A General Synthesis of Structurally Diverse ... - ACS Publications

Rational, yet simple, design and synthesis of an antifreeze-protein inspired polymer for cellular cryopreservation. Daniel E. Mitchell , Neil R. Camer...
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Bioconjugate Chem. 2001, 12, 817−823

817

A General Synthesis of Structurally Diverse Building Blocks for Preparing Analogues of C-Linked Antifreeze Glycoproteins Adewale Eniade, Anastasia V. Murphy, Geraldine Landreau,† and Robert N. Ben* Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 13902. Received March 26, 2001

A synthetic methodology to afford unusual glycoconjugate building blocks useful for the solid-phase synthesis of C-linked antifreeze glycoprotein (AFGP) analogues is described. Such compounds are urgently required in order to elucidate the molecular mechanism by which AFGPs function. All reactions are general in nature and accommodate structural variation in the carbohydrate moiety, polypeptide backbone, and amino acid side chain.

INTRODUCTION

Biological antifreezes are a diverse class of compounds that have the unusual ability to inhibit the growth of ice crystals in organisms inhabiting subzero environments. Consequently, these organisms are protected against cryoinjury. Antifreeze glycoproteins (AFGPs1) are one class of biological antifreezes found in Arctic and Antarctic teleost fish. The structure of a typical AFGP is shown in Figure 1. The core unit consists of a L-threonylL-alanyl-L-alanyl tripeptide where the secondary hydroxyl group of the threonine is glycosylated with the β-Dgalactosyl-(1,3)-R-D-N-acetylgalactosamine subunit. There are eight classes of AFGP and each differs in molecular weight (1-3). For instance, AFGP 1-4 are 20-33 kDa while AFGP 5-8 are in the 19-2.2 kDa range. Minor variations in primary structure are often found in low molecular weight AFGPs (i.e., AFGP 7-8) (4-6). During the last 30 years, the macromolecular mechanism of action for AFGPs has been extensively investigated and is largely regarded as an adsorption-inhibition phenomenon in which the biological antifreeze binds to the surface of a growing ice crystal (7, 8). After adsorption, ice growth occurs between adjacent AFGPs, and the surface grows outward in a convex fashion. As the radius of curvature increases, it becomes energetically unfavorable to add water molecules to the ice lattice, and consequently a localized freezing point depression is observed. This is referred to as the Kelvin Effect (9). The difference between the melting and freezing points is defined as thermal hysteresis (TH). In contrast to the macromolecular mechanism, a definitive molecular mechanism of action has not emerged and remains a source of debate among experts in the field. This has restricted the use of AFGPs in many medical, industrial, and commercial applications (1013). Additional complications include the fact that isolation/purification of AFGPs from natural sources remains a costly and laborious process (14) while the chemical and * To whom correspondence should be addressed. Phone: (607) 777-4400. Fax: (607) 777-4478. E-mail: [email protected]. † Visiting Scholar, University of Orleans, France. 1 Abbreviations: AFGP, antifreeze glycoprotein; SPS, solidphase synthesis; NMR, nuclear magnetic resonance; CDI, 1,1carbonyldiimidazole; DIPEA, N,N-diisopropylethylamine; TBS, tert-butyldimethylsilyl.

Figure 1. A typical antifreeze glycoprotein (AFGP).

Figure 2. Generalized building block structure.

biological instability of natural AFGPs make them unsuitable for most applications (15). The chemical synthesis of AFGP and AFGP analogues is an attractive alternative to the difficult isolation from natural sources. With the many recent advances in peptide and carbohydrate chemistry, the rational design of chemically and biologically stable AFGP analogues possessing enhanced activity should be feasible. However, before such compounds can be designed, a thorough understanding of how AFGPs function at the molecular level must be attained (14). In an attempt to address these issues, our laboratory has been the first to synthesize low molecular weight C-linked AFGP analogues (16). While this may seem like a dramatic structural modification, recent studies have demonstrated that C-linked glycoconjugates bind substrates with nearly identical conformations and affinities as O-linked glycoconjugates (17, 18). C-linked structures are attractive and offer significant advantages with respect to natural O-linked glycoconjugates as they are stable to a wide range of chemical and biological conditions. Our approach centers on the preparation of glycosylated tripeptide building blocks (Figure 2) that are assembled into C-linked AFGP analogues using conventional solid-phase synthesis (SPS). We recently reported the synthesis of a C-linked AFGP analogue with a molecular weight of 1.5 kDa using this strategy (Figure 3) (19, 20). An attractive advantage of the solid-phase approach is that C-glycopeptide polymers

10.1021/bc0155059 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/18/2001

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Eniade et al.

Figure 3. Solid-phase synthesis of a C-linked AFGP mimic.

of a predetermined length are easily prepared. In contrast, solution-phase polymerization approaches to AFGP often produce mixtures of varying molecular weight (21). This article describes the rationale for and preparation of several structurally diverse glycoconjugate building blocks necessary to synthesize C-linked AFGP analogues using a linear solid-phase approach. EXPERIMENTAL PROCEDURES:

General Methods. Infrared (IR) spectra were recorded using a Perkin-Elmer 1600 Series FTIR. Unless otherwise noted 1H and 13C NMR spectra were recorded using a Bruker AM 360 or a Bruker AC 300 in CDCl3. Chemical shift values are reported in ppm downfield from TMS as an internal standard. Multiplicities are reported as s, singlet; bs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. Low-resolution mass spectra were obtained using a Hewlett-Packard series 1100 MSD instrument equipped with an API-ES ionization chamber. High-resolution mass spectra were obtained using a Micromass QTof-II high-resolution mass spectrometer and sodium iodide as an internal standard. Chromatographic separations were performed using 230400 mesh silica gel from Natland Inc. All solvents were dried and distilled prior to utilization; THF was distilled over sodium-benzophenone, and methylene chloride was distilled over calcium hydride. 2-(2,3,4,6-Tetra-O-acetyl-r-D-galactopyranosyl)acetaldehyde (3). Ozone was bubbled into a solution of C-allylated β-D-galactose pentaacetate (866 mg, 2.33 mmol) in dry dichloromethane (12 mL) under nitrogen at -78 °C until the solution turned blue in color. Nitrogen was then bubbled through the solution until it turned colorless. Triphenylphosphine (1.5 g, 5.7 mmol) was then added, and the solution was then allowed to warm to room temperature over a period of 3 h. Dichloromethane was removed under reduced pressure, and the residue was purified by column chromatography (ethyl acetate/ hexanes 1:1) to afford 632 mg (73% yield) of 3 as a colorless oil. IR (CH2Cl2): 1738 cm-1; 1H NMR (360 MHz, CDCl3) δ: 9.64 (1H, s), 5.15 (1H, dd, J ) 5.8, 2.8 Hz), 5.02 (1H, dd, J ) 8.8, 4.7 Hz,), 4.96 (1H, dd, J ) 8.8, 3.1 Hz), 4.62 (1H, m), 4.04 (1H, dd, J ) 11.1, 7.5 Hz), 3.9 (1H, m), 3.84 (1H, dd, J ) 11.2, 4.6 Hz), 2.5 (2H, m), 1.87 (3H, s), 1.81 (3H, s), 1.78 (6H, s); 13C NMR (90 MHz, CDCl3) δ: 198.4, 169.6, 169.3, 169.1, 169.0, 68.8, 67.2, 66.6, 66.4, 61.0, 41.0, 19.9. Benzyl Fluorenomethyloxycarbonyl-L-lysine[(2,3,4,6-tetra-O-acetyl-r-D-galactopyranosyl)ethyl]glycylglycinate (5). Aldehyde 3 (100 mg, 0.47 mmol) was dissolved in 10 mL of dry THF under nitrogen atmosphere at room temperature. Compound 4 (60 mg, 0.85 mmol) was dissolved in 3 mL of dry THF and added to the solution of 3. Sodium triacetoxyborohydride (100 mg, 0.47 mmol) was then added, and the solution was stirred

at room temperature for 12 h. The solution was concentrated under reduced pressure and the residue dissolved in methanol and stirred for 20 min. This solution was concentrated under reduced pressure and the residue purified by column chromatography (CH2Cl2/MeOH 10: 1) to afford 80 mg (55% yield) of 5 as a colorless oil. IR (CH2Cl2): 3366, 1744, 1672 cm-1; 1H NMR (360 MHz, CDCl3) δ: 7.62 (2H, d, J ) 7.4 Hz,), 7.47 (2H, br s), 7.21 (9H, m), 6.50 (1H, br s), 5.30 (1H, br s), 5.10 (2H, br s), 4.91 (3H, br s), 4.37 (1H, m), 4.22 (4H, br s), 4.07 (3H, br s), 3.91 (6H, br s), 3.03 (2H, br s), 2.93 (2H, br s), 1.95 (14H, m), 1.73 (4H, br s), 1.47 (2H, br s); 13C NMR (90 MHz, CDCl3) δ: 175.5, 173.17, 170.9, 169.8, 169.7, 169.6, 169.5, 156.6, 143.7, 141.1, 135.1, 128.4, 128.3, 128.0, 127.6, 127.0, 125.1, 119.8, 69.8, 68.6, 68.3, 67.5, 67.2, 67.0, 66.9, 60.9, 54.7, 47.9, 47.0, 45.1, 42.9, 41.2, 31.3, 25.3, 23.6, 22.2, 20.9, 20.7, 20.7, 20.6. LRMS (electrospray, MeOH, positive ion mode, m/z) calcd. for C48H58N4O15 (M+) 930.38; found 931.40 (M+ + H). HRMS calcd for C43H38N4O15 (M+ + H) 931.3977, found 931.3976. Benzyl Fluorenomethyloxycarbonyl-L-lysine[bis[(2,3,4,6-tetra-O-acetyl-r-D-galactopyranosyl)ethyl]]glycylglycinate (6). Aldehyde 3 (120 mg, 0.32 mmol) was dissolved in 3 mL of dry THF and allowed to stir at room temperature under nitrogen atmosphere. Compound 4 (100 mg, 0.15 mmol) was dissolved in 6 mL of dry THF and added to the solution of 3. Sodium triacetoxyborohydride (100 mg, 0.47 mmol) and 0.1 mL of acetic acid were added directly to this solution, and the mixture was allowed to stir at room temperature for 12 h. The solvent was removed under reduced pressure, and the resulting residue was redissolved in methanol and stirred for 20 minutes. This solution was then concentrated and the residue was redissolved in ethyl acetate. The ethyl acetate solution was washed successively with saturated sodium bicarbonate, water, and brine and the solution dried over MgSO4 and concentrated under reduced pressure. The crude product was then purified by column chromatography (CH2Cl2/MeOH 30:1) to afford 117 mg (63% yield) of 6 as a colorless oil. IR (CH2Cl2): 1733, 1655 cm-1; 1H NMR (360 MHz, CDCl3) δ: 7.71 (2H, d, J ) 7.4 Hz), 7.56 (2H, t, J ) 6.2 Hz), 7.35 (2H, t, J ) 7.2 Hz), 7.26 (7H, m), 7.14 (1H, br s), 5.81 (1H, br s). 5.38 (2H, br s), 5.26 (1H, s), 5.20 (3H, br s), 5.06 (2H, s), 4.36 (2H, br s), 4.26 (4H, m), 4.16 (2H, t, J ) 6.9 Hz), 4.00 (8H, m), 2.42 (4H, br s), 2.34 (2H, br s), 2.07 (6H, s), 2.02 (6H, s), 2.00 (6H, s), 1.99 (6H, s), 1.80 (2H, br s), 1.64 (2H, br s), 1.54 (2H, br s), 1.38 (4H, br s); 13C NMR (90 MHz, CDCl3) δ: 172.4, 170.6, 169.9, 169.8, 169.4, 169.0, 156.4, 143.8, 141.1, 135.1, 128.4, 128.3, 128.1, 127.6, 126.9, 124.9, 124.9, 119.8, 70.1, 68.3, 67.9, 67.8, 67.4, 66.9, 61.4, 61.2, 54.9, 49.6, 47.0, 42.8, 41.0, 32.0, 26.6, 25.4, 23.1, 20.6, 20.5, 20.5, 20.2. HRMS calcd for C64H80N4O24 (M+ + Na) 1311.5062, found 1311.5069.

Analogues of C-Linked Antifreeze Glycoproteins

Benzyl tert-Butoxycarbonyl-L-alaninyl-L-alaninate (10). Boc-L-Ala-OBn (842 mg, 3.0 mmol) was dissolved in 10 mL of dry dichloromethane at stirred at room temperature. Trifluoroacetic acid (5 mL) was added, and the solution was stirred for 1 h after which time the mixture was concentrated under reduced pressure. The resulting residue was then added to a flask containing a dichloromethane solution of t-Boc-Ala-OH (586 mg, 3.0 mmol) and 1,1′-carbonyldiimidazole (CDI) (535 mg, 3.1 mmol) which had been stirring for 10 min under nitrogen. The entire solution was allowed to react for 1 h at room temperature and then washed successively with 5% aqueous hydrochloric acid, saturated sodium bicarbonate, and brine. The combined organic extracts were then dried over magnesium sulfate and concentrated. The crude product was then purified by flash chromatography (ethyl acetate/hexanes 1:3) to afford 517 mg (96% yield) of 10 as a colorless oil. IR (CH2Cl2): 3311, 1738, 1661 cm-1; 1H NMR (360 MHz, CDCl ) δ: 7.29 (5H, br s), 7.04 (1H, 3 br s), 5.37 (1H, d, J ) 7.4 Hz), 5.14 (1H, d, J ) 12.3 Hz), 5.08 (1H, d, J ) 12.3 Hz,), 4.56 (1H, quintet, J ) 7.1 Hz), 4.21 (1H, br s), 1.39 (9H, s), 1.35 (3H, d, J ) 7.2 Hz), 1.28 (3H, d, J ) 7.2 Hz); 13C NMR (90 MHz, CDCl3) δ: 172.4, 172.4, 155.3, 135.2, 128.4, 128.2, 127.9, 79.7, 66.8, 49.7, 47.9, 28.1, 18.3, 17.8. Benzyl Fluorenylmethoxycarbonyl-L-lysine(tertbutyloxycarbonyl)-L-alaninyl-L-alaninate (11). Compound 10 (0.624 g, 1.78 mmol) was dissolved in dry dichloromethane, and trifluoroacetic acid (8 mL) was added. The solution was stirred for 2 h at room temperature under nitrogen atmosphere after which time the reaction mixture was concentrated under reduced pressure. The resulting deprotected dipeptide was then added to a solution of Fmoc-Lys(Boc)OH (0.84 g, 1.8 mmol) and CDI (0.33 g, 2 mmol) dissolved in 10 mL of dichloromethane. The solution was stirred overnight at under nitrogen atmosphere and then washed successively with 5% HCl, water, and brine. The combined organic layers were dried using MgSO4, filtered, and concentrated under reduced pressure. Column chromatography was performed using hexanes:EtOAc (1:2) as eluant and afforded 1.0 g (74% yield) of the desired tripeptide 11. IR (CH2Cl2): 3288, 1738, 1683, 1644 cm-1; 1H NMR (360 MHz, CDCl3) δ: 7.68 (2H, d, J ) 7.4 Hz), 7.52 (2H, d, J ) 7.3 Hz), 7.26 (9H, m), 7.03 (1H, dd, J ) 11.1, 3.0 Hz) 5.85 (1H, br d, J ) 5.8 Hz), 5.11 (1H d, J ) 12.2 Hz), 5.05 (1H, d, J ) 12.3 Hz), 4.52 (2H, m), 4.31 (3H, m), 4.11 (3H, m), 3.03 (2H, br s), 1.74 (2H, br s), 1.51 (2H, br s), 1.37 (11H, br s), 1.31 (3H, d, J ) 5.9 Hz), 1.21 (3H, d, J ) 7.1 Hz); 13C NMR (360 MHz, CDCl3) δ: 172.4, 170.4, 156.7, 156.6, 144.5, 142.3, 135.2, 128.8, 128.7, 128.4, 127.9, 127.2, 125.2, 120.1, 67.6, 54.3, 49.3, 48.6, 47.3, 32.2, 29.8, 28.6, 22.5, 18.7, 18.1. LRMS (electrospray, MeOH, positive ion mode, m/z) calcd. for C39H48N4O8 (M+) 700.34; found 723.31 (M+ + Na). Benzyl Fluorenylmethoxycarbonyl-L-lysine[2-(2,3,4,6-tetra-O-tert-butyldimethylsilyl-r-D-galactopyranosyl)acetyl]-L-alaninyl-L-alaninate (12). Tripeptide 11 (370 mg, 0.528 mmol) was dissolved in CH2Cl2 (3 mL), and 2 mL of trifluoroacetic acetic was added. The solution was stirred at room temperature, under nitrogen atmosphere for 2 h, and then concentrated under reduced pressure. A solution of 14 (110 mg, 0.161 mmol) and CDI (40 mg, 0.242 mmol) dissolved in 10 mL of dichloromethane was stirred at room temperature, under nitrogen atmosphere for 1 h. Then, the deprotected tripeptide (0.37 g, 0.528 mmol) and N,N-diisopropylethylamine (0.2 mL) were added to the reaction mixture, and the solution was stirred overnight at room temperature. The crude

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reaction mixture was washed successively with 5% HCl, water, and brine. The organic extract was dried over MgSO4, filtered, and concentrated under reduced pressure. Purification of the crude product was accomplished using CH2Cl2/MeOH (50:1) and afforded 120 mg (57% yield) of the desired glycosylated tripeptide as a colorless oil. IR (CH2Cl2): 3288, 1716, 1644 cm-1; 1H NMR (300 MHz, CDCl3) δ: 7.74 (2H, d, J ) 7.4 Hz), 7.61 (2H, m), 7.31 (9H, m), 6.85 (1H, br d, J ) 7.7 Hz), 5.89 (1H, br d, J ) 4.6 Hz), 5.15 (1H, d, J ) 12.3 Hz), 5.12 (1H, d, J ) 12.4 Hz), 4.51 (1H, m), 4.46-4.12 (6H, m), 3.73 (2H, m), 2.70 (1H, m), 2.42-2.07 (1H, m), 1.50 (1H, br s), 1.35 (3H, d, J ) 6.0 Hz), 1.22 (3H, s), 0.88 (44H, m), 0.06 (24 H, m); 13C NMR (360 MHz, CDCl3) δ: 174.5, 172.5, 167.8, 156.3, 143.9, 141.3, 138.0, 132.4, 130.9, 127.0, 125.2, 121.0, 119.9, 107.7, 79.5, 73.1, 68.1, 66.9, 58.6, 47.1, 38.7, 31.9, 28.9, 25.7, 23.7, 23.0, 18.9, 18.5, 12.0, 10.9. 2-(2,3,4,6-Tetra-O-tert-butyldimethylsilyl-r-D-galactopyranosyl)acetic Acid (14). Ozone was bubbled into a solution of 13 (4.5 g, 6.8 mmol) dissolved in dry dichloromethane and cooled to -78 °C. Once the solution turned blue in color, nitrogen was bubbled through the solution until the blue color disappeared. Triphenylphosphine (2.0 g, 7.6 mmol) was added, and the solution was then allowed to warm to room temperature over a period of 3 h. This solution was then concentrated under reduced pressure, and the residue was redissolved in 10 mL of tert-butyl alcohol and 10 mL of 2-methyl-2-butene. Monobasic potassium phosphate (9.3 g, 68 mmol) and sodium chlorite (6.1 g, 68 mmol) were dissolved in 20 mL of water and added to the above solution. After stirring for 4 h at 0 °C, the solution was extracted with ethyl acetate. The organic extracts were washed successively with water and brine, and the solution was then dried over magnesium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (neat dichloromethane) to afford 3.6 g (78% yield) of 14 as a white solid. IR (CH2Cl2): 1722 cm-1; 1H NMR (360 MHz, CDCl ): 4.23 (3H, m,), 4.02 (1H, dd, 3 J ) 13.4,6.8 Hz), 3.74 (1H, t, J ) 3.0 Hz), 3.70 (1H, dd, J ) 12.0, 2.2 Hz), 3.51 (1H, d, J ) 3.7 Hz), 2.70 (1H, dd, J ) 16.6, 9.7 Hz), 2.38 (1H, dd, J ) 16.6, 3.9 Hz), 0.89 (9H, s), 0.87 (18H, s), 0.85 (9H, s), 0.03 (24H, m); 13C NMR (90 MHz, CDCl3) δ: 174.0, 79.2, 73.1, 72.8, 66.7, 63.3, 58.4, 36.0, 26.3, 25.9, 25.8, 25.6, 18.2, 18.0, 17.8, -4.5, -4.6, -4.99, -5.1, -5.3, -5.5. HRMS calcd for C32H70O7Si4 (M+ + H) 679.4277, found 679.4262. Benzyl Fluorenomethyloxycarbonyl-L-lysine[2(2,3,4,6-tetra-O-tert-butyldimethylsilyl-r-D-galactopyranosyl)acetyl]-L-prolylglycinate (15). Fmoc-Lys(Boc)-Pro-Gly-OBn (0.1582 g0.22 mmol) was dissolved in dry dichloromethane (5 mL), and TFA (0.15 mL) was stirred at room temperature for 1 h. The solution was concentrated under reduced pressure. Compound 14 (0.1513 g, 0.22 mmol) and CDI (0.0392 g, 0.25 mmol) were added to dry dichloromethane (5 mL). The resulting solution was stirred at room temperature, under nitrogen atmosphere. After 1 h, the deprotected tripeptide was dissolved in dry dichloromethane (3 mL) followed by the addition of diisopropyl ethylamine (0.3 mL). The solution was stirred for 12 h at room temperature and then washed successively with saturated ammonium chloride, water, and brine. The solution was dried over magnesium sulfate, filtered, and concentrated under vacuum. The crude product was purified by column chromatography (CH2Cl2:MeOH 30:1) and furnished 146 mg (60%yield) of 15 as a colorless oil. IR (CH2Cl2): 3333, 1716, 1644 cm-1; 1H NMR (300 MHz, CDCl3) δ: 7.65 (2H, d, J ) 7.3 Hz), 7.48 (2H, d, J ) 6.6 Hz), 7.27 (5H, m), 5.04 (2H, s),

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4.25 (4H, m), 4.18 (2H, d, J ) 7.8 Hz), 4.09 (6H, m), 3.91 (6H, m), 3.62 (8H, br s), 3.38 (2H, d, J ) 3.7 Hz), 1.39 (8H, m), 1.19 (1H, s), 0.80 (36H, m), -0.05 (24H, m,). 13C NMR (360 MHz, CDCl3) δ: 172.5, 172.0, 171.0, 169.4, 155.9, 143.7, 143.6, 141.1, 135.0, 134.6, 130.7, 128.6, 128.4, 128.4, 128.3, 128.2, 128.1, 127.5, 126.9, 124.9, 120.3, 119.8, 78.9, 73.6, 73.4, 72.9, 66.9, 66.8, 66.5, 63.1, 58.1, 52.0, 47.0, 41.2, 36.2, 29.5, 28.2, 25.9, 25.8, 25.8, 25.7, 25.6, 25.5, 25.5, 25.5, 25.4, 18.1, 17.9, 17.8, 17.8, 17.7, -3.7, -4.5, -4.6, -4.7, -4.8, -5.0, -5.0, -5.1, -5.2, -5.2, -5.7. LRMS (electrospray, MeOH, positive ion mode, m/z) calcd. for C67H108N4O12Si4 (M+) 1274.72; found 1274.71 (M+). HRMS calcd for C67H108N4O12Si4 (M+ + Na) 1295.6939, found 1295.6940. Benzyl Fluorenomethyloxycarbonyl-L-ornithine[2-(2,3,4,6-tetra-O-tert-butyldimethylsilyl-r-D-galactopyranosyl)acetyl]glycylglycinate (16). A solution of the Fmoc-Orn(Boc)-Gly-Gly-OBn (0.1037 g, 0.16 mmol) and dry dichloromethane (5 mL) and TFA (0.12 mL) was stirred at room temperature for 1 h, and then concentrated under reduced pressure. Compound 14 (0.1070 g, 0.16 mmol) and CDI (0.028 g, 0.17 mmol) were dissolved in dry dichloromethane (10 mL) and stirred at room temperature, under nitrogen atmosphere. After 1 h, the deprotected tripeptide was dissolved in dry dichloromethane (3 mL) and added to this reaction mixture, followed by diisopropyl ethylamine (0.27 mL). The solution was stirred overnight at room temperature, under nitrogen, and then washed successively with saturated ammonium chloride, water, and brine. The solution was dried over magnesium sulfate and concentrated under vacuum. The crude product was purified using column chromatography (CH2Cl2:MeOH 30:1) to yield 87 mg (53%) of 16. IR (CH2Cl2): 3311, 1738, 1677 cm-1; 1H NMR (300 MHz, CDCl3) δ: 7.65 (2H, d, J ) 7.44 Hz), 7.49 (2H, m), 7.28 (10H, m), 4.98 (2H, s), 4.23 (15H, m), 3.91 (8H, m), 3.66 (6H, m), 3.36 (2H, d, J ) 3.77 Hz), 2.71 (2H, dd, J ) 7.5 Hz), 2.25 (2H, dd, J ) 4.8 Hz), 0.81 (36H, m), -0.05 (24H, m); 13C NMR (360 MHz, CDCl3) δ: 172.9, 172.6, 172.3, 169.6, 169.3, 169.1, 169.0, 167.5, 156.4, 143.7, 143.6, 141.1, 136.3, 130.7, 128.4, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 127.5, 127.5, 126.9, 125.0, 125.0, 124.9, 119.8, 119.7, 115.8, 79.7, 73.0, 72.4, 67. 0, 66.9, 66.8, 66.8, 66.7, 63.5, 58.3, 58.2, 47.0, 41.0, 41.0, 36.9, 28.2, 28.1, 25.9, 25.9, 25.8, 25.7, 25.5, 25.5, 25.4, 18.1, 17.9, 17.9, 17.8, 17.7, 17.7, -4.5, -4.5, -4.6, -4.7, -4.8, -4.9, -5.0, -5.1, -5.4, -5.5, -5.6, -5.7. HRMS calcd for C63H102N4O12Si4 (M+ + Na) 1241.6469, found 1241.6465. RESULTS AND DISCUSSION

Building block 1 has been at the center of our preliminary work. The structure of 1 is different than that of the core repeating tripeptide unit in Figure 1 and serves to function as a structural analogue for the tripeptide unit in low molecular weight AFGP 7-8 which has the threonine substituted with an arginine residue (5, 6). The amide bond between the C-linked galactose and -amino group of the lysine side chain mimics the terminal guanidinium group of arginine. This may seem like a dramatic structural modification since lysine is not native to AFGPs; however, recent results have shown that polypeptides containing repeating L-lysine residues possess weak antifreeze protein-specific activity (22). In addition to the above modification, the R-D-galacosyl-βD-N-acetylgalactosamine found in AFGP has been truncated in 1. This is based upon early structure-function studies where the cis-3,4-dihydroxy group in the terminal

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galacatose residue was shown to be especially important for antifreeze protein-specific activity (23). This result is reminiscent of biologically significant oligosaccharides where in most cases, only the terminal residue(s) are necessary for tight interactions with a receptor (24). Traditionally, the driving force for adsorption of AFGP onto the ice surface is thought to involve hydrophilic interactions (hydrogen bonds) between specific hydroxyl groups of the carbohydrate moiety and the ice surface. While hydrogen bond formation is a very general phenomenon, molecular modeling studies of potential interactions between the R-D-galacosyl-β-D-N-acetylgalactosamine moiety and the ice surface imply that only two hydroxyl groups are ideally positioned to hydrogen bond to the ice lattice (25). This suggests that AFGP 8 (with four glycosylated tripeptide units) will form only eight hydrogen bonds with the ice surface. Given that individual hydrogen bonds are relatively weak in nature, it is difficult to rationalize the irreversible binding of AFGP to ice, and consequently it has been proposed that the hydroxyl groups are actually incorporated into the ice lattice. While the above hypothesis is based upon the notion that hydrophilic interactions are a dominant force at the protein-ice interface, recent evidence has shown that hydrogen bonding might be only a secondary interaction and hydrophobic interactions might predominate (26, 27). The assessment of AFGP analogues with structurally diverse carbohydrate moieties will lend valuable insight into hydrophilic interactions and the role they play in the molecular mechanism of action. Consequently, our laboratory has undertaken the preparation of C-linked AFGP analogues in which both the structure and orientation of a C-linked carbohydrate may be easily modified. One way to modify orientation of the saccharide residue is to alter the linkage between the saccharide and tripeptide. We are also interested in preparing AFGP analogues possessing multivalent saccharide derivatives of the native AFGP saccharide moiety. Multivalent saccharide derivatives have found numerous therapeutic applications as effective inhibitors of various carbohydrate protein interactions (28-30). As we wish to synthesize C-linked AFGP building blocks possessing different carbon-linked carbohydrate structures, the preparative methodology must be high yielding and general in nature. To accomplish this, we employ a general and highly stereoselective allylation reaction (Scheme 1) (31). Unfortunately, the resulting olefin was produced as an 88:12 mixture of R: β anomers, and such a mixture proved very difficult to separate. However, replacement of the acetate protecting groups with the less polar tert-butyldimethylsilyl (TBS) ethers allowed for clean separation of the desired R-anomer (19). After exchange of the TBS protecting groups with acetates, the resulting olefin was subjected to ozonolysis and furnished aldehyde 3 in 73% yield after purification. This sequence of reactions has been successfully utilized to prepare both the R- and/or β-D-mannose and glucose derivatives of 3. As previously discussed, one way to alter orientation of the carbohydrate residue in our building block is to utilize different covalent linkages between the carbohydrate and amino acid side chain. Consequently, we chose to modify the amide linkage in 1. To accomplish this we sought to employ sodium cyanoborohydride in a reductive amination protocol reported by Campbell et al. (32) where reaction of tripeptide 4 (19) with aldehyde 3 was expected to produce glycosylated tripeptide 5 (Scheme 2). However, this approach failed to produce even trace quantities of

Analogues of C-Linked Antifreeze Glycoproteins

Bioconjugate Chem., Vol. 12, No. 5, 2001 821

Scheme 1. Synthesis of C-Linked Saccharide

Scheme 2. Synthesis of Mono- and Divalent Building Blocks

5. Alternatively, sodium triacetoxy borohydride worked very well. As illustrated in Scheme 2, reaction of tripeptide 4 with 0.8 equiv of 3 in the presence of sodium triacetoxy borohydride furnished 5 in 55% yield. Twodimensional NMR studies (NOESY and ROESY) confirmed that the C-linked galactose residue in 5 adopts a rigid orientation (relative to peptide backbone) that is different than in 1. The glycosylated tripeptide building block 5 is a precursor to structurally diverse divalent building blocks. As illustrated in Scheme 2, reaction of 4 with 2.2 equiv of 3 and sodium triacetoxy borohydride (2.2 equiv) furnished divalent tripeptide 6 in 63% yield after purification. We also sought to synthesize divalent derivatives possessing both the amide and amine linkages between the saccharide residue and polypeptide backbone as represented by compound 8 in Scheme 2. However, reaction of 5 with 1.2 equiv of carboxylic acid 7 (19) using 1,1′-carbonyldiimidazole (CDI) failed to produce any trace of the desired product even at elevated temperatures. This reaction also failed in the presence of uronium coupling agents such as HBTU (O-benzotriazol-1-ylN,N,N′,N′,-tetramethyluronium hexafluorophophate) and HATU (O-(7-aza-benzotriazol-1-yl)-N,N,N′,N′,-tetramethyluronium hexafluorophophate).

Studies of the solution structure of AFGP 8 have implied the existence of a polyproline type II helix (33, 34). While this structural motif is believed to be essential for antifreeze activity, definitive evidence to support this hypothesis has not been published. Thus, a preparative methodology that affords structural diversity in the polyamide backbone is highly desirable. Of potential interest are polypeptide backbones that possess the L-alanyl-L-alanyl dipeptide of native AFGP and derivatives that incorporate L-proline residues. Since the glycylglycine dipeptide residue in the tripeptide sequence of 1, 5, 6, 8 is structurally dissimilar to the L-alanyl-L-alanyl dipeptide found in native AFGP, we prepared the glycosylated L-lysyl-L-alaninyl-L-alanine polypeptide backbone. Unfortunately, initial attempts to synthesize L-alanine benzyl ester 9 using standard procedures failed to produce 9 in high optical purity, and we were forced to employ an alternate approach. As illustrated in Scheme 3, reaction of commercially available N-tert-butoxycarbonylalanine with benzyl alcohol (2.5 equiv) in the presence of CDI furnished 9 as the trifluoroacetate salt in 82% yield after removal of the tert-butyl carbamate with trifluoroacetic acid. Analysis of the product by polarimetry confirmed that no racemization had taken place. Subsequent reaction of 9 with N-tert-butoxycar-

822 Bioconjugate Chem., Vol. 12, No. 5, 2001

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Scheme 3. Synthesis of Monovalent Building Block

Scheme 4. Synthesis of C-Linked Saccharide

Figure 4. Other structurally diverse C-linked AFGP building blocks.

bonylalanine using CDI and N,N-diisoproplyethylamine (DIPEA) in dichloromethane yielded dipeptide 10 in 96% isolated yield. Repetition of this sequence using Fmoclys(Boc)-CO2H furnished tripeptide 11 in 74% yield and a final CDI-mediated coupling between galactose derivative 14 and the deprotected derivative of 11 furnished the glycosylated tripeptide 12 in 57% yield after purification. The C-linked galactose derivative 14 was prepared as outlined in Scheme 4 (35) from 13 (19). Building block 12 is a structural analogue of the glycosylated tripeptide in low molecular weight AFGP where the L-threonine residue is substituted with arginine (5, 6). This preparative methodology affords endless structural variations in the polypeptide backbone using other natural amino acids or even synthetic amino acid derivatives that may serve as a peptide scaffold. To further demonstrate this, building block 15 (with L-proline incorporated into the polyamide backbone, Figure 4) was easily prepared in a convergent fashion using procedures and reagents analogous to that described in Schemes 3 and 4. This is important because it has been observed that one or both L-alanine residues may be substituted with L-proline in AFGP 7-8 (4). The reason for this substitution is not understood. It is likely that the structure and length of the amino acid side chain bearing the carbohydrate residue affects conformation and ultimately antifreeze activity. This is a complex issue since protein conformation is governed

by primary structure, glycosylation, and solvation (3638). The relationship between changes in the solution conformation of AFGP and antifreeze protein-specific activity has not been adequately investigated. Studies probing solution conformations of AFGP 8 have postulated the existence of an intramolecular hydrogen bond between the N-Ac of N-acetylgalactosamine and one of the carbonyl groups of the polyamide backbone (39). While the existence of such an interaction remains uncertain (40), an optimal distance between the carbohydrate residue and polyamide backbone may exist, and close proximity of the carbohydrate residue may have a profound effect upon the solution conformation of AFGP. Consequently, structurally diverse tripeptide units possessing different amino acid side chain lengths will be useful to study possible intramolecular interactions and probe solution conformations. Our preparative methodology can easily accommodate these structural modifications as evidenced by the convergent synthesis of 16 (Figure 4) in 66% overall yield (two steps). CONCLUSIONS

In summary, we have presented a preparative methodology for the synthesis of structurally diverse C-linked glycoconjugates useful for the solid-phase synthesis of chemically and biologically stable AFGP analogues. The approach centers on the preparation of carbon-linked glycosylated building blocks and permits structural modification of the saccharide component, polyamide backbone, and amino acid side chain. These building blocks are currently being assembled into discrete Clinked AFGP analogues. The in vitro assessment for antifreeze protein-specific activity will provide valuable insight into the structural features necessary to inhibit ice crystal growth. ACKNOWLEDGMENT

The authors wish to thank Petroleum Research Fund (ACS-PRF# 35280-G1), National Institutes of Health (grant no. GM60319), and A/F Protein Inc. for funding,

Analogues of C-Linked Antifreeze Glycoproteins

the National High Magnetic Field Laboratory (NHMFL) in Tallahassee, FL, for access to high-field NMR, SUNY College of Environmental Science and Forestry (ESF) for ES-MS analysis, and Dr. Jim Kerwin (Cornell University) for MADLI-MS. LITERATURE CITED (1) Yeh, Y., and Feeney, R. E. (1996) Structures and mechanisms of action. Chem. Rev. 96, 2. (2) Davies, P. L., and Sykes, B. D. (1997) Antifreeze proteins. Curr. Opinion Struct. Biol. 7, 828. (3) Ben, R. N. (2001) Antifreeze glycoproteins: preventing the growth of ice. ChemBioChem 2, 161. (4) Morris, H. R., Thompson, M. R., Osuga, D. T., Ahmed, A. I., Chan, S. M., Vandenheede, J. R., and Feeney, R. E. (1978) Antifreeze glycoproteins from the blood of an antarctic fish. J. Biol. Chem. 253, 5155. (5) Hew, C. L., Slaughter, D., Fletcher, G., and Shashikant, J. B. (1981) Antifreeze glycoproteins in the plasma of Newfoundland Atlantic Cod. Can. J. Zool. 59, 2186. (6) Raymond, J. A., Lin, Y., and DeVries, A. L. (1975) Glycoprotein and protein antifreezes in tow Alaskan fishes. J. Exp. Zool. 193, 125. (7) Ewart, K. V., Lin, Q., and Hew, C. L. (1999) Structure, function and evolution of antifreeze proteins. CMLS, Cell. Mol. Life. Sci. 55, 271. (8) Knight, C. A. (2000) Adding to the antifreeze agenda. Nature 406, 249. (9) Wilson, P. (1993) Explaining thermal hysteresis by the Kelvin Effect Cryo-Lett. 14, 31. (10) Hays, L. M., Feeney, R. E., Crowe, L. M., Crowe, J. E., and Oliver, A. E. (1996) Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions. Proc. Natl. Acad. Sci. U.S.A. 93, 6835. (11) Tablin, F., Oliver, A. E., Walker, N. J., Crowe, L. M., and Crowe, J. H. (1996) Membrane phase transition of intact human platelets: correlation with cold-induced activation. J. Cell. Phys. 165, 305. (12) Hansen, T. N., Smith, K. M., and Brockbank, K. G. M. (1993) Type I antifreeze protein attenuates cell recoveries following cryopreservation. Transplant. Proc. 25, 3182. (13) Griffith, M., and Ewart, K. V. (1995) Antifreeze proteins and their potential use in frozen foods. Biotechnol. Adv. 13, 375. (14) Jiaang, J. W., Hsiao, K. F., Chen, S. T., and Wang, K. T. (1999) Facile synthesis of a glycoprotein building block of antifreeze glycoprotein. Synthesis 9, 1687. (15) Elofsson, M., Salvador, L. A., and Kihlberg, J. (1997) Preparation of Tn and sialyl Tn building blocks used in Fmoc solid-phase synthesis of glycopeptide fragments from HIV gp120. Tetrahedron 53, 369 (and references therein). (16) For a recent comprehensive review on different glycopeptide mimetics see: Marcaurelle, L. A., and Bertozzi, C. R. (1999) New directions in the synthesis of glycopeptide mimetics. Chem. Eur. J. 5, 1384. (17) Ravishankar, R., Surdia, A., Vijuyan, M., Lim, S., and Kishi, Y. (1998) Preferred conformations of C-lactose at the free and peanut lectin bound states. J. Am. Chem. Soc. 120, 11297. (18) Wang, J., Kovac, P., Sinay, P., and Gluademans, C. P. (1998) Synthetic C-oligsaccharides mimic their natural, analogous immunodeterminants in binding to three monoclonal immunoglobulins. J. Carbohydr. Res. 308, 191. (19) Ben, R. N., Eniade, A. A., and Hauer, L. (1999) Synthesis of a C-linked antifreeze glycoprotein (AFGP) mimic: probes for investigating the mechanism of action. Org. Lett. 1, 1759. (20) For a convergent approach to C-linked AFGP analogues see: Eniade, A., and Ben, R. N. (2001) A fully convergent solid-phase synthesis of antifreeze glycoprotein analogues. Biomacromolecules 2, 557.

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