Three-Dimensional Arrangement of Sugar Residues along a Helical

Three-Dimensional Arrangement of Sugar Residues along a Helical Polypeptide Backbone: Synthesis of a New Type of Periodic Glycopeptide by ...
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Biomacromolecules 2002, 3, 775-782

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Three-Dimensional Arrangement of Sugar Residues along a Helical Polypeptide Backbone: Synthesis of a New Type of Periodic Glycopeptide by Polymerization of a β-O-Glycosylated Tripeptide Containing r-Aminoisobutyric Acid Akinori Takasu,* Takuma Houjyou, Yoshihito Inai, and Tadamichi Hirabayashi Department of Environmental Technology and Urban Planning, Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan Received January 30, 2002; Revised Manuscript Received April 23, 2002

A new type of glycopeptide having a periodic sequence of -[L-Glu(OMe)-Ser(β-D-GlcNAc)-Aib]- was synthesized by polymerization of a glycosylated tripeptide with diphenylphosphoryl azide (DPPA) and active ester methods using H-L-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]-Aib-OH (13) and H-L-Glu(OMe)-Ser[β-DGlcNAc(Ac)3]-Aib-ONp (15, Np ) p-nitrophenyl) as the monomers, respectively. Number-average molecular weights were determined by size exclusion chromatography (SEC) and matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometry, those in the latter method were higher than those in the former one. CD and FT IR spectra of poly(13) and poly(15) indicated that they form righthanded helical conformations. Deacetylation of the acetylated glycopeptide was established without racemization using hydrazine/methanol. CD spectra of the deacetylated glycopeptides 16 (21 and 24 residues) in water showed negative Cotton effect at wavelength of 208 and 222 nm indicating an R-helical conformation, i.e., N-acetyl-D-glucosamine (GlcNAc) moieties were arranged spatially along the R-helical peptide keeping a specific distance and orientation in water. Addition of ethanol to aqueous solutions of the periodic glycopolymer 16 resulted in an increase in the R-helix content. Semiempirical molecular orbital calculation also supported the R-helical conformation of 16. Introduction Recent progress in glycobiology has showed that cell surface oligosaccharides play essential roles in various biological recognition process, including intercellular recognition, adhesion, cell growth, and differentiation.1 Although the recognition process is essentially based on carbohydrateprotein interactions, individual interactions are generally low. Some glycopolymers in which saccharide residues are incorporated to polymer backbones induce enhancement of binding affinity toward proteins ascribed to multivalent recognition, i.e., the “cluster effect”2 when the density and relative spatial arrangement of the carbohydrate residues are appropriate. Even the glycopolymers in which the spaces between saccharides are random showed stronger recognitions.2 Therefore, if the three-dimensional arrangement, i.e., interval and direction of the pendant carbohydrate could be regulated, remarkable enhancement of the biding ability would be promised. However, there are few reports of glycopolymer with a definite geometrical pattern to date.3,4 Aoi et al.3 applied dendrimer skeltone for the three-dimensional arrangement of saccharide as a “sugar ball”. Recently, Matsuura et al.4b proposed a new strategy to prepare periodic glycosylated oligonucleotide (20-mers). There are only two examples of intersugar space regulated glycopolymers having a much * To whom correspondence should be addressed: Telephone: +81-52735-7159. Fax: +81-52-735-5488. E-mail [email protected].

Figure 1. (a) Repeating unit of a new type of periodic glycopeptide and (b) schematic representation of helical glycopeptide.

improved binding ability toward proteins to the best of our knowledge. Therefore, it is still a challenging subject to synthesize a new type of glycopolymers with an ordered carbohydrate array. A polypeptide chain, being rigid and regular such as R-helix and β-sheet, is considered to be an excellent framework to support sugar residue, keeping a specific distance and orientation between the neighboring ones. Polymerization of glycosylated tripeptide will realize construction of three-dimensional carbohydrate ligands. Moreover, preparation of glycopeptide through a polymerization process offers a potential advantage over the tedious stepwise solid-state method. This paper reports the first application of sequential helical polypeptide backbone for three-dimensional regulation of carbohydrate.

10.1021/bm020014s CCC: $22.00 © 2002 American Chemical Society Published on Web 06/06/2002

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Recently, the novel intracellular carbohydrate modification by O-glycosidically linked N-acetyl-D-glucosamine (GlcNAc) has been shown to be ubiquitous among eukaryotes,5 on cytoplasmic6 or nuclear membrane,7 and plasma membrane associated8 proteins. The specific function of O-GlcNAc attachment to serine residue may often have a reciprocal relationship to the regulatory effect of protein phosphorylation.7,9 The O-GlcNAc expression significantly exists in an Alzheimer brain over that of age-matched control brains.10 However, the proteins have not yet been fully elucidated. Synthesis of a series of O-glycosylated Aib-containing peptides and a conformational investigation would address the effect of O-glycosylation on the biological activities of the peptides with a defined conformational preference as well as the molecular design of a new type of three-dimensional carbohydrate ligand. Experimental Section Materials. Z-Ser-OH, Di-tert-butyl carbonate (Boc2O), γ-methyl L-glutamate [H-Glu(OMe)-OH], and diphenylphosphoryl azide (DPPA) were purchased from Kokusan Chemical Works Ltd. (Tokyo, Japan). Trioctylmethylammonium chloride, R-aminoisobutyric acid (Aib), 1-hydroxybenzotriazole monohydrate (HOBt‚H2O), and triethylamine (TEA) were obtained from Tokyo Kasei Co. (Tokyo, Japan). N,Ndicyclohexylcarbodiimide (DCC), 10% palladium-carbon (Pd-C), formic acid, p-nitrophenol (NpOH), triethylamine (TEA), and N-methylmorphorin (NMM) were purchased from Nacalai Tesque (Kyoto, Japan). Chloroform, dichloromethane, and dimethyl sulfoxide (DMSO) were distilled from calcium hydride. The methanol, ethanol, acetonitrile, and water used were purified by distillation. Measurements. FT IR spectra were recorded in KBr disks using a JASCO FT/IR-430 spectrometer. 1H and 13C NMR spectra were measured at 27 °C using a Bruker DPX200 spectrometer (200 MHz for 1H NMR). All chemical shifts were expressed as δ downfield from tetramethylsilane (TMS). Number-average molecular weights (Mn) and the polydispersity indexes (Mw/Mn) of polymers were estimated by size exclusion chromatography (SEC) calibrated with polystyrene or poly(ethylene oxide) standards using systems of Tosoh HLC 803D with a Tosoh RI-8020 detector, Tosoh G2000-, G3000-, G4000-, and G5000-HXL columns (eluent, THF; flow rate, 1.0 mL/min; temperature, 40 °C), and JASCO model PU-1580 with JASCO RI-1530 and Amersham Pharmacia Biotech Superdex Peptide HR 10/30 (eluent, 0.05 M aqueous K2HPO4; flow rate, 0.5 mL/min; temperature, 27 °C), respectively. CD and UV absorption spectra were simultaneously recorded using a JASCO J-600 spectrometer in acetonitrile or water/ethanol mixed solvents at 27 °C. The path length of the quartz cell was 1.0 mm, and the concentration of peptide was 1.0 mM (0.5 mg/mL). These solvents were purified by distillation before use. Matrixassisted laser desorption/ionization time-of-flight (MALDITOF) mass spectrometry was performed on a Voyager DEPRO (Applied Biosystems) using 2,5-dihydroxybenzoic acid (DHB) as a matrix reagent, respectively. To generate sodiumcationized ions ([M + Na]+), NaI was used as a cationization salt.

Takasu et al.

Preparation of Z-Ser[β-D-GlcNAc(Ac)3]-OBn (3). In a flask, 5.4 g (16 mmol) of Z-Ser-OBn (1) and 7.1 g (20 mmol) of 2-acetamido-3,4,6-triacetyl-R-D-glucopyranosyl chloride (2) were dissolved in dichloromethane (87 mL) and stirred for 20 min. To the solution, 5.0 g (20 mmol) of silver triflate (AgOTf) was added. The solution was stirred at 40 °C for 21 h. The resultant purple mixture was neutralized with triethylamine, diluted with dichloromethane (240 mL), and filtered. The filtrate was washed with ice-cold saturated aqueous NaHCO3 and water, dried with MgSO4, and concentrated to give brown solid. 3 was obtained as white crystals from 2-propanol (4.0 g, 37% yield). The spectra data were identified with those prepared by Hg(CN)2 catalyst in toluene (reflux, 8 h) reported by Grag et al.11 Preparation of Boc-Ser[β-D-GlcNAc(Ac)3]-OH (5). Into a tube were added 3.0 g (4.6 mmol) of 412 and 6.0 mL of 1,4-dioxane. Di-tert-butyl carbonate (0.62 g, 3.0 mmol) and NaHCO3 (0.26 g, 3.0 mmol) were added to the solution at 0 °C successively. The mixture was stirred for 36 h and then evaporated. The pale-yellow powder was dissolved in 5% NaHCO3 aqueous solution and washed with diethyl ether. The water layer was adjusted at pH 2-3 by adding 5% aqueous KHSO4 from which 5 was extracted with ethyl acetate (1.3 g, 90% yield). The spectra data were identified with those prepared by another synthetic route reported by Lavielle et al.12 Preparation of Boc-Ser[β-D-GlcNAc(Ac)3]-ONp (6). At 0 °C, 5 (215 mg, 0.402 mmol), NpOH (62 mg, 0.442 mmol), and DCC (91 mg, 0.442 mmol) were dissolved in dichloromethane (0.7 mL) and stirred at 0 °C for 28 h. After the reaction, acetic acid (11 µL, 0.192 mol) was added to the mixture in order to remove unreacted DCC and ethyl acetate was added to precipitate dicyclohexylurea. The precipitate was collected and dried to give ester 6 (220 mg, 83%); Rf ) 0.55 (ethyl acetate). 1H NMR (δ, CDCl3): 1.47 (9H, s, CH3), 1.95 (3H, s, NHCOCH3), 2.03, 2.04, 2.08 (9H, 3s, OCOCH3), 3.67-3.75 (1H, m, H-2), 3.88-3.97 (2H, m, CHCH2O), 4.08-4.31 (2H, m, H-6a, H-6b), 4.44 (1H, m, H-5), 4.624.76 (1H, m, CHCH2O), 4.72 (1H, d, 8.3 Hz, H-1β), 5.08 (1H, t, 9.9 Hz, H-4), 5.23 (1H, t, 9.5 Hz, H-3), 5.50-5.70 (2H, br, t-BuOCONH, NHCOCH3), 7.34 (2H, d, 9.2 Hz, aromatic), 8.28 (2H, d, 9.2 Hz, aromatic). Preparation of Boc-Ser[β-D-GlcNAc(Ac)3]-Aib-OBn (8). To a solution of 5 (550 mg, 1.0 mmol) and HOBt‚H2O (190 mg, 1.1 mmol) in DMF (2.5 mL) was added DCC (234 mg, 1.1 mmol) at 0 °C. After addition of TsOH‚EAib-OBn (7)13 (0.38 g, 1.0 mmol), the reaction mixture was neutralized by addition of NMM (125 µL, 1.1 mmol) and stirred at 27 °C for 88 h. After the reaction, the solution was concentrated and the residue was redissolved in ethyl acetate. Dicyclohexylurea was removed by filtration and the filtrate was washed with 10% NaCl, 5% KHSO4, 10% NaCl, and 5% NaHCO3 aqueous solutions successively and dried over MgSO4. The organic layer was evaporated to give 8 (0.49 g, 66% yield); Rf ) 0.12 (ethyl acetate). 1H NMR (δ, CDCl3): 1.43 (6H, s, CH3), 1.56 (9H, s, CH3), 1.92 (3H, s, NHCOCH3), 2.03, 2.04, 2.06 (9H, 3s, OCOCH3), 3.67-4.30 (7H, m, H-2, 5, 6a, 6b, CH, CH2), 4.67 (1H, d, 8.4 Hz, H-1β),

Biomacromolecules, Vol. 3, No. 4, 2002 777

Three-Dimensional Arrangement of Sugar Residues Scheme 1. Synthesis of Glycosylated Tripeptide Containing Aib Residuea

5.05 (1H, t, 10.0 Hz, H-4), 5.15-5.26 (3H, m, CH2Ph, H-3), 5.34 (1H, d, 7.1 Hz, CONH), 5.77 (1H, d, J ) 10.0 Hz, CONH), 7.02 (1H, s, CONH), 7.30-7.35 (5H, br, aromatic). FT IR (cm-1, KBr): 3432 (νN-H), 2957 (νC-H), 1780 (νCdO, ester), 1635 (νCdO, amide I), 1540 (δN-H, amide II), 1255 and 1051 (νC-O), 755 (νC-O, phenyl). Preparation of H-Ser[β-D-GlcNAc(Ac)3]-Aib-OBn (9). Protected dipeptide 8 (0.23 g, 1.14 mmol) was dissolved in formic acid (10 mL) and stirred at 27 °C for 8 h. Unreacted formic acid was removed by evaporation and the residue was neutralized by 5% aqueous NaHCO3. N-terminal deprotected dipeptide 9 was extracted with ethyl acetate (0.19 g, 91% yield). Preparation of Boc-Glu(ΟMe)-OH (10). In a tube, 1.00 g (6.21 mmol) of H-Glu(OMe)-OH was dissolved in water (4.0 mL) at 0 °C. Subsequently, 7.0 mL of 1,4-dioxane was added to the solution and stirred for 30 min. At 0 °C, Boc2O (1.38 g, 6.83 mmol) and NaHCO3 (0.57 g, 6.83 mmol) was added to the solution and stirred at 27 °C for 27 h. The mixture was dried under reduced pressure, and the residue was dissolved in 5% aqueous KHSO4, and washed with diethyl ether. The organic layer was kept at pH 2-3, from

which N-terminal protected Glu(OMe) (10) was extracted with ethyl acetate (1.59 g, 98% yield); Rf ) 0.62 (methanol). 1 H NMR (CDCl3): δ 1.45 (9H, s, CH3), 2.20-2.26 (2H, m, CHCH2), 2.43-2.52 (2H, m, CH2CO), 3.69 (3H, s, COOCH3), 4.26-4.40 (1H, m, CH), 5.20 and 6.53 (1H, brd and br, 6 Hz, NH). Preparation of Boc-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]Aib-OBn (11). As shown in Scheme 1, protected tripeptide 11 was prepared by similar procedure to that for dipeptide 8 (85% yield); Rf ) 0.14 (ethyl acetate). 1H NMR (δ, CDCl3): 1.45 (9H, s, CH3), 1.55 (6H, s, CH3), 1.95 (3H, s, NHCOCH3), 2.02, 2.06, 2.08 (9H, 3s, OCOCH3), 2.11-2.16 (2H, m, CH2CH2CO), 2.41-2.50 (2H, m, CH2CH2CO), 3.68, 3.70 (3H, s, OCH3), 3.73-4.30 (7H, m, H-2, 5, 6a, 6b, CHCH2O, CHCH2O), 4.52-4.55 (1H, m, CHCH2CH2CO), 4.66 (1H, d, 8.4 Hz, H-1β), 5.05 (1H, t, 10.0 Hz, H-4), 5.16 (2H, s, CH2Ph), 5.24 (1H, t, 10.0 Hz, H-3), 5.52 (1H, d, 7.5 Hz, CONH), 6.11 (1H, d, J ) 10.0 Hz, CONH), 7.03 (1H, s, CONH), 7.07 (1H, s, CONH), 7.35 (5H, br, aromatic). FT IR (cm-1, KBr): 3382 (νN-H), 2933 (νC-H), 1743 (νCdO, ester), 1665 (νCdO, amide I), 1525 (δN-H, amide II), 1368 (νC-H), 1242 and 1047 (νC-O).

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Preparation of Boc-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]Aib-OH (12). In a flask, tripeptide 11 (220 mg, 0.258 mmol) was dissolved in methanol (10 mL). To the solution, water and 10% Pd-C (25 mg) were added successively and stirred at 27 °C for 18 h. Pd-C was removed by filtration. The filtrate was evaporated to give 12 in an excellent yield (194 mg, 99%); Rf ) 0.11 (acetone/ethyl acetate ) 1/2, v/v). Preparation of H-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]Aib-OH (13). Deprotection of 12 was carried out under the same condition as that for 8 (yield, 76%). 1H NMR [CDCl3 + DMSO (1drop)]: δ 1.48 (6H, s, CH3), 1.92 (3H, s, NHCOCH3), 1.99, 2.01, 2.08 (9H, 3s, OCOCH3), 2.10-2.56 (4H, br, CH2CH2CO, CH2CH2CO), 3.66 (3H, s, OCH3), 3.45-4.38 (7H, m, H-2, 5, 6a, 6b, CHCH2O, CHCH2O), 4.58 (1H, br, CHCH2CH2CO), 4.75 (1H, brd, 7.79 Hz, H-1β), 5.02 (1H, t, 10.3 Hz, H-4), 5.23 (1H, t, 9.6 Hz, H-3), 7.638.02 (2H, br, CONH), 8.13-8.45 (1H, br, CONH). Polymerization of H-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]Aib-OH (13) by DPPA method. To a mixture of 13 (30 mg, 0.045 mmol) and solvent (DMSO, 60 µL) were added DPPA (24 µL, 0.12 mmol) and triethylamine (16 µL, 0.12 mmol) at 5-10 °C. The reaction mixture was stirred vigorously at 5-10 °C for 2 h, and stirring was continued for 6 days at 27 °C. To the mixture was added a large volume of water, and the precipitate was collected by centrifugation. The obtained peptide was fractionated by chromatography on a column of Sephadex LH-60/DMF. Preparation of Boc-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]Aib-ONp (14). p-Nitrophenyl esterification of 12 was established by same way as that for 5 to give 14 in an excellent yield (98%); Rf ) 0.37 (ethyl acetate). 1H NMR (δ, CDCl3): 1.46 (9H, s, CH3), 1.65 (6H, s, CH3), 1.97 (3H, s, NHCOCH3), 2.02, 2.04, 2.07 (9H, 3s, OCOCH3), 2.052.12 (2H, m, CH2CH2CO), 2.30-2.40 (2H, m, CH2CH2CO), 3.68 (3H, s, OCH3), 3.70-4.24 (7H, m, H-2, 5, 6a, 6b, CHCH2O, CHCH2O), 4.57-4.61 (2H, m, CHCH2CH2CO, H-1β), 5.08 (1H, t, 9.5 Hz, H-4), 5.13 (1H, t, 10.0 Hz, H-3), 5.47 (1H, d, 5.4 Hz, t-BuOCONH), 5.87 (1H, d, 9.0 Hz, NHCOCH3), 7.04 (1H, d, 7.5 Hz, CONH), 7.20 [1H, s, CONH (Aib)], 7.31 (2H, d, 9.1 Hz, aromatic), 8.25 (2H, d, 9.1 Hz, aromatic). Preparation of HCl‚H-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]-Aib-ONp (15). In a tube, 14 (86 mg, 0.0973 mmol) was dissolved in 1,4-dioxane (1.2 mL), and 4 N HCl/1,4dioxane (0.4 mL) was added to the solution at 0 °C. The mixture was stirred at 27 °C for 8 h. Excess diethyl ether was added to the solution. The precipitate was washed with diethyl ether and dried to give 15 (77 mg, 96%). Polymerization of HCl‚H-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]-Aib-ONp (15). Tripeptide 15 (124 mg, 0.15 mmol) was dissolved in dimethyl sulfoxide (140 µL), and the solution was vigorously stirred. Triethylamine (28 µL, 0.20 mmol) was added slowly. Polymerization was allowed to proceed at 27 °C for 7 days. To the solution, excess water was added to precipitate the polypeptide. The precipitate was washed with water and diethyl ether successively and dried to give pale yellow powder (44 mg, yield, 45%). 1H NMR (δ, CDCl3): 1.26-1.66 (6H, CH3), 1.80-2.25 (12H, COCH3), 2.30-2.51 (2H, CH2CH2CO), 2.51-2.81 (2H, CH2CH2CO),

Takasu et al.

3.66 (3H, OCH3), 3.78-4.50 (7H, m, H-2, 5, 6a, 6b, CHCH2O, CHCH2O), 4.50-4.65 (1H, CHCH2CH2CO), 4.75-4.92 (1H, H-1β), 5.08 (1H, br, H-4), 5.39 (1H, br, H-3), 7.63, 7.88, 8.20 (4H, br, NHCO). FT IR (cm-1, KBr): 3311 (νN-H), 2954 (νC-H), 1750 (νCdO, ester), 1662 (νCdO, amide I), 1540 (δN-H, amide II), 1233 and 1047 (νC-O). Deacetylation of Poly(15). Deprotection of acetylated glycopeptide 15 was performed according to previous reports.14,15 Into an ice-cooled solution of 15 (50 mg, 0.078 mmol) in methanol (4.3 mL) was dropped with stirring 300 µL (6.20 mmol) of hydrazine monohydrate. After the reaction was mixed at 27 °C for 6 h, 4.3 mL of acetone was added to the solution to quench hydrazine with cooling at 0 °C in an ice-bath. The mixture was evaporated, and then the product was purified by repeated reprecipitations from water to ethanol. After the product was dried in vacuo, periodic glycopeptide 16 was isolated in 96% yield (38 mg). 1H NMR (D2O): δ 1.43 (6H, CH3), 1.99 (3H, s, COCH3), 2.15-2.30 (2H, CH2CH2CO), 2.43 (2H, br, CH2CH2CO), 3.64 (3H, s, OCH3), 3.35-3.59 (3H, H-3, 4, 5), 3.69 (1H, H-2), 3.88, 3.93 (2H, H-6a and 6b), 4.01 (1H, CHCH2O) 4.40 (1H, CHCH2CH2CO), 4.55 (1H, brd, 7.7 Hz, H-1β). FT IR (cm-1, KBr): 3371 (νN-H), 2945 (νC-H), 1727 (νCdO, ester), 1658 (νCdO, amide I), 1540 (δN-H, amide II), 1051 (νC-O). Semiempirical Molecular Orbital Calculation. Ac-[LGlu(OMe)-Ser(β-D-GlcNAc)-Aib]7-NHMe was energy-minimized by AM1 molecular orbital (MO) method16 in MOPAC 2000 (MOPAC 2000 version 1.0, Fujitsu Ltd., Tokyo, Japan, 1999). The minimization was carried out with the variables of all bond length, bond angles, and torsion angles using a standard right-handed R-helix (φ ) -57°, ψ ) -47°, ω ) 180°)17 for the starting conformation. The starting conformation for the AM1 calculation was obtained using PEPCON18 program based on geometric parameters in ECEPP system.18a The geometry of the GlcNAc moiety was obtained from a previous paper reported by Mo et al.19 Results and Discussion In our strategy, polymerizable glycosylated tripeptides were synthesized in solution, by a step-by-step approach, starting from a triacetylated GlcNAc-substituted L-serine, Z-Ser[β-D-GlcNAc(Ac)3]-OBn (3) (Z, benzyloxycarbonyl; Bn, benzyl) (Scheme 1). The glycosylated serine 3 was prepared by the Koenigs-Knorr reaction20 of Z-L-Ser(OH)OBn (1) and triacetylated GlcNAc chloride 2 in the presence of silver triflate (AgOTf)21 catalyst (yield, 37%). The β-selective glycosidation was confirmed by 1H NMR resonance ascribed to anomeric proton (4.68 ppm, J1,2 ) 8.4 Hz). The sugar-substituted L-serine 5, which was prepared by hydrogenation of 311 and subsequent N-terminal protection, was coupled with H-Aib-OBn to afford a new glycodipeptide 8 in 66% yield using DCC/HOBt coupling strategy. Deprotection of Boc, followed by coupling with Boc-L-Glu(OMe)OH (10) by DCC/HOBt method gave terminally protected, tripeptide, Boc-L-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]-AibOBn (11). The glycotripeptide 11 obtained was deprotected to produce a new polymerizable, O-glycosylated, and Aib-

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Three-Dimensional Arrangement of Sugar Residues Table 1. Polymerization of Glycosylated Tripeptide 13 by DPPA Methoda monomer run

no.

mg (mmol)

DPPA (µL (mmol))

triethylamine (µL (mmol))

solvent

time (day)

yield (mg (%))

Mnb × 10-3

Mw/Mnb

1 2 3

13 13 4

30 (0.045) 25 (0.038) 150 (3.5)

24 (0.118) 10 (0.049) 94 (4.5)

16 (0.118) 7 (0.049) 62 (4.5)

DMSO CDCl3 DMSO

6 6 4

13 (44) 10 (40) trace

1.4 1.1 1.6

1.11 1.10 1.20

a

[M]0 ) 1.0 mol/L; temp, 27 °C. Feed molar ratio of DPPA to amine ) 1.0. b Determined by SEC in THF relative to polystyrene.

Scheme 2. Synthesis of Glycosylated Tripeptide 13 by the DPPA Method

containing glycotripeptide H-L-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]-Aib-OH (13). The structure of 13 was confirmed by 1 H NMR and IR spectra. In CDCl3, the 1H NMR signals of 13 were broadened and turned to be clearer by addition of DMSO-d6 (1 drop). The result suggested that intermolecular association of glycosidated tripeptide took place in CDCl3.22 We expected that the association of the monomer would influence the polymerization behavior. Polymerizations of 13 with DPPA method23 were attempted in DMSO or CHCl3. The results are summarized in Table 1. Conversion and Mn in CHCl3 (40%, 1.1 × 103, run 2) were almost the same as those in DMSO (44%, 1.4 × 103, run 1), which were not influenced by the solvent. Polymerization of 4 by the DPPA method was also carried out under the same conditions (in DMSO at 27 °C, run 3). The Mn and n values of poly(4) obtained were 1.6 × 103 and n ) 4. The low polymerizabilities of tripeptide 13 and sugar-substitute L-serine 4 are ascribed to steric repulsion of C-terminal Aib residue (R,Rdisubstituted residue) for 13 and that of bulky acetylated sugar for 4, respectively. The structure of oligo(13) was identified by IR and 1H NMR spectra. The conformation was investigated by FT IR and CD spectroscopies, which are the most extensively used tools to elucidate secondary structure of peptides. In the FT IR spectrum of oligo(13), analysis of the CdO stretching region reveals an amide I mode at 1662 cm-1, indicating typical 310- and/or R-helical conformations (1663-1657 cm-1).24 The Aib residue is well-known to be a strong inducer that forms a 310-helix (sometimes an R-helix depending on peptide sequence, chain length, or environment).24,25,26 The 310-helix is just slightly more elongated than the R-helix.25 The CD patterns of oligo(13) (ca. six residues) were characterized by a negative Cotton effect (∆) (broken line in Figure 2). ∆ ()L - R) is expressed with respect to the molar concentration of the amino acid residue. Aib-containing glycosidated tripeptide 13 gave no signals (only a noisy baseline) in the CD spectrum (200-260 nm). Accordingly, CD spectra of oligo(13) were not caused by artifacts such as light-scattering. The CD spectrum showed intense maxima (λmax) around 205 nm (band I) with a shoulder at 222 nm

assignable to amide residue in backbone. Whereas in the right-handed R-helix the intensities of the two negative maxima centered at 208 nm (parallel component of the πfπ* transition) and 222 nm (n f π* transition) are very close,27 it is worth recalling that in the 310-helix the n f π* transition exhibits a drastically reduced intensity with respect to that of the π f π* transition and tends to undergo a modest blue shift.25 More specially, a value of 0.15-0.40 for the R ([θ]222/[θ]205 ) [∆]222/[∆]205) ratio is considered to be diagnostic for 310-helical conformation.25c In the CD spectrum of oligo(13), the R value was 0.36. On this basis, the oligo(13) examined in this work seemed to prefer the right-handed helical conformation to a substantial extent in acetonitrile, but it was difficult to distinguish R- from 310helixes. To obtain higher molecular weight glycopeptide, we tried an active ester method, which is an alternative method effective for polypeptide synthesis. p-Nitrophenyl ester is one of the most important active esters.28 Therefore, we prepared HCl‚H-L-Glu(OMe)-Ser[β-D-GlcNAc(Ac)3]-AibONp (15) (Np, p-nitrophenyl) by C-terminal deprotection

Figure 2. CD (top) and UV (bottom) spectra of oligo(13) (broken line) and poly(15) (solid line) in acetonitrile at 27 °C.

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Scheme 3. Synthesis of Glycosylated Tripeptide 15 by the Active Ester Method

Table 2. Polymerization of Glycosylated Tripeptide 15 by Active Ester Methoda monomer run

no.

mg (mmol)

amine

time (day)

yield (mg (%))

1 2 3 4

15 15 15 6

77 (0.094) 124 (0.15) 70 (0.085) 150 (0.35)

TEA TEA NMM TEA

5 7 7 5

14 (26) 44 (45) 14 (26) 14 (45)

deacetylationc

Mnb × 10-3

Mw/Mnb

2.5 (4.4e)

1.26

2.5

1.36

yield (%)

Mnd × 10-3

Mw/Mnd

75 97 88 53

3.9 4.1 3.6 (3.6e) 2.5

1.21 1.38 1.32 1.25

[M]0 ) 0.8 mol/L in DMSO; temp, 27 °C. Feed molar ratio of monomer to amine ) 1.3. Determined by SEC in THF relative to polystyrene. c [H2NNH2]0 ) 1.3 mol/L in methanol at 0 °C for 6 h; [H2NNH2]0/[acetyl group]0 ) 26. d Determined by SEC in 0.05 M aqueous K2HPO4 relative to poly(ethylene oxide). e Determined by MALDI-TOF mass analysis. a

of 11, esterification with p-nitrophenol, and N-terminal deprotection successively. Polymerizations of 15 were carried out in DMSO at 27 °C for 5-7 days using TEA and NMM as the bases (Table 2). TEA was more effective for the polymerization (run 2). The structure was confirmed by IR, NMR (see Experimental Section), and MALDI-TOF mass spectra. In the MALDI-TOF mass measurement of poly(15) (Mn ) 2.5 × 103, run 2), there was one main series of peaks whose interval was regular, 644 m/z, corresponding to the mass of repeating unit of poly(15). Mn of poly(15) by SEC using poly(styrene) standards was 2.5 × 103 (run 2), which was higher than that in the DPPA method (1.06 × 103-1.39 × 103, Table 1). We consider that the lower reactivity of the tripeptide 13 than 15 is ascribed to the low active esterification. Using SEC with a polystyrene calibration provides for relative Mn data, because the hydrodynamic volume theory developed for statistical coil will not be applicable to these polypeptides. Therefore, we calculate the absolute Mn from MALDI-TOF mass spectrometry, in which separate oligomers are clearly resolved. The peak top mass was 4048 m/z and the calculated absolute Mn was 4.4 × 103. The result indicated that SEC measurement using polystyrene standard gave lower Mn (2.5 × 103) than that from absolute Mn. In the CD measurement of poly(15) (absolute Mn ) 4.4 × 103, solid line in Figure 2), the CD pattern corresponds to a right-handed arrangement of the transition moment; i.e., protected GlcNAc residues are arranged regularly along a right-handed helical main chain. The amplitude of negative Cotton effect for poly(15) with

b

longer chain length was markedly larger than that for poly(13). The results indicated the structural stability (righthanded helical conformation) of the peptide was enhanced by increase of chain length, because CD amplitudes, in general, tend to increase with decreasing irregularity and thermal fluctuations in a specific conformation.23b Acetyl protecting groups of poly(15) consisting of base sensitive glycosylated serine units can be removed very safely and with no racemization using hydrazine in methanol14,15 at 27 °C for 6 h to give periodic glycopeptide 16 in an excellent yield (96%). Complete deprotection was confirmed by IR, 1H NMR, and MALDI-TOF mass measurements. In the TOF mass spectrum, the peaks are separated by 518 m/z corresponding to the mass of repeating unit of glycopeptide 16. Unexpectedly, the methyl ester of Glu(OMe) unit in poly(15) was not deprotected at all under these experimental conditions. In a deprotection of 13 as the model reaction, not only deacetylation but also carbonyl attack of all the methyl ester of Glu(OMe) unit occurred to give the hydrazide under the same conditions. It might be due to the helical structure of poly(15), in which the methyl ester groups were surrounded by acetylated sugar moieties, so that hydrazine could not attack the methyl ester. Mn of periodic glycopeptide 16 estimated by SEC using poly(ethylene oxide) standards was 3.6 × 103-4.1 × 103 (ca. 21-24 residues), in which Mn of 16 (3.6 × 103, run 3 in Table 2) was identified well with that determined by MALDI-TOF mass measurement (Mn ) 3.6 × 103). FT IR spectrum of 16 (run 3 in Table 2, Mn ) 3.6 × 103, 21 residues) showed the

Three-Dimensional Arrangement of Sugar Residues

Figure 3. CD (top) and UV (bottom) spectra of periodic glycopeptide 16 (21 residues, n ) 7, run 3 in Table 2) in water-ethanol mixtures of varying compositions (v/v %) at 27 °C.

absorptions at 1658 and 1540 cm-1, which are assigned to amide I and II ascribed to R-helical polypeptide,24,25 respectively. As shown in Figure 3, CD spectra of GlcNAcsubstituted periodic glycopeptide 16 showed the double minima (204 and 228 nm) of negative Cotton effects in water, which coincided with the FT IR analysis. These results reveal that glycopeptide 16 prefers R-type helical conformations in water. This CD pattern remained unchanged as the concentration of 16 was varied from 0.5 to 2 mg/mL, showing that 16 is not aggregated in water. Polymerization of 6 was also carried out in the same procedure (run 4 in Table 2). Subsequent deacetylation afforded a GlcNAccarrying poly(L-serine) (Mn ) 2.5 × 103, eight residues). In the CD spectrum, remarkable negative Cotton effect at 197 nm (∆197 ) -2.0) indicating random coil conformation. In general, glycopeptides tend to prefer random coil structures to helical structures in water. Aoi et al.15 reported a synthesis of glucose-carrying poly(L-serine) (12-46 residue) via ringopening polymerization of O-(tetra-O-acetyl-β-D-glucopyranosyl)-L-serine N-carboxyanhydride (NCA), and Rocchi et al.29 synthesized -[Ala-Ala-Thr(β-D-Gal)]n- (n ) 2-7) by solid-phase synthesis, in which both the peptides preferred a randomly coiled conformation in water. The results reveal that the Aib unit in glycopolymer 16 seems to act as a strong inducer for the helical conformation. This is the first example of periodic glycopeptide having a regular helical backbone in aqueous solutions as far as we know. As for the main-chain conformation, the ∆222 value calculated with respect to the amide group is a good measure for the helix content.30 The ∆222 value of glycopeptide (-0.6) at 27 °C was smaller than that expected for a long polypeptide chain in a 100% helix conformation (-10.6 to -12.1),30 and the value hardly depended on temperature ranging from 4 to 35 °C. FT IR and CD measurements of longer 16 (run 2 in Table 2, Mn ) 4.1 × 103, 24 residues) were also carried out. In the FT IR spectra, absorptions at 1658 and 1540 cm-1 were observed, and the CD spectra in water showed double minima (204 and 228 nm) of negative Cotton effects indicating an R-helical conformation the same as that for 16 (21 residues). However remarkable increase

Biomacromolecules, Vol. 3, No. 4, 2002 781

Figure 4. Helical conformation of periodic glycopeptide 16 (21 residues, n ) 7) energy-minimized by AM1 method.

of the Cotton effect was not confirmed (∆222 ) -0.5). It revealed that the intensities of the negative Cotton effects were affected strongly by solvent. As solution composition was varied from pure water to increasing percentages of ethanol, R-helix content of glycopeptide 16 (21 residues) increased (Figure 3). In water/ethanol (30/70, v/v), the ∆222 value was increased (-3.7), indicating 31-35% R-helix content. At a concentration above 70 wt % of ethanol content, CD measurement was impossible because 16 was not dissolved in the solvent. Addition of methanol (higher polar than ethanol) also induced an increase in the ∆222 value, but the effect was lower than ethanol. In water, solvent interactions with sugar residue are strong enough to disrupt the H-bonding found in the folded structure. This is reasonable since it is known that sugar residue interacts strongly with water molecules through formation of an ordered H-bonded structure, which can counterbalance the loss of amide H-bonding. Furthermore, the energy minimization was also performed for Ac-[L-Glu(OMe)-Ser(β-D-GlcNAc)-Aib]7-NHMe (21 residue) by the semiempirical molecular orbital method16 to identify the type of helical structure of periodic glycopeptide 16. A perspective view of the energy-minimized glycopeptide 16 (21-mer) is shown in Figure 4. The average torsion angles in the Aib residue were φ ) -53°, ψ ) -44°, and ψ ) -178°, corresponding to a typical R-helical conformation (φ ) -57°, ψ ) -47°, ω ) 180°).17 In the model, intramolecular hydrogen bonds between (i) CO and (i + 4) NH were confirmed. The results supported R-helical conformation of glycopeptide 16, which was identified with FT IR and CD analyses. From the model, the nearest intersugar distances were estimated from the length between C-4 carbons of the terminal sugar residue. As shown in Figure 4, it was 10 Å along the helical backbone (Figure 4, right) and the GlcNAc moiety would be positioned on the same side along the helical peptide each 18 residues, i.e., sugar residue in Ser(β-D-GlcNAc) (second residue from N-terminal) and that in Ser(β-D-GlcNAc) (20th residue from N-terminal) are overlapped along the helical axis. The perpendicular distance between sugar residues (sugar pitch) was 27 Å (Figure 4, left).

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Biomacromolecules, Vol. 3, No. 4, 2002

In this article, we describe the synthesis of a new type of periodic glycopeptide 16 (21-24 residues) having a GlcNAc residue. Conformational analysis of the glycopeptide 16 by CD and FT-IR spectroscopies and semiempirical molecular orbital calculations indicated that 16 has an R-helical conformation in aqueous solutions, in which a threedimensional regular arrangement of sugar residue along the R-helical peptide was established. The helix content changed depending on water-alcohol mixture of varying concentrations. The solvent-dependent conformational properties of 16 would provide a means for change of interval between sugar residues. This fundamental data would provide a new guideline to regulate three-dimensional arrangement of carbohydrate ligand. Acknowledgment. The authors express their sincere gratitude to Professor M. Kawai and Dr. K. Yamashita, Nagoya Institute of Technology for their permission to use the CD apparatus. Financial support to A.T. from the Ministry of Education, Science, and Culture of Japan (Grantin-Aid for Development Scientific Research, No. 13750816) is gratefully acknowledged. A.T. also acknowledges Dr. Hiroaki Sato (Meijo University) for his technical support in MALDI-TOF mass measurements. References and Notes (1) (a) Kobata, A. Eur. J. Biochem. 1992, 209, 483. (b) Dwek, R. A. Chem. ReV. 1996, 96, 683. (2) (a) Neoglycoconjugates: Preparation and Applications; Lee Y. C., Lee, R. T., Eds.; Academic Press: San Diego, CA, 1994. (b) Kobayashi, A.; Akaike, T.; Kobayashi, K.; Sumitomo, H. Makromol. Chem. Rapid Commun. 1986, 7, 645. (c) Roy, R.; Tropper, F. C. J. Chem. Soc., Chem. Commun. 1988, 1058. (d) Manning, D. D.; Hu, X.; Beck, P.; Kiessling, L. L. J. Am. Chem. Soc. 1997, 119, 3161. (e) Nishimura, S.-I.; Furuike, T.; Matsuoka, K.; Maruyama, K.; Nagata, K.; Nishi, N.; Tokura, S. Macromolecules 1994, 27, 4876. (f) Wulff, G.; Schmid, J.; Venhoff, T. Macromol. Chem. Phys. 1996, 197, 259. (3) (a) Aoi, K.; Ito, K.; Okada, M. Macromolecules 1995, 28, 5391. (b) Aoi, K.; Tsutsumiuchi, K.; Yamamoto, A.; Okada, M. Tetrahedron 1997, 53, 15415. (4) (a) Hasegawa, T.; Shunsuke, K.; Matsuura, K.; Kobayashi, K. Macromolecules 1999, 32, 6595. (b) Matsuura, K.; Hibino, M.; Yamada, Y.; Kobayashi, K. J. Am. Chem. Sci. 2001, 11, 1281. (5) Hart, G. W.; Greis, K. D.; Dong, D. L.-Y.; Blomberg, M. A.; Chou, T.-Y.; Jiang, M. S.; Roquemore, E. P.; Snow, D. M.; Kreppel, L. K.; Cole, R. C.; Comer, F. I.; Arnold, C. S.; Hayes, B. K. Glycosylation Dis. 1994, 1, 214. (6) Holt, G. D.; Haltiwanger, R. S.; Torres, C.-R.; Hart, G. W. J. Biol. Chem. 1987, 262, 14847.

Takasu et al. (7) Haltiwanger, R. S.; Kelly, W. G.; Roquemore, E. P.; Blomberg, M. A.; Dong, D. L.-Y.; Kreppel, L.; Chou, T.-Y.; Hart, G. W. Biochem. Soc. Trans. 1992, 20, 264. (8) Griffith, L. S.; Mathes, M.; Schmitz, B. J. Neurosci. Res. 1992, 41, 270. (9) Hart, G. W.; Haltiwanger, R. S.; Holz, G. D.; Kelly, W. K. Annu. ReV. Biol. 1989, 58, 841. (10) Griffith, L. S.; Schmitz, B. Biochem. Biophys. Res. Commun. 1995, 213, 424. (11) Grag, H. G.; Jeanloz, R. W. Carbohydr. Res. 1976, 49, 482. (12) Lavielle, S.; Ling, N. C.; Saltman, R.; Guillemin, R. C. Carbohydr. Res. 1981, 89, 229. (13) Balasubramanian, T. M.; Nancy, C. E.; Kendrick, M.; Taylor, M.; Marshall, G. R.; Hall, J. E.; Vodyanoy, I.; Reusser, F. J. Am. Chem. Soc. 1981, 103, 6127. (14) Kunz, H. Angew. Chem., Int. Ed. Engl. 1987, 26, 294. (15) Aoi, K.; Tsutsumiuchi, K.; Okada, M. Macromolecules 1994, 27, 875. (16) Dewar, M. J. S.; Zoebisch, E. G.; Healy, E. F.; Stewart, J. J. P. J. Am. Chem. Soc. 1985, 107, 3902. (17) Arnott, S.; Wonacott, A. J. J. Mol. Biol. 1966, 21, 371. (18) (a) Momany, F.; McGuire, R. F.; Burgess, A. W.; Scheraga, H. A. J. Phys. Chem. 1975, 79, 231. (b) Beppu, Y. Comput. Chem. 1989, 13, 101. (c) Sisido, M. Pept. Chem. 1991 1992, 29, 105. (19) Mo, F.; Jensen, L. H. Acta Crystallogr. 1978, B34, 1562. (20) Koenigs, W.; Knorr, E. Ber. Dtsch. Chem. Ges. 1901, 34, 957. (21) Jensen, K. J.; Hansen, P. R.; Venugopal, D.; Barany, G. J. Am. Chem. Soc. 1996, 118, 3148. (22) Takasu, A.; Houjyou, T.; Inai, Y.; Hirabayashi, T. Polym. Prepr., Jpn. 2000, 49, E243. (23) (a) Nishi, N.; Naruse, T.; Hagiwara, K.; Nakajima, B.; Tokura, S. Makromol. Chem. 1991, 192, 1799. (b) Inai, Y.; Ito, T.; Hirabayashi, T.; Yokota, K. Polym. J. 1995, 27, 846. (24) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry 1991, 30, 6541. (25) (a) Toniolo, C.; Polese, A.; Formaggio; F.; Crisma, M.; Kamphuis, J. J. Am. Chem. Soc. 1996, 119, 10278. (b) Yorder, G.; Polese, A.; Silva, R. A. G. D.; Formaggio, F.; Crisma, M.; Broxterman, Q. B.; Kamphuis, J.; Toniolo, C.; Keiderling, T. A. J. Am. Chem. Soc. 1997, 118, 22744. (c) Formaggio, F.; Crisma, M.; Rossi, P.; Scrimin, P.; Kaptein, B.; Broxterman, Q. B.; Kamphuis, J. Chem.sEur. J. 2000, 6, 4498. (26) (a) Inai, Y.; Kurokawa, Y.; Hirabayashi, T. Biopolymers 1999, 49, 551. (b) Inai, Y.; Ashitaka, S.; Hirabayashi, T. Polym. J. 1999, 31, 246. (c) Inai, Y.; Kurokawa, Y.; Hirabayashi, T. Macromolecules 1999, 32, 4575. (27) Manning, M.; Woody, R. W. Biopolymers 1991, 31, 569. (28) (a) Papaka, R. S.; Urry, D. W. Int. J. Peptide Protein Res. 1978, 11, 97. (b) Papaka, R. S.; Okamoto, K.; Urry, D. W. Int. J. Peptide Protein Res. 1978, 11, 109. (29) Filira, F.; Biondi, L.; Scolaro, B.; Foffani, M. T.; Mammi, S.; Peggion, E.; Rocchi, R. Int. J. Biol. Macromol. 1990, 12, 41. (30) (a) Sisido, M. Macromolecules 1989, 22, 3280. (b) Scholtz, J. M.; Qian, H.; York, E. J.; Stewart, J. M.; Baldwin, R. L. Biopolymers 1991, 31, 1463.

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