NMR spectrum in Figure 4 showed peaks due to both the main chain curdlan (peaks 1
6) and the side branch N-acetyl-Dglucosamine (peaks 10 -60 , CH3, and CdO). The results of deprotection of various glycosylated derivatives 7 having different ds values to the corresponding 2 are shown in Figure 5. As apparent there, the ds values of products 2 were close

to those of starting materials 7, indicating that the side branches were not removed under these deprotection conditions. The molecular weights of typical samples were measured in an aqueous solvent, and the Mn values were a little over 10 000 (Table S3 in the Supporting Information). Solubility. Intermediates 3, 4, and 5, and glycosylated derivative 7 were readily soluble in organic solvents such as DMSO, DMF, and pyridine, which enabled a series of chemical modifications to proceed facilely and quantitatively in solution. Final product 2 exhibited much higher solubility and was soluble even in water at neutral pH in addition to common organic solvents. The solubility data are reported in Table S4 in the Supporting Information. The solubility of 2 in a wide variety of solvents is rather surprising, considering the fact that curdlan swells only to low extents in water and most organic solvents except DMSO, and lentinan 1, a curdlan derivative having D-glucose branches, is scarcely soluble in water, ethanol, and acetone. These data imply that N-acetyl-D-glucosamine is much superior to D-glucose as branches to improve solubility. Endotoxins Possibly Contained in Branched Curdlans. Prior to the evaluation of immunomodulating activity of branched curdlans 2, the possibility of endotoxin contamination should be examined because endotoxins stimulate macrophages to induce secretion of NO and cytokines including TNF-R. The branched curdlan solutions were hence incubated with the LAL reagent at 37 °C for 60 min, but no aggregation was observed. Because the detection limit of the LAL kit was