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Bioconjugate Chem. 1998, 9, 268−276
Novel Glycosynthons for Glycoconjugate Preparation: Oligosaccharylpyroglutamylanilide Derivatives Christophe Que´tard,† Sylvain Bourgerie,† Nadia Normand-Sdiqui,†,‡ Roger Mayer,† Ge´rard Strecker,§ Patrick Midoux,† Annie-Claude Roche,† and Michel Monsigny*,† Glycobiologie, Centre de Biophysique Mole´culaire, CNRS and Universite´ d’Orle´ans, F-45071 Orle´ans Cedex 2, France, and Laboratoire de Chimie Biologique, CNRS, Universite´ des Sciences et Technologies de Lille, F-59655 Villeneuve d’Ascq Cedex, France. Received July 2, 1997; Revised Manuscript Received October 15, 1997
The reducing sugar of an oligosaccharide reacting with the R-amino group of an amino acid is converted to an N-oligosaccharylamino acid which can then be stabilized by N-acylation. Oligosaccharides in solution in N,N-dimethylformamide reacted with R-glutamyl-p-nitroanilide at 50 °C for a few hours, leading to an N-oligosaccharylglutamyl-p-nitroanilide. Then, the γ-carboxylic group of the glutamyl moiety, activated by adding (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), reacted with the substituted R-amino group of the glutamyl residue, leading to an N-oligosaccharylpyroglutamyl-p-nitroanilide within 0.5 h. Such a one-pot two-step reaction was shown to be very efficient in the case of a disaccharide such as lactose, or pentasaccharides such as lactoN-fucopentaoses, Lewisa or Lewisx. The glycosynthons were characterized by chromatography (HPAEC and HPLC); their molecular mass was determined by electrospray ionization mass spectrometry, and the glycosylamides were shown to have a β-anomeric configuration on the basis of their proton NMR. The N-oligosaccharylpyroglutamyl-p-nitroanilides are quite stable at room temperature over a large pH range. They are easily converted to N-oligosaccharylpyroglutamyl-p-isothiocyanatoanilides which can be used to prepare glycoconjugates such as cationic glycosylated polylysines suitable for specifically delivering genes or oligonucleotides in a sugar-dependent manner.
INTRODUCTION
The sugar moieties of glycoconjugates are recognition signals which are involved in several important physiological phenomena [for reviews, see Monsigny et al. (1988b,c, 1994b), Lis and Sharon (1991), Drickamer and Taylor (1993), Varki (1993), Karlsson (1995), and Crocker and Feizi (1996)]. To make easier the investigation of the role of those recognition signals, synthetic glycoconjugates have been prepared, including neoglycoproteins, serum albumin substituted with about 25 simple sugar residues as well as glycosylated neutral polymers [for reviews, see Aplin and Wriston (1981), Stowell and Lee (1980), Duncan and Kopecek (1984), Monsigny et al. (1983, 1989, 1994a,b), Lee and Lee (1994a-c), Wong (1995), Roy (1996), and Gabius and Gabius (1997)]. While free mono- or disaccharides usually have a low affinity for lectins, those synthetic glycoconjugates have a high apparent affinity for lectins [Privat et al., 1974; Krantz et al., 1976; Townsend and Stahl, 1981; for reviews, see Stowell and Lee (1980), Monsigny et al. (1994a), Lee and Lee (1994), and Kiessling and Pohl (1996)] because of the presence of a large number of sugar units inducing a multiplicity effect related to avidity. Similarly, complex oligosaccharides have a much higher affinity than their various components. For instance, the galactose-specific hepatocyte lectin binds triantennary oligosaccharides with three Galβ-4GlcNAcβ moieties in * To whom correspondence shoud be addressed. Telephone: 33 2 38 25 55 57. Fax: 33 2 38 69 00 94. E-mail: monsigny@ cnrs-orleans.fr. † CNRS and Universite ´ d’Orle´ans. ‡ Current address: Innovir Ltd., Cambridge CB4 4FJ, United Kingdom. § Universite ´ des Sciences et Technologies de Lille.
a terminal nonreducing position, with an affinity 1000 times higher than that of monoantennary oligosaccharides with a single Galβ-4GlcNAcβ moiety in a terminal nonreducing position (Lee et al., 1983). On theses bases, neoglycoproteins and glycopolymers bearing a small number of complex oligosaccharides should be more efficient and more specific than those bearing simple sugars. To prepare such glycoconjugates, several methods leading to activated oligosaccharide derivatives suitable to be covalently linked to either a protein, a polymer, or a matrix have been described. These methods belong to three main categories. The first one is a total de novo synthesis [for reviews, see Garg et al. (1994), Sinay¨ (1991), Lee and Lee (1992, 1994a-c), Meldal (1994a,b), Roy (1996), Whitfield and Douglas (1996), and Von Itzstein and Colman (1996)] which requires many protection, coupling, and deprotection steps; this approach is time-consuming, and the overall yield remains relatively low. In the second approach, the oligosaccharide can be reversibly converted into a glycosylamine by incubation in the presence of a high concentration of ammonia, an ammonium salt, an aliphatic amine, or an aromatic amine [for reviews, see Ellis and Honeyman (1955), Isbell and Frush (1980), Aplin and Wriston (1981), Kallin (1994), Rice and Corradi Da Silva (1996), and Roy (1996)]. The glycosylamines must be further stabilized by acylation [see, for instance, Isbell and Frush (1958), Thomas (1970), and Likhosherstov et al. (1986)], the acylating agent being usually an organic compound containing an activated carboxylic group. However, this method suffers from two major disadvantages. One is that it requires several intermediary reactions and purification steps. The second is that there is at least one step in alkaline medium which may release any acyl substituent borne
S1043-1802(97)00122-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/13/1998
Glycosynthons for Glycoconjugate Synthesis
on the oligosaccharide. The third approach proposed by Gray (1974) is a reductive amination, which allows a onestep glycoconjugate synthesis. This method leads to an altered oligosaccharide, the reducing sugar being transformed into a linear polyol amine derivative. In a previous paper (Sdiqui et al., 1995), we reported a one-pot two-step synthesis of N-glycoamino acids: the first step was the coupling of an oligosaccharide with R-glycyl-p-nitroanilide (Gly-pNA1 ), and the second step was an acylation of the glycosylamine formed. Here, we describe the synthesis of novel glycosylamine derivatives by coupling R-glutamyl-p-nitroanilide (Glu-pNA) to reducing oligosaccharides. With regard to the previous method described by Sdiqui et al. (1995), this new method has two main advantages. First, the formation of the glycosylamine is faster. Second, the acylation step is an intramolecular reaction involving the γ-carboxylic group of glutamic acid, and as a consequence, it is rapid, efficient, and highly specific. The glycoamino acids obtained can be further coupled to any molecule bearing an amino group. EXPERIMENTAL PROCEDURES
Materials. R-Glutamyl-p-nitroanilide (Glu-pNA) was purchased from Bachem (Bubendorf, Switzerland). Poly(L-lysine) (pLK) HBr salt (Mr ) 95 100) with a mean degree of polymerization of 455 (pLK455) and -aminocaproic acid (Aca) were purchased from Sigma (St. Quentin Fallavier, France); pLK, HBr was transformed into pLK, p-toluenesulfonate (Derrien et al., 1989) to increase the solubility of the polymer in organic solvents and to avoid the cytotoxic effect of the bromide anion. Lactose and N,N-diisopropylethylamine (DIEA) were from Janssen Chimica (Beerse, Belgium); imidazole and dimethyl sulfoxide (DMSO) were from Merck (Darmstadt, Germany). (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) was from Richelieu Biotechnologies (Saint Hyacinthe, Canada). N,N-Dimethylformamide (DMF) was freshly distilled in the presence of 0.2 mg/mL (benzyloxycarbonyl)glycyl-p-nitrophenyl ester. N,N′-Thiocarbamylbisimidazole (TCBI), p-toluenesulfonic acid, and δ-gluconolactone were purchased from Aldrich (St. Quentin Fallavier, France); lacto-N-fucopentaose II [Lewisa, Galβ-(FucR4)3GlcNAcβ-3Galβ-4Glc] and III [Lewisx, Galβ-(FucR3)4GlcNAcβ-3Galβ-4Glc] (as a mixture) were isolated from human milk (G. Strecker, unpublished data). Chromatography. Conjugates were purified by gel filtration on a column (2.3 × 90 cm) of Trisacryl GF 05 (Biosepra, Villeneuve-la-Garenne, France) stabilized in and eluted with 0.1 M acetic acid containing 3% n-butanol (flow rate, 8 mL/h). High-performance anion-exchange chromatography (HPAEC) was performed on a Dionex DX-300 chromatography system (Sunnyvale, CA) which includes a quaternary gradient pump, an eluent degas (He) module, a (4 × 250 mm) CarboPac PA1 column with a matching guard column, a pulsed amperometric detector (PAD), and a PD40 diode array detector. The flow rate was 1 1 Abbreviations: Aca, -aminocaproic acid; BOP, (benzotriazol1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Glu-pNA, R-glutamyl-p-nitroanilide; Gly-pNA, R-glycyl-p-nitroanilide; HOBt, 1-hydroxybenzotriazole; HPAEC, high-performance anion-exchange chromatography; paA, p-aminoanilide; PITC, phenyl isothiocyanate; pLK, poly(L-lysine); pNA, p-nitroanilide; TCBI, N,N′thiocarbamylbisimidazole; TEAA, triethylammonium acetate.
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mL/min. The gradient was obtained by using two solutions (solvent A, 0.1 N sodium hydroxide; and solvent B, 1 M sodium acetate in 0.1 N sodium hydroxide). The column was equilibrated with solution A and eluted with solution A for 5 min and linear gradients of solution B up to 20% over the course of 10 min and then up to 100% over the course of 15 min. High-performance liquid chromatography (HPLC) analysis was performed on a reverse phase (4 × 250 mm) column (LiChrospher 100 RP-18, 5 µm, Merck) to monitor the reduction of N-(β-lactosyl)-pGlu-pNA into N-(β-lactosyl)-pGlu-p-aminoanilide and its conversion into N-(βlactosyl)-pGlu-p-amidophenyl isothiocyanate [N-(β-lactosyl)-pGlu-p-amidoPITC]; a reverse phase (3.9 × 150 mm) cartridge column (Nova-Pak C18, 4 µm, Waters) was used to monitor the coupling of Aca with N-(β-lactosyl)pGlu-p-amidoPITC. The HPLC system included a dual pump system monitored by a Waters 600 Controller linked to a Waters 996 photodiode array detector. The mobile phase was solvent C [0.1 M triethylammonium acetate (TEAA, pH 7.0) and 5% acetonitrile] and solvent D [acetonitrile and 0.1 M TEAA (5%)]. The flow rate was 1 mL/min. The column was equilibrated with a mixture of solvents C and D (95:5, v/v). After the sample (20 µL) was injected, the column was eluted for 5 min with a 95% C/5% D mixture; solvent D then increased linearly from 5 up to 30% over the course of 30 min. Electrospray Ionization Mass Spectrometry (ESIMS). The positive ion electrospray mass spectra were obtained using a Platform quadrupole mass spectrometer (VG Biotech, Fisons Instruments, Altrincham, U.K.) equipped with an electrospray atmospheric pressure ionization source. The capillary voltage was set at 3 kV, and the cone voltage was adjusted at 25 V. The analyzer was calibrated for m/z from 400 to 1200 in the positive ion mode using a cesium iodide solution. The sample was dissolved in an acetonitrile/1% formic acid mixture (1:1, v/v) at 20-50 pmol/mL. 1H NMR Spectroscopy. For 1H NMR analysis, compounds were dissolved at room temperature in deuterated water at 5 mg/mL, once with D2O containing 99.9% D and, after freeze-drying, a second time with D2O containing 99.96% D (Sigma). 1H NMR spectroscopy was performed at 300 MHz on a Bruker AM-300 spectrometer (Wissembourg, France). Chemical shifts were measured with acetone (δ ) 2.225 ppm in D2O) used as an internal reference and were expressed in parts per million downfield from internal sodium 4,4-dimethyl-4-silapentane1-sulfonate (DSS). Synthesis, Purification, and Characterization of N-β-Oligosaccharyl-pGlu-pNA. (1) N-(β-Lactosyl)pGlu-pNA (4). In a typical experiment, 5 mg (15 µmol) of lactose (2, peak 2 in Figure 2A), 7.8 mg (30 µmol) of Glu-pNA, and 4 mg (60 µmol) of imidazole (1, peak 1 in Figure 2A) were dissolved in 250 µL of DMF. The solution was kept at 50 °C for 8 h. Without any purification, the N-(β-lactosyl)-Glu-pNA (3, peak 3 in Figure 2) was further intramolecularly N-acylated at room temperature by adding 14.6 mg (33 µmol) of BOP and 4 mg (60 µmol) of imidazole. The formation of lactosylpyroglutamyl-p-nitroanilide, N-(βlactosyl)-pGlu-pNA (4, peak 4 in Figure 2), monitored by HPAEC, was found to be complete within 30 min (Figure 2). N-(β-Lactosyl)-pGlu-pNA (4) was purified by gel filtration. The reaction mixture diluted up to 10 mL with solvent E (0.1 M acetic acid containing 3% n-butanol) was introduced onto a Trisacryl GF 05 column (2.3 × 90 cm) using solvent E as an eluent at a flow rate of 8 mL/h.
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Figure 1. Synthesis of glycoamino acid species: I, coupling; and II, cyclization. (a) Reducing oligosaccharide. (b) R-Glu derivative, Glu-pNA when R is p-nitroanilide (NHC6H4NO2). (c) N-Oligosaccharyl-Glu-pNA. (d) N-Oligosaccharyl-pGlu-pNA.
The glycoamino acid came out first; the other reagents and the coupling solvent came out later. As shown by HPAEC, the purified product still contained some imidazole. The glycoconjugate was made free of imidazole by precipitation from its water solution upon adding 9 volumes of ethanol. Using electrospray ionization mass spectrometry, the mass of the molecular ion was 574.2 (Figure 3A), equal to that calculated for C23H31N3O14 [(M + H)+, 574.1]: 1H NMR (300 MHz, D2O) δ 8.29 and 7.75 (4H, 2d, H aromatic), 5.23 (1H, d, J1,2 ) 8.25 Hz, H-1, β-D-Glc), 4.65 (1H, d, J1,2 ) 7.83 Hz, R CH pGlu), 4.37 (1H, d, J1,2 ) 7.51 Hz, H-1 β-D-Gal), 2.83 (1H, m, γ CH2 pGlu), 2.56 (2H, m, γ′ CH2 and β CH2 pGlu), 2.28 (1H, m, β′ CH2 pGlu). (2) Lewisa/Lewisx-pGlu-pNA. Lewisa/Lewisx oligosaccharides were treated under the conditions described for the synthesis of lactosyl-pGlu-pNA. The mass of the molecular ion, using an electrospray ionization mass spectrometer [calculated for C43H64N4O28 (M + H)+, 1085.4], was found to be 1085.6 (Figure 3B). The assigment of the Lewisa/Lewisx-pGlu-pNA 1H NMR spectrum in D2O was as follows: resonances common to the two glycopeptides δ 8.33 and 7.79 (4H, 2d, H aromatic), 4.90 (2H, m, H-5 R-L-Fuc), 4.67 (1H, d, J1,2 ) 7.32 Hz, H-1 β-D-GlcNAc), 4.63 (1H, d, R CH pGlu) 4.37 (1H, d, J1,2 ) 7.42 Hz, H-1 β-D-Galint), 4.16 (1H, s, H-4 β-D-Galint), 2.83 (H, m, γ CH2 pGlu), 2.58 (2H, m, γ′ and β CH2 pGlu), 2.33 (H, m, β′ CH2 pGlu), 2.05 and 2.04 (6H, 2s, CH3 Ac β-D-GlcNAc), 1.22 and 1.21 (6H, 2s, CH3 R-LFuc); Lewisa-specific δ 5.05 (1H, d, H-1 R-L-Fuc); 4.53 (1H, d, H-1 β-D-Gal); Lewisx-specific δ 5.24 (1H, d, J1,2 ) 7.2 Hz, H-1 R-L-Fuc); 4.50 (1H, d, H-1 β-D-Gal). Stability of Glycosyl-pGlu-pNA. To assess the stability of the glycosyl-pGlu-pNA derivatives, we exposed the lactosyl-pGlu-pNA (4) at various pHs. The
Figure 2. Conversion of lactose to N-(β-lactosyl)-pGlu-pNA. HPAE chromatography analysis monitored using (A) amperometry and (B) absorbance at 316 nm: (a) after 10 min, (b) after incubation for 6 h, and (c) after the cyclization step. Peak 1, imidazole; peak 2, lactose; peak 3, N-(β-lactosyl)-Glu-pNA; peak 4, N-(β-lactosyl)-pGlu-pNA; and peak 5, HOBt.
Figure 3. Electrospray ionization mass spectra of (A) N-(βlactosyl)-pGlu-pNA (4) and (B) Lewisa/Lewisx-pGlu-pNA.
formation of putative degradation products was monitored by HPAEC, using sorbitol as an internal standard. The conjugate (1 mg/mL) and sorbitol (500 µg/mL) were maintained up to 48 h at either 25 or 90 °C in an acidic environment (50 mM sodium acetate buffer at pH 3.6), in a neutral medium (50 mM sodium phosphate buffer at pH 7.0), or in a basic environment (50 mM sodium carbonate buffer at pH 9.0). Reduction of N-(β-Lactosyl)-pGlu-pNA (4) and Conversion into N-(β-Lactosyl)-pGlu-p-amidophen-
Glycosynthons for Glycoconjugate Synthesis
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Figure 4. Time course of the formation of (b) N-(β-lactosyl)Glu-pNA and comparison with the formation of (1) N-(βlactosyl)-Gly-pNA (inset). Each reaction was performed in DMF at 50 °C in the presence of imidazole. The conjugate formation was monitored by HPAE chromatography. Figure 6. Time course of the coupling of -aminocaproic acid (5 equiv) with N-(β-lactosyl)-pGlu-p-amidoPITC (1 equiv) monitored by HPLC at 290 nm: after (a) 1 min, (b) 45 min, and (c) 100 min. Peak 7 is N-(β-lactosyl)-pGlu-p-amidoPITC, and peak 8 is -[N-(β-lactosyl)-pGlu-p-amidophenylthioureido]caproic acid.
Figure 5. Preparation of N-(β-lactosyl)-pGlu-p-amidoPITC, monitored by HPLC analysis at 290 nm. Reduction of N-(βlactosyl)-pGlu-pNA (4) into N-(β-lactosyl)-pGlu-paA (6) and conversion to N-(β-lactosyl)-pGlu-p-amidoPITC (7).
yl Isothiocyanate [7, N-(β-Lactosyl)-pGlu-p-amidoPITC]. N-(β-Lactosyl)-pGlu-pNA (4) (5 mg, 8.7 µmol) was dissolved in 1 mL of a water/methanol mixture (1:1, v/v). To this solution was added 2 mg of 10% palladium on charcoal (Merck). The suspension was stirred for 3 h under hydrogen (1 atm) at room temperature. Both N-(βlactosyl)-pGlu-pNA (4, peak 4 in Figure 5) and its reduced derivative N-(β-lactosyl)-pGlu-p-aminoanilide [N-(β-lactosyl)-pGlu-paA] (6, peak 6 in Figure 5) were monitored by HPLC at 290 nm. The palladium on charcoal was removed by centrifugation. The methanolic solution was treated at room temperature with 23 µL of a TCBI (13 µmol) solution at 100 mg/mL in chloroform. After the mixture was stirred for 30 min, the N-(β-lactosyl)-pGlu-p-amidoPITC (7, peak 7 in Figure 5) was obtained in a good yield, as shown by HPLC at 290 nm (Figure 5). One volume of chloroform was added to extract the excess of TCBI. The organic phase was removed upon centrifugation (3000g for 5 min). Then methanol was removed under reduced pressure. Lewisa/Lewisx-pGlu-pNA was reduced according to the same procedure. Coupling of E-Aminocaproic Acid (Aca) with N-(βLactosyl)-pGlu-p-amidoPITC (7). Three milligrams
(5.2 µmol) of N-(β-lactosyl)-pGlu-p-amidoPITC (7, peak 7 in Figure 6) was added to Aca (3.4 mg, 25 µmol) in 800 µL of 0.3 M NaCl, 0.1 M sodium bicarbonate buffer at pH 9.3. The mixture was stirred at room temperature and monitored by HPLC at 290 nm. The glycosylated Aca, -[N-(β-lactosyl)-pGlu-p-amidophenylthioureido]caproic acid (8, peak 8 in Figure 6) was obtained after 100 min in 90% yield as calculated from HPLC. The pure compound was isolated by collecting the corresponding HPLC peak and evaporation of the solvent. The mass of the molecular ion using an electrospray ionization mass spectrometer [calculated for C30H44N4O14S1 (M + H)+, 717.3] was 717.2: 1H NMR (300 MHz, D2O) δ 7.56 and 7.34 (4H, 2d, H aromatic), 5.22 (1H, d, H-1 β-D-Glc), 4.63 (1H, d, R CH pGlu), 4.40 (1H, d, H-1 β-D-Gal), 3.57 (2H, m, CH2-6 Aca), 2.83 (1H, m, γ CH2 pGlu), 2.58 (2H, m, γ′ CH2 and β CH2 pGlu), 2.28 (1H, m, β′ CH2 pGlu), 2.22 (2H, t, CH2-2 Aca), 1.61 (4H, m, CH2-3 and 5 Aca), 1.38 (2H, m, CH2-4 Aca). Preparation of the (Lewisa/Lewisx)100-pLK455 Conjugate. Lewisa/Lewisx-pGlu-p-amidoPITC (8 mg, 7.4 µmol) was added to p-toluenesulfonate poly(L-lysine) (9.0 mg, 0.0655 µmol) dissolved in 1 mL of DMSO in the presence of DIEA (8.2 µL, 58 µmol). The mixture was stirred overnight at room temperature. The glycosylated pLK was precipitated by adding 10 volumes of 2-propanol and centrifuged (1800g for 15 min). The pellet was washed with 2-propanol, collected upon centrifugation (1800g for 15 min), solubilized in distilled water, and freeze-dried. The average number of Lewisa/Lewisx residues bound per pLK molecule was determined using the colorimetric resorcinol sulfuric acid micromethod (Monsigny et al., 1988a). RESULTS
Synthesis of N-(β-Lactosyl)-pGlu-pNA. The general scheme for the synthesis is shown in Figure 1. The first step (I) was the coupling between a reducing oligosaccharide (a) and R-glutamyl-p-nitroanilide (b) in the presence of imidazole. Efficient and rapid coupling between the hemiacetal hydroxyl group of the reducing saccharide and the R-amino group of glutamic acid occurred by dehydration. The second step (II) was an intramolecular N-acylation (d) of the glycoamino acid
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derivative. The reaction was monitored by HPAEC by using two detectors (amperometric and UV). Chromatograms in panels A and B of Figure 2 showed that the amount of N-(β-lactosyl)-Glu-pNA (3, peak 3 in Figure 2A,B), eluted at 29.2 min, increased as the reaction proceeded; concomitantly, the amount of lactose (2, peak 2 in Figure 2A), which came out at 4.9 min, decreased. The time course of the coupling between Glu-pNA and lactose, as monitored by HPAEC, showed that the formation of N-lactosyl-Glu-pNA was fast; after 2 h, 80% of the lactose was already converted into lactosyl-Glu-pNA, and after 6 h, 95% was converted (Figure 4). Comparatively, the time course of the coupling between lactose and GlypNA was much slower (Figure 4, inset); 120 h was required to reach the 95% level of reaction (Sdiqui et al., 1995). Stabilization of the glycopeptide with 1.1 equiv of BOP and an additional 2 equiv of imidazole led to N-(βlactosyl)-pGlu-pNA (4, peak 4 in Figure 2A,B) which came out at 23 min (Figure 2A,B, chromatograms c). Stability of N-(β-Lactosyl)-pGlu-pNA (4). The lactosyl-pyroglutamate conjugate maintained at 25 °C for 48 h at pH 7.0, 3.6, or 9.0 was quite stable; neither a decrease in the N-(β-lactosyl)-pGlu-pNA (4) nor the appearance of a new compound could be detected. At 90 °C, the N-(β-lactosyl)-pGlu-pNA (4) was stable as long as the pH was either slightly acidic (pH 3.6) or neutral (pH 7.0), and no degradation product could be detected even after 5 h. In contrast, in alkaline medium (pH 9.0), the concentration of N-(β-lactosyl)-pGlu-pNA (4) decreased according to a first-order kinetics with a constant of 1.25 × 10-4 s-1; a second, still unidentified compound (retention time, 11.5 min) appeared, and its amount increased correlatively. Reduction of N-(β-Lactosyl)-pGlu-pNA (4), Conversion into N-(β-Lactosyl)-pGlu-p-amidoPITC (7), and Its Reaction with E-Aminocaproic Acid. The pure product 4 [N-(β-lactosyl)-pGlu-pNA, peak 4 in Figure 5] was quantitatively reduced into 6 [N-(βlactosyl)-pyroglutamyl-p-aminoanilide [N-(β-lactosyl)pGlu-paA, peak 6], in the presence of palladium (10% Pd) on charcoal under a hydrogen pressure of 1 atm. The reduced derivative 6 then reacted with TCBI and was converted into N-(β-lactosyl)-pyroglutamyl-p-isothiocyanatoanilide [N-(β-lactosyl)-pGlu-p-amidoPITC], compound 7 (peak 7 in Figure 5), within 30 min. The arylisothiocyanate group was further corroborated by infrared spectroscopy (ν, 2120 cm -1) and by UV spectroscopy on the basis of its maximal absorbance at 290 nm. To test the reactivity of this compound with an ω-alkylamine, the coupling reaction between 7 [N-(βlactosyl)-pGlu-p-amidoPITC] and -aminocaproic acid (Aca) was monitored by HPLC at 290 nm (Figure 6). A complete disappearance of 7, N-(β-lactosyl)-pGlu-p-amidoPITC, occurred within 2 h, leading to the formation of 8 [-[N-(β-lactosyl)-pGlu-p-amidophenylthioureido]caproic acid]. Compound 8 was collected and characterized by electrospray ionization mass spectrometry and proton magnetic resonance (1H NMR). Both spectra were consistent with the expected product (see Experimental Procedures). Synthesis of Lewisa/Lewisx-pGlu-pNA and Preparation of pLK-Lewisa/Lewisx. Lewisa/Lewisx oligosaccharides were reacted with R-glutamyl-p-nitroanilide using the conditions described for the N-(β-lactosyl)-pGlupNA synthesis. This one-pot two-step procedure was also found to be very successful with such relatively complex oligosaccharides. The purified products were obtained, as described in detail in Experimental Procedures, upon gel filtration on a Trisacryl GF05 column and ethanolic
Que´tard et al.
precipitation. The purified compound, Lewisa/LewisxpGlu-pNA, was shown to be the expected glycoamino acid derivative by 1H NMR spectroscopy (see Experimental Procedures) and ESI-MS analysis, with a molecular ion peak at m/z 1085.6 (Figure 3B) corresponding to the calculated value. Under the experimental conditions used for N-(βlactosyl)-pGlu-pNA, Lewisa/Lewisx-pGlu-pNA was quantitatively reduced to the Lewisa/Lewisx-pGlu-p-aminoanilide and then, upon reaction with TCBI, was converted to the Lewisa/Lewisx-pGlu-p-amidoPITC. The coupling of Lewisa/Lewisx-pGlu-p-amidoPITC with pLK455 under the conditions described above was achieved with a yield leading to a poly(L-lysine) substituted with 100 Lewisa/Lewisx per pLK molecule. DISCUSSION
Natural oligosaccharides isolated from biological fluids or released by enzymatic or chemical means from natural glycoconjugates can be used for many purposes, especially to synthesize conjugates such as glycopolymers, neoglycoproteins, neoglycolipids, or even glycosylated prodrugs. Among the various described methods, the transformation of reducing oligosaccharides to glycosylamines or glycosylamine derivatives is quite attractive. Glycosylamines have been obtained as crystalline substances arising from sugars dissolved in methanol containing ammonia (Lobry de Bruyn, 1895; Lobry de Bruyn and Franchimont, 1893; Lobry de Bruyn and Van Leent, 1895, 1896). Glycosylamines were also prepared upon incubation of sugars in the presence of a saturated ammonium bicarbonate solution for up to 7 days (Likhosherstov et al., 1986; Kallin et al., 1989; Otvos et al., 1989; Urge et al., 1991, 1992; Manger et al., 1992a,b; Vetter and Gallop, 1995) or in the presence of 0.2 M ammonium bicarbonate in 16 M aqueous ammonia for 36 h (Lubineau et al., 1995). Alternatively, β-glycosylamines may be obtained from N-type glycoproteins upon hydrolysis of the linkage between the oligosaccharylamide and the β-carboxylic group of the aspartyl residue by N-glycanase under alkaline conditions (Rasmussen et al., 1992; Tarentino et al., 1993). Unfortunately, glycosylamines are readily hydrolyzed in neutral or slightly acidic aqueous solution (Lobry de Bruyn and Van Leent, 1895; Frush and Isbell, 1951; Isbell and Frush, 1958). Glycosylamines were stabilized by acylation using various simple organic acids activated as acid chloride, anhydride, mixed anhydride, N-succinimide ester, etc. [for reviews, see Ellis and Honeyman (1955), Thomas (1977), and Garg et al. (1994)]. Alternatively, glycosylamines were stabilized as a glycoamino acid or a glycopeptide [for reviews, see Ellis and Honeyman (1955) and Garg et al. (1994)] by acylation with an activated amino acid (Otvos et al., 1989; Urge et al., 1991, 1992; Gupta and Surolia, 1994; Tamura et al., 1994; Arsequel et al., 1994; Wadhwa et al., 1995) and/or a peptide bearing a single activated carboxylic group (Anisfeld and Lansbury, 1990; Cohen-Anisfeld and Lansbury, 1993; Wong et al., 1993). Glycosylamines were also stabilized by acylation with a bifunctional organic compound such as acryloyl chloride (Kallin et al., 1989) or disuccinimidyl suberate (Vetter et al., 1995) or with an activated halogeno (bromoacetic, chloroacetic, or iodoacetic) acid (Thomas, 1970; Manger et al., 1992a,b; Wong et al., 1994) or chloroacetic anhydride (Manger et al., 1992a,b), the second function being used later to achieve a further substitution. Glycosylamines may also be acylated by enzymatic β-aspartylation (Mononen et al., 1996).
Glycosynthons for Glycoconjugate Synthesis
Glycopeptides may also be obtained by direct coupling of a reducing sugar and a peptide. Dixon (1972) found that NR-glucosylpeptides were formed upon incubation of glucose and short peptides such as Val-His or Ile-Tyr in the presence of pyridine and acetic acid for 3 days. The conjugate was stable enough to be separated by paper electrophoresis. The equilibrium constant for the dissociation of Glc-Val-His to Val-His in aqueous solution at pH 6.2 was found to be 0.3 M. The relatively high stability of such glucopeptides may be related to the steric hindrance of the side chain of the N-terminal amino acid (Val or Ile) substituted by the glucosyl residue or to the Amadori rearrangement [for reviews, see Hodge (1955), Hodge and Fisher (1963), and Lee and Cerami (1992)]. In contrast, glycosylamino acid such an oligosaccharidylGly-pNA containing a glycyl residue was found to be readily hydrolyzed in neutral aqueous medium (Normand-Sdiqui, 1995; Sdiqui et al., 1995), and an Nacylation was required to stabilize it. To simplify the stabilization step, we developed another class of glycoamino acid derivatives in which a glutamic acid derivative was used. The γ-COOH function of the glutamic acid side chain, upon activation, readily reacts with the R-amino group, leading to a pyroglutamate derivative; this intramolecular cyclization occurred with an excellent yield. The N-glycopyroglutamyl conjugate is perfectly stable at room temperature in aqueous medium tested at any pH in the 3.6-9.0 range. It is also quite stable at high temperatures in a slightly acidic or a neutral medium; no degradation was observed after exposure at 90 °C for 5 h. In contrast, the compound was degraded in alkaline medium (pH 9.0) at 90 °C with a half-life of 85 min. The main advantage of using a glutamic acid derivative is that the intramolecular cyclization step does not require any additional acylating reagent; the addition of a carboxylic group activator is sufficient. The formation of the pyroglutamyl derivative is very fast and actually stoichiometric, and no other acylation reaction could be detected. This contrasts with the N-acylation of the glyco-Gly-pNA which requires the addition of an acetylating agent, leading in some cases to a partial Oacetylation in addition to the expected N-acylation. The reaction conditions used here to couple lactose with Glu-pNA are similar to those used previously for the coupling of lactose with Gly-pNA; i.e., the reaction occurs in DMF at 50 °C with 2 equiv of Glu-pNA and 4 equiv of imidazole. However, the coupling reaction was faster in the case of Glu-pNA than in that of Gly-pNA. Indeed, the times required to reach 50% of the reaction leading to N-(β-lactosyl)-Gly-pNA and to N-(β-lactosyl)-Glu-pNA were 30 and
